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Protection of the proteolytic activity of crude papain and chemical modification of papain by tetrathionate Arteaga Mac Kinney, Guillermo Eleazar 1988

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PROTECTION OF THE PROTEOLYTIC ACTIVITY OF CRUDE PAPAIN AND CHEMICAL MODIFICATION OF PAPAIN BY TETRATHIONATE by G u i l l e r m o E l e a z a r Arteaga Mac Kinney Biochemical Engineer Manager i n Food P r o c e s s i n g , The I n s t i t u t e of Technology and Higher S t u d i e s of Monterrey, Mexico, 1984 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n THE FACULTY OF GRADUATE STUDIES (The Department of Food Science) We accept t h i s t h e s i s as conforming to the r e q u i r e d standard THE UNIVERSITY OF BRITISH COLUMBIA JULY 1988 © G u i l l e r m o E. Arteaga Mac Kinney , 1988 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of f-poci ^ c i e w c g The University of British Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 ABSTRACT In the f i r s t c hapter, sodium t e t r a t h i o n a t e (TT), a s u l f h y d r y l b l o c k i n g agent, i s assessed f o r i t s a b i l i t y to p r o t e c t the p r o t e o l y t i c a c t i v i t y (PA) of papaya l a t e x d u r i n g a i r , sun or vacuum d r y i n g , and of crude papain d u r i n g s t o r a g e . By means of Taguchi's L 2 ? ( 3 X 3 ) f r a c t i o n a l f a c t o r i a l d e s ign, i t was found t h a t the a d d i t i o n of 1% TT s i g n i f i c a n t l y i n c r e a s e d the r e t e n t i o n of PA of papaya l a t e x when i t was a i r d r i e d a t a temperature of 55°C. T h i s p r o t e c t i o n of PA was found to be 23% higher than the one given by the a d d i t i o n of 1% sodium m e t a b i s u l f i t e , the compound commonly used i n the commercial p r o c e s s i n g of papaya l a t e x . When d r y i n g was c a r r i e d out e i t h e r under 27 inches vacuum a t 50°C or i n the sun, the p r o t e c t i v e e f f e c t of TT on the PA was not s i g n i f i c a n t l y d i f f e r e n t from t h a t of m e t a b i s u l f i t e . The PA of crude papain d u r i n g storage at room temperature was a l s o p r o t e c t e d by TT. A l o s s of 20% of the o r i g i n a l PA occurred over a p e r i o d of 13 wk when crude papain contained 1% TT, compared to a l o s s of 45% when the crude enzyme p r e p a r a t i o n c o n tained 1% m e t a b i s u l f i t e . In the same chapter f i v e d i f f e r e n t oxidants f o r s y n t h e s i s of TT from t h i o s u l f a t e are compared, namely: i o d i n e , hydrogen peroxide, f e r r i c c h l o r i d e , c u p r i c s u l f a t e and sodium vanadate. The r e s u l t s i n d i c a t e d t h a t hydrogen peroxide or sodium vanadate were not onl y e f f e c t i v e i n the o x i d a t i o n but a l s o much l e s s expensive than i o d i n e , which i s the most popular oxidant f o r the s y n t h e s i s of TT. The r e s u l t s obtained i n t h i s chapter warrant the use of TT i n the commercial p r o d u c t i o n of commercial papain to prevent the d e s t r u c t i o n of the enzymes d u r i n g h a r v e s t i n g , s t o r a g e , t r a n s p o r t a t i o n and p r o c e s s i n g . In the second chapter, chemical m o d i f i c a t i o n of pure papain by TT i s d i s c u s s e d . O p t i m i z a t i o n techniques were a p p l i e d f o r improving the p r e c i s i o n of two methods used i n t h i s study: c i r c u l a r d i c h r o i s m (CD) and p r o t e o l y t i c a c t i v i t y d e t e r m i n a t i o n . Simplex o p t i m i z a t i o n s i g n i f i c a n t l y improved r e p e a t a b i l i t y and s i g n a l to noise r a t i o of the CD scan of papain. A new o p t i m i z a t i o n approach, which was a combination of a c e n t r a l composite r o t a t a b l e design and simplex o p t i m i z a t i o n , was s u c c e s s f u l l y a p p l i e d to achieve maximum p r e c i s i o n f o r the p r o t e o l y t i c a c t i v i t y assay of papain using c a s e i n as a s u b s t r a t e . T h i s approach may a l s o be a p p l i e d to other a n a l y t i c a l methods t o improve the r e l i a b i l i t y of the experimental d a t a . I n f l u e n t i a l f a c t o r s i n the i n a c t i v a t i o n of PA of papain by using TT and r e a c t i v a t i o n of the i n a c t i v a t e d papain by c y s t e i n e were c a r r i e d out us i n g two Taguchi's L i e ( 2 1 S ) f r a c t i o n a l f a c t o r i a l d e s i g n s . The r e s u l t s i n d i c a t e d t h a t when i n a c t i v a t i o n was c a r r i e d out at pH 6.8, with a r e a c t i o n time of 5 min a t 22<>C, and a molar r a t i o of TT to papain of 10, the i n a c t i v a t i o n r e a c t i o n was h i g h l y r e v e r s i b l e upon a d d i t i o n of 20 mM c y s t e i n e . - i i i -Although some i n t e r a c t i o n s of the f a c t o r s were s i g n i f i c a n t , 70% r e a c t i v a t i o n was achieved i n most cases. A n a l y s i s of UV absorbance, near-UV and far-UV CD s p e c t r a i n d i c a t e d t h a t there were no major changes i n the s p e c t r a i n papain upon the chemical m o d i f i c a t i o n of the enzyme with TT. Secondary s t r u c t u r e computed from far-UV CD s p e c t r a a l s o demonstrated no s i g n i f i c a n t changes upon t h i s m o d i f i c a t i o n . S u l f h y d r y l data and pH-fluorescence p r o f i l e s of the modified papain support the hypothesis t h a t r e v e r s i b l e b l o c k i n g by TT r e s u l t s from b i n d i n g with the s i n g l e r e a c t i v e c y s t e i n e r e s i d u e present i n papain. Quenching of the i n t r i n s i c f l u o r e s c e n c e of papain when the m o d i f i c a t i o n was c a r r i e d out u s i n g high molar r a t i o s of TT to papain was s u g g e s t i v e of m o d i f i c a t i o n of tryptophan r e s i d u e s i n the enzyme d u r i n g the o x i d a t i o n r e a c t i o n with TT. P r e c i p i t a t i o n or i n s o l u b i l i z a t i o n of pure papain, and of the p r o t e i n s of papaya l a t e x and commercial papain was observed upon the chemical m o d i f i c a t i o n with TT under c e r t a i n c o n d i t i o n s . A d d i t i o n of fl-mercaptoethanol and TT a t l e v e l s of 100 mM and 50 mM, r e s p e c t i v e l y , p r e c i p i t a t e d 90% of pure papain. S o l u b i l i t y s t u d i e s together with e l e c t r o p h o r e t i c a n a l y s i s of the p r e c i p i t a t e d papain suggested formation of i n s o l u b l e aggregates due to the i n s o l u b l e a g g r e g a t i o n as a r e s u l t of i n t e r - m o l e c u l a r d i s u l f i d e bonds formation. TT was found to be a c o m p e t i t i v e i n h i b i t o r of both r e v e r s i b l e and i r r e v e r s i b l e i n h i b i t i o n of the enzyme a c t i o n , when - i v -carbobenzoxyglycine p - n i t r o p h e n y l e s t e r was used as a s u b s t r a t e . The second order i n a c t i v a t i o n constant i n the absence of s u b s t r a t e was computed to be 16,919 M^sec" 1, i n d i c a t i n g t h a t the r e a c t i o n had a high r a t e . -v-TABLE OP CONTENTS Page ABSTRACT i i TABLE OF CONTENTS v i LIST OF TABLES x i i LIST OF FIGURES XV LIST OF APPENDICES x x i ACKNOWLEDGEMENTS XXi i GENERAL INTRODUCTION 1 CHAPTER 1. P r o t e c t i o n of the p r o t e o l y t i c a c t i v i t y of papaya l a t e x and crude papain by t e t r a t h i o n a t e LITERATURE REVIEW 4 A. D e f i n i t i o n s 4 B. P r o d u c t i o n of papaya l a t e x 5 1. Agronomic f a c t o r s 5 2. Papaya l a t e x h a r v e s t i n g 6 3. Y i e l d s of papaya l a t e x 7 C. P r o d u c t i o n of crude papain and commercial papain 8 1. Drying 8 2. R e f i n i n g 10 3. Grades and p r i c e s of crude and commercial papain 11 D. Losses of p r o t e o l y t i c a c t i v i t y of papaya l a t e x due to d r y i n g and d u r i n g storage of crude and commercial papain 13 E. Process to improve the s t a b i l i t y of crude and commercial papain 15 1. Improvement of tapp i n g and c o l l e c t i n g procedures 16 2. The Boudart process 16 3. A d d i t i o n of reducing agents 17 F. Sodium t e t r a t h i o n a t e as a s t a b i l i z i n g agent of s u l f h y d r y l proteases 18 1. Mechanism of r e a c t i o n 18 2. R e v e r s i b l e i n a c t i v a t i o n 19 G. Chemical p r o p e r t i e s of t e t r a t h i o n a t e 21 1. Some p r o p e r t i e s of t e t r a t h i o n a t e 22 2. S t r u c t u r e of t e t r a t h i o n a t e . . . 23 3. Uses 23 4 . T o x i c i t y 25 - v i -Page H. P r e p a r a t i o n of t e t r a t h i o n a t e 27 1. Iodine o x i d a t i o n 29 2. O x i d a t i o n with other compounds 31 (a) O x i d a t i o n with hydrogen peroxide 31 (b) O x i d a t i o n with metal s a l t s 32 (c) O x i d a t i o n with vanadate 32 3. Other methods of s y n t h e s i s of t e t r a t h i o n a t e 33 MATERIALS AND METHODS 35 A. M a t e r i a l s 35 B. Rehydration of crude papain 35 C. Drying c h a r a c t e r i s t i c s of papaya l a t e x 36 D. Determination of i n f l u e n t i a l f a c t o r s on the l o s s e s of p r o t e o l y t i c a c t i v i t y due to d r y i n g of papaya latex..36 E. E f f e c t of d i f f e r e n t types of d r y i n g and a d d i t i v e s on the l o s s e s of p r o t e o l y t i c a c t i v i t y of papaya latex..40 P. Losses of the p r o t e o l y t i c a c t i v i t y of crude papain d u r i n g storage 41 G. P r o t e o l y t i c a c t i v i t y assay 41 H. P r e p a r a t i o n of sodium t e t r a t h i o n a t e 43 1. Iodine o x i d a t i o n 43 2. Hydrogen peroxide o x i d a t i o n 43 3. F e r r i c o x i d a t i o n 44 4. C u p r i c o x i d a t i o n 45 5. Vanadate o x i d a t i o n 46 I. Determination of p u r i t y and y i e l d 46 1. I o d a t e - i o d i n e t i t r a t i o n 46 2. A l k a l i n e c y a n o l y s i s 49 3. M e l t i n g p o i n t d e t e r m i n a t i o n 50 4. C a l c u l a t i o n of p u r i t y and y i e l d 50 J . Cost e v a l u a t i o n 51 K. I n a c t i v a t i o n / a c t i v a t i o n e f f i c i e n c y of the s y n t h e s i z e d t e t r a t h i o n a t e s 51 RESULTS AND DISCUSSION 53 A. Drying Rates of papaya l a t e x 53 B. Determination of i n f l u e n t i a l f a c t o r s on the l o s s e s of p r o t e o l y t i c a c t i v i t y due to d r y i n g of papaya l a t e x 57 C. E f f e c t of d i f f e r e n t types of d r y i n g and a d d i t i v e s on the l o s s of p r o t e o l y t i c a c t i v i t y of papaya l a t e x 62 D. E f f e c t of a d d i t i v e s on l o s s of the p r o t e o l y t i c a c t i v i t y of crude papain d u r i n g storage 64 E. Comparison of the d i f f e r e n t methods to s y n t h e s i z e t e t r a t h i o n a t e 66 - v i i -Page F. Cost e v a l u a t i o n of the d i f f e r e n t methods of t e t r a t h i o n a t e s y n t h e s i s 68 G. M e l t i n g p o i n t d e t e r m i n a t i o n 74 H. I n a c t i v a t i o n / a c t i v a t i o n e f f i c i e n c y of the s y n t h e s i z e d t e t r a t h i o n a t e s 96 CONCLUSION 98 CHAPTER 2. Chemical m o d i f i c a t i o n of papain by t e t r a t h i o n a t e LITERATURE REVIEW 100 A. Chemical m o d i f i c a t i o n of p r o t e i n s 100 B. Chemical m o d i f i c a t i o n of food r e l a t e d p r o t e i n s 102 C. M o d i f i c a t i o n of s u l f h y d r y l groups i n p r o t e i n s 103 1, O x i d a t i o n 104 (a) M o d i f i c a t i o n by aromatic d i s u l f i d e s 106 (b) M o d i f i c a t i o n by t e t r a t h i o n a t e 107 I. T e t r a t h i o n a t e as a s t a b i l i z i n g agent f o r s u l f h y d r y l proteases 108 I I . T e t r a t h i o n a t e as a b l o c k i n g agent of c y s t e i n e r e s i d u e s 108 I I I . T e t r a t h i o n a t e as a chemical m o d i f i c a t i o n agent of c y s t e i n e r e s i d u e s 109 (c) M o d i f i c a t i o n by iodobenzoates and mercurials....112 2. A l k y l a t i o n 113 D. Papain 114 1. D e f i n i t i o n and i s o l a t i o n 114 2. Physicochemical p r o p e r t i e s and s t r u c t u r e 115 3. S t a b i l i t y 118 4. A c t i v i t y 119 E. Chemical m o d i f i c a t i o n of papain 119 1. M o d i f i c a t i o n of Cys-25 120 2. M o d i f i c a t i o n of other r e s i d u e s 121 F. Enzyme k i n e t i c s 125 1. Reactions r a t e s 125 (a) Zero order r e a c t i o n s 125 (b) F i r s t order r e a c t i o n s 126 (c) Second order r e a c t i o n s 126 I. Type I 127 I I . Type II 127 I I I . Type I I I 127 2. S t a t e s of an enzymatic r e a c t i o n 128 (a) The pre-steady s t a t e 128 (b) The steady s t a t e 130 (c) The n o n l i n e a r s t a t e 130 - v i i i -Page 3. Measurement of v e l o c i t y of enzyme c a t a l y z e d r e a c t i o n s 130 (a) E f f e c t of s u b s t r a t e c o n c e n t r a t i o n on the i n i t i a l v e l o c i t y 131 (b) Determination of Km and Vmax 133 G. Determination of i n i t i a l v e l o c i t i e s 134 H. N i t r o p h e n y l e s t e r s as s u b s t r a t e s f o r papain 136 1. Steady s t a t e k i n e t i c s f o r the papain c a t a l y z e d h y d r o l y s i s of carbobenzoxyglycine p - n i t r o p h e n y l e s t e r 137 2. Pre-steady s t a t e k i n e t i c s f o r the papain c a t a l y z e d h y d r o l y s i s of carbobenzoxyglycine p - n i t r o p h e n y l e s t e r 138 I. Enzyme i n h i b i t o r s 140 1. D e f i n i t i o n and c l a s s i f i c a t i o n .140 (a) I r r e v e r s i b l e i n h i b i t o r s 140 I. K i n e t i c s of i r r e v e r s i b l e i n h i b i t o r s 141 I I . I r r e v e r s i b l e i n h i b i t o r s of s u l f h y d r y l enzymes 144 (b) R e v e r s i b l e i n h i b i t i o n 144 I. K i n e t i c s or r e v e r s i b l e i n h i b i t i o n 145 I I . Competitive i n h i b i t i o n 145 I I I . Uncompetitive i n h i b i t i o n 146 IV. Noncompetitive i n h i b i t i o n 146 V. R e v e r s i b l e i n h i b i t i o n of s u l f h y d r y l enzymes 146 MATERIALS AND METHODS 149 A. M a t e r i a l s 149 B. Determination of p r o t e i n c o n c e n t r a t i o n 149 C. Determination of p r o t e o l y t i c a c t i v i t y 150 1. O p t i m i z a t i o n of the c o n d i t i o n s to measure p r o t e o l y t i c a c t i v i t y of papain 150 2. P r o t e o l y t i c a c t i v i t y d e t e r m i n a t i o n 151 D. Determination of the i n f l u e n c i a l f a c t o r s f o r maximum i n h i b i t i o n and r e a c t i v a t i o n of the p r o t e o l y t i c a c t i v i t y of papain by t e t r a t h i o n a t e 154 E. P r e p a r a t i o n of t e t r a t h i o n a t e modified papain 158 F. C i r c u l a r Dichroism ....159 1. O p t i m i z a t i o n of the c o n d i t i o n s f o r measuring the CD s p e c t r a of papain 160 2. Far-UV CD Spectra (190-240 nm) 161 3. Near-UV Spectra (250 -350 nm) 162 G. Secondary s t r u c t u r e p r e d i c t i o n 163 H. Flu o r e s c e n c e and d i f f e r e n t i a l a b s o r p t i o n s p e c t r o s c o p y 163 I. Determination of t o t a l -SH groups of papain 165 - i x -Page J . I n s o l u b i l i z a t i o n of papain with t e t r a t h i o n a t e 166 1. E f f e c t of c o n c e n t r a t i o n of t e t r a t h i o n a t e and 0-mercaptoethanol on the p r e c i p i t a t i o n of papain 166 2. E f f e c t of pH and temperature on the p r e c i p i t a t i o n of papain by t e t r a t h i o n a t e 168 3. R e s o l u b i l i z a t i o n of the p r e c i p i t a t e d p r o t e i n 168 4. A n a l y s i s of the p r e c i p i t a t e d p r o t e i n 169 K. Determination of Vmax and Km f o r the pa p a i n - c a t a l y z e d h y d r o l y s i s of carbobenzoxyglycine p - n i t r o p h e n y l e s t e r 171 L. I n h i b i t i o n experiments 173 1. R e v e r s i b l e i n h i b i t i o n experiments 173 2. I r r e v e r s i b l e i n h i b i t i o n experiments 174 (a) In the absence of s u b s t r a t e 174 (b) In the presence of s u b s t r a t e 175 M. S t a t i s t i c a l a n a l y s i s 176 RESULTS AND DISCUSSION 177 A. O p t i m i z a t i o n of the c o n d i t i o n s to measure p r o t e o l y t i c a c t i v i t y of papain 177 1. L o c a l i z a t i o n of optimum c o n d i t i o n s 180 B. Determination of the i n f l u e n c i a l f a c t o r s f o r maximum i n h i b i t i o n by t e t r a t h i o n a t e and r e a c t i v a t i o n by c y s t e i n e of the p r o t e o l y t i c a c t i v i t y of papain 189 C. O p t i m i z a t i o n of the c o n d i t i o n s f o r measuring the CD s p e c t r a of papain 201 D. CD of n a t i v e and t e t r a t h i o n a t e m o d i f i e d papain 208 E. Secondary s t r u c t u r e of n a t i v e and t e t r a t h i o n a t e - m o d i f i e d papain 212 F. F l u o r e s c e n c e and UV-absorption of n a t i v e and t e t r a t h i o n a t e m o d i f i e d papain 214 G. Determination of -SH groups i n n a t i v e and t e t r a t h i o n a t e modified papain 219 H. I n s o l u b i l i z a t i o n of papain with t e t r a t h i o n a t e 221 I. K i n e t i c s parameters f o r the p a p a i n - c a t a l y z e d h y d r o l y s i s of carbobenzoxyglycine p - n i t r o p h e n y l e s t e r 236 J . C h a r a c t e r i z a t i o n of the i n h i b i t i o n e f f e c t of t e t r a t h i o n a t e 244 1. R e v e r s i b l e i n h i b i t i o n 244 2. I r r e v e r s i b l e i n h i b i t i o n 245 (a) In the presence of s u b s t r a t e 245 (b) In the absence of s u b s t r a t e 248 -x-Page CONCLUSIONS 255 REFERENCES CITED 259 APPENDIX 275 -xi-LIST OF TABLES Page Table 1. C o n d i t i o n s r e p o r t e d f o r oven d r y i n g of papaya l a t e x 9 Table 2. P r i c e s of crude, commercial and pure papain 12 Table 3. Standard e l e c t r o d e p o t e n t i a l (E°) f o r some chemical r e a c t i o n s 28 Table 4. Chemical r e a c t i o n s i n the s y n t h e s i s of t e t r a t h i o n a t e 30 Table 5. F a c t o r s and t h e i r assigned l e v e l s i n v e s t i g a t e d f o r t h e i r p o s s i b l e i n f l u e n c e on the l o s s e s of p r o t e o l y t i c a c t i v i t y of papaya l a t e x 38 Table 6. A n a l y s i s of v a r i a n c e (Taguchi's L»» 3") f o r p r o t e o l y t i c a c t i v i t y values of papaya l a t e x obtained from 27 d r y i n g experiments 58 Table 7. Mean r e t e n t i o n of the p r o t e o l y t i c a c t i v i t y of the crude papain r e s u l t i n g from papaya l a t e x t r e a t e d with 1% t e t r a t h i o n a t e or 1% m e t a b i s u l f i t e and d r i e d using d i f f e r e n t methods 63 Table 8. Y i e l d s and p u r i t y of t e t r a t h i o n a t e s y n t h e s i z e d by the d i f f e r e n t methods 67 Table 9. Commercial p r i c e of t e t r a t h i o n a t e , m e t a b i s u l f i t e and chemicals used to s y n t h e s i z e t e t r a t h i o n a t e 70 Table 10. I n a c t i v a t i o n and a c t i v a t i o n e f f i c i e n c y of t e t r a t h i o n a t e s s y n t h e s i z e d u s i n g d i f f e r e n t methods 97 Table 11. O x i d a t i o n products of s u l f h y d r y l groups 105 Table 12. Phys i c o c h e m i c a l p r o p e r t i e s of some c y s t e i n e proteases 116 Table 13. Michaelis-Menten parameters f o r the papain c a t a l y z e d h y d r o l y s i s of carbobenzoxyglycine p - n i t r o p h e n y l e s t e r 139 Table 14. Upper and lower l i m i t s f o r the three f a c t o r s used f o r o p t i m i z a t i o n of the p r o t e o l y t i c a c t i v i t y d e t e r m i n a t i o n of papain 151 - x i i -Page Table 15. F a c t o r s and t h e i r a s s i g n e d l e v e l s i n v e s t i g a t e d f o r t h e i r p o s s i b l e i n f l u e n c e on the i n h i b i t i o n of the p r o t e o l y t i c a c t i v i t y of papain, and on the subsequent r e a c t i v a t i o n of the a c t i v i t y by c y s t e i n e 155 Table 16. L e v e l s of the f a c t o r s used i n the RSM experiment of the p r e c i p i t a t i o n of papain by t e t r a t h i o n a t e 167 Table 17. C e n t r a l compositive r o t a t a b l e design matrix used f o r the o p t i m i z a t i o n of the c o n d i t i o n s t o measure p r o t e o l y t i c a c t i v i t y of papain, and r e s u l t s f o r each experiment 178 Table 18. A n a l y s i s of v a r i a n c e of the second order model 179 Table 19. A n a l y s i s of v a r i a n c e f o r the modified second order model 181 Table 20. A n a l y s i s of v a r i a n c e (Taguchi's Lx« 2") f o r the i n h i b i t i o n of the p r o t e o l y t i c a c t i v i t y of papain by t e t r a t h i o n a t e 191 Table 21. A n a l y s i s of v a r i a n c e (Taguchi's Li« 21*) f o r the r e a c t i v a t i o n by c y s t e i n e of the p r o t e o l y t i c a c t i v i t y of t e t r a t h i o n a t e - i n a c t i v a t e d papain 193 Table 22. S i m p l e x - c e n t r o i d o p t i m i z a t i o n of the c o n d i t i o n s f o r CD spectrophotometry of papain 203 Table 23. Values of mean r e s i d u e e l l i p t i c i t y and corresp o n d i n g c o e f f i c i e n t of v a r i a t i o n at three wavelengths f o r the d i f f e r e n t v e r t i c e s of the s i m p l e x - c e n t r o i d o p t i m i z a t i o n 207 Table 24. P r e d i c t e d secondary s t r u c t u r e f r a c t i o n s of n a t i v e and t e t r a t h i o n a t e modified papain based on CD data, u s i n g two a l g o r i t h m s , and X-ray determine secondary f r a c t i o n s 213 Table 25. S u l f h y d r y l content of n a t i v e and t e t r a t h i o n a t e m o d i f i e d papain 220 - x i i i -Page Tab le 26. Experimental data f o r the two-factor, f i v e - l e v e l response s u r f a c e a n a l y s i s of the e f f e c t of t e t r a t h i o n a t e and 0-mercaptoethanol c o n c e n t r a t i o n on the p r e c i p i t a t i o n of papain 223 Tab le 27. A n a l y s i s of v a r i a n c e f o r the second order model f o r the p r e c i p i t a t i o n of papain by t e t r a t h i o n a t e , obtained u s i n g backward m u l t i p l e r e g r e s s i o n 224 Tab le 28. E f f e c t of temperature and pH on the p r e c i p i t a t i o n of papain by t e t r a t h i o n a t e 226 Tab le 29. E f f e c t of d i f f e r e n t reagents on the r e s o l u b i l i z a t i o n of p r e c i p i t a t e d papain 228 Tab le 30. P r o t e o l y t i c a c t i v i t y of n a t i v e and i n s o l u b l e papain 230 Tab le 31. Values of B and corresponding e m p i r i c a l r e a c t i o n order with r e s p e c t t o time a t d i f f e r e n t i n i t i a l s u b s t r a t e c o n c e n t r a t i o n s f o r the r e a c t i o n of papain-carbobenzoxyglycine p - n i t r o p h e n y l e s t e r 240 Table 32. R e s u l t s of curve f i t t i n g the k i n e t i c s data f o r the r e a c t i o n of papain-carbobenzoxyglycine p - n i t r o p h e n y l e s t e r a t v a r i o u s i n i t i a l s u b s t r a t e c o n c e n t r a t i o n s . I n i t i a l v e l o c i t e s (Vo) determined by the f i x e d time assay method are i n c l u d e d f o r comparative purposes 242 Tab le 33. K i n e t i c s parameters of the r e a c t i o n of the papain-carbobenzoxyglycine p - n i t r o p h e n y l e s t e r r e a c t i o n (+ standard e r r o r ) , computed with the program of O e s t r e i c h e r and P i n t o (1983) u s i n g i n i t i a l v e l o c i t i e s estimated by f i x e d time assays or d e r i v e d from e x p e r i m e n t a l l y determined curves. 243 Tab le 34. Second order r a t e constants f o r i n a c t i v a t i o n f o r some papain i n h i b i t o r s and f o r t e t r a t h i o n a t e with two d i f f e r e n t enzymes 254 - x i v -L I S T O F F I G U R E S P A G E F i g u r e 1. Scheme used i n the Taguchi L i ? f r a c t i o n a l f a c t o r i a l experiment 39 F i g u r e 2. The e f f e c t of three d i f f e r e n t d r y i n g temperatures on the d r y i n g r a t e of papaya l a t e x a t a d r y i n g load of 2,381 g/m* 54 F i g u r e 3. The e f f e c t of d r y i n g load on the d r y i n g r a t e of papaya l a t e x a t a d r y i n g temperature of 80°C 55 F i g u r e 4. The e f f e c t of the a d d i t i o n of 1% t e t r a t h i o n a t e or m e t a b i s u l f i t e on the d r y i n g r a t e of papaya l a t e x a t 5 5 0 C and d r y i n g load of 2,381 g/m* 56 F i g u r e 5. E f f e c t curve f o r the i n t e r a c t i o n between d r y i n g temperature and treatment p r i o r to d r y i n g on the p r o t e o l y t i c a c t i v i t y r e t a i n e d by crude papain. (Meanj-Confidence l i m i t s c a l c u l a t e d a t p<0.05) 59 F i g u r e 6. E f f e c t curve f o r the i n t e r a c t i o n between d r y i n g load and treatment p r i o r to d r y i n g on the p r o t e o l y t i c a c t i v i t y r e t a i n e d by crude papain 61 F i g u r e 7. Change i n the p r o t e o l y t i c a c t i v i t y of crude papain with or without a d d i t i v e s d u r i n g storage at room temperature 65 F i g u r e 8a. The c o s t of s y n t h e s i s of t e t r a t h i o n a t e f o r the d i f f e r e n t methods e v a l u a t e d . Cost i n c l u d i n g t h a t of the s o l v e n t s 71 F i g u r e 8b. The c o s t of s y n t h e s i s of t e t r a t h i o n a t e f o r the d i f f e r e n t methods e v a l u a t e d . Cost without i n c l u d i n g t h a t of the s o l v e n t s 72 F i g u r e 9. D i s t r i b u t i o n of the c o s t of s y n t h e s i s of t e t r a t h i o n a t e u s i n g the i o d i n e and the hydrogen peroxide methods 73 F i g u r e 10. T y p i c a l DSC thermogram of a commercial t e t r a t h i o n a t e sample from ICN Pharmaceutical, Inc. T e t r a t h i o n a t e content (%) determined us i n g the i o d a t e - i o d i n e r e a c t i o n (Mean+S.D n=3)= 102.0 + 5% 76 -xv-P A G E F i g u r e 11. T y p i c a l DSC thermogram of t e t r a t h i o n a t e s y n t h e s i z e d u s i n g the i o d i n e r e a c t i o n . T e t r a t h i o n a t e content (%) determined using the i o d a t e - i o d i n e r e a c t i o n (Mean+S.D n=3)= 98.31+6% 78 F i g u r e 12. T y p i c a l DSC thermogram of t e t r a t h i o n a t e s y n t h e s i z e d u s i n g the peroxide method. T e t r a t h i o n a t e content (%) determined u s i n g the i o d a t e - i o d i n e r e a c t i o n (Mean+S.D n=3)= 37.9 + 6% 80 F i g u r e 13. T y p i c a l DSC thermogram of t e t r a t h i o n a t e s y n t h e s i z e d u s i n g the c u p r i c method. T e t r a t h i o n a t e content (%) determined using the i o d a t e - i o d i n e r e a c t i o n (Mean+S.D n=3)= 20.4 + 8% 82 F i g u r e 14. T y p i c a l DSC thermogram of t e t r a t h i o n a t e s y n t h e s i z e d u s i n g the f e r r i c method. T e t r a t h i o n a t e content (%) determined using the i o d a t e - i o d i n e r e a c t i o n (Mean+S.D n=3)= 18.3 + 5% 84 F i g u r e 15. T y p i c a l DSC thermogram of t e t r a t h i o n a t e s y n t h e s i z e d u s i n g the vanadate method with h y d r o c h l o r i c a c i d . T e t r a t h i o n a t e content (%) determined u s i n g the i o d a t e - i o d i n e r e a c t i o n (Mean+S.D n=3)= 38.6 + 7% 86 F i g u r e 16. T y p i c a l DSC thermogram of t e t r a t h i o n a t e s y n t h e s i z e d u s i n g the vanadate method with a c e t i c a c i d . T e t r a t h i o n a t e content (%) determined u s i n g the i o d a t e - i o d i n e r e a c t i o n (Mean + S.D n=3)= 35.0 + 7% 88 F i g u r e 17. T y p i c a l DSC thermograms of a sample of pure t h i o s u l f a t e from F i s h e r S c i e n t i f i c Comp 91 F i g u r e 18. T y p i c a l DSC thermogram of a mixture of pure t h i o s u l f a t e and t e t r a t h i o n a t e at a 1:1 (w/w) r a t i o 93 F i g u r e 19. E f f e c t of a d d i t i o n of t h i o s u l f a t e on the area of two endothermic peaks of t e t r a t h i o n a t e 94 F i g u r e 20. E f f e c t of a d d i t i o n of t h i o s u l f a t e on the m e l t i n g p o i n t of t e t r a t h i o n a t e 95 -xv i -PAGE F i g u r e 2 1 . Progress r e a c t i o n curve f o r an i d e a l enzyme r e a c t i o n 1 2 9 F i g u r e 2 2 . T y p i c a l graph of the i n i t i a l v e l o c i t y of an enzymatic r e a c t i o n as a f u n c t i o n of i n i t i a l s u b s t r a t e c o n c e n t r a t i o n 1 3 2 F i g u r e 2 3 . Scheme used i n the Taguchi L i t f r a c t i o n a l f a c t o r i a l experiment 1 5 6 F i g u r e 2 4 . P l o t of p r e d i c t e d values a c c o r d i n g to the modified second order model of the SD of the d e t e r m i n a t i o n of the p r o t e o l y t i c a c t i v i t y of papain a g a i n s t the corresponding experimental ones 1 8 2 F i g u r e 2 5 . P l o t of r e s i d u a l s f o r the modified second order model 1 8 3 F i g u r e 2 6 . Computational o p t i m i z a t i o n used to o b t a i n the best experimental c o n d i t i o n s f o r p r o t e o l y t i c a c t i v i t y d e t e r m i n a t i o n of papain. The response i s the standard d e v i a t i o n c a l c u l a t e d u s i n g the modified second order model 1 8 5 F i g u r e 2 7 . Response s u r f a c e of standard d e v i a t i o n a g a i n s t i n c u b a t i o n time and enzyme c o n c e n t r a t i o n , a t a constant i n c u b a t i o n temperature of 35<>C 1 8 6 F i g u r e 2 8 . Response s u r f a c e of standard d e v i a t i o n a g a i n s t i n c u b a t i o n time and temperature, a t a constant papain c o n c e n t r a t i o n of 0 . 1 mg/ml 1 8 7 F i g u r e 2 9 . Response s u r f a c e of standard d e v i a t i o n a g a i n s t i n c u b a t i o n temperature and enzyme c o n c e n t r a t i o n , at a constant i n c u b a t i o n time of 5 min 1 8 8 F i g u r e 3 0 . E f f e c t of two l e v e l s of the molar r a t i o of t e t r a t h i o n a t e to papain i n the i n a c t i v a t i o n of papain 1 9 2 F i g u r e 3 1 . E f f e c t curve f o r the i n t e r a c t i o n between the molar r a t i o of t e t r a t h i o n a t e t o papain and r e a c t i o n time of i n a c t i v a t i o n r e a c t i o n on the % r e a c t i v a t i o n of the p r o t e o l y t i c a c t i v i t y of papain 1 9 5 - x v i i -PAGE F i g u r e 3 2 . E f f e c t curve f o r the i n t e r a c t i o n between the molar r a t i o of t e t r a t h i o n a t e to papain and pH of the i n a c t i v a t i o n r e a c t i o n on the % r e a c t i v a t i o n of the p r o t e o l y t i c a c t i v i t y of papain 197 F i g u r e 3 3 . E f f e c t curve f o r the i n t e r a c t i o n between the c y s t e i n e c o n c e n t r a t i o n d u r i n g the r e a c t i v a t i o n and pH of the i n a c t i v a t i o n on the % r e a c t i v a t i o n of the p r o t e o l y t i c a c t i v i t y of papain ....198 F i g u r e 3 4 . E f f e c t curve f o r the i n t e r a c t i o n between the c y s t e i n e c o n c e n t r a t i o n d u r i n g r e a c t i v a t i o n and temperature of i n a c t i v a t i o n on the % r e a c t i v a t i o n of the p r o t e o l y t i c a c t i v i t y of papain 199 F i g u r e 3 5 . E f f e c t curve f o r the i n t e r a c t i o n between the c y s t e i n e c o n c e n t r a t i o n d u r i n g r e a c t i v a t i o n and time of i n a c t i v a t i o n on the % r e a c t i v a t i o n of the p r o t e o l y t i c a c t i v i t y of papain 200 F i g u r e 3 6 . E f f e c t curve f o r the i n t e r a c t i o n between the pH and temperature of i n a c t i v a t i o n on the % r e a c t i v a t i o n of the p r o t e o l y t i c a c t i v i t y of papain 202 F i g u r e 3 7 . Trace of the far-UV CD spectrum of papain measured under d i f f e r e n t c o n d i t i o n s . (A) CD spectrum measured under the c o n d i t i o n s of v e r t e x 1. (B) CD spectrum measured under the optimum c o n d i t i o n s (Vertex 15) 205 F i g u r e 3 8 . Far-UV CD s p e c t r a of n a t i v e and TT-papain (molar r a t i o t e t r a t h i o n a t e to papain d u r i n g r e a c t i o n = 200). Note: both p r o t e i n s gave the same s p e c t r a 209 F i g u r e 3 9 . Near-UV CD s p e c t r a of n a t i v e and TT-papain (molar r a t i o t e t r a t h i o n a t e to papain d u r i n g r e a c t i o n = 200)... 211 F i g u r e 4 0 . E f f e c t of pH on the r e l a t i v e f l u o r e s c e n c e i n t e n s i t y a t 22°C of n a t i v e papain 215 - x v i i i -PAGE F i g u r e 41. E f f e c t of pH on the r e l a t i v e f l u o r e s c e n c e i n t e n s i t y a t 2 2 ° C of TT-papain (molar r a t i o t e t r a t h i o n a t e to papain d u r i n g r e a c t i o n = 2 0 0 ) 2 1 6 F i g u r e 4 2 . E f f e c t of the molar r a t i o of t e t r a t h i o n a t e to papain d u r i n g the chemical m o d i f i c a t i o n on the r e l a t i v e f l u o r e s c e n c e i n t e n s i t y a t 3 5 2 nm (pH 6 . 8 and 22<>c) 2 1 8 F i g u r e 4 3 . Response curve f o r the e f f e c t of t e t r a t h i o n a t e and 0-mercaptoethanol c o n c e n t r a t i o n on the p r e c i p i t a t i o n of papain 2 2 2 F i g u r e 4 4 . Change of pH d u r i n g the r e a c t i o n between t e t r a t h i o n a t e , 0-mercaptoethanol and papain. F i n a l [ t e t r a t h i o n a t e ] = [B-mercaptoethanolJ = 1 0 0 mM, f i n a l [papain]= 1 0 mg/ml 2 2 7 F i g u r e 4 5 . Electrophoretogram of n a t i v e and i n s o l u b l e papain 2 3 1 F i g u r e 4 6 . Electrophoretogram of s o l u b l e and i n s o l u b l e papaya l a t e x and commercial papain 2 3 3 F i g u r e 4 7 . Electrophoretogram under non-reduced c o n d i t i o n s of s o l u b l e t e t r a t h i o n a t e - r a o d i d i f i e d pure papain and of t e t r a t h i o n a t e - t r e a t e d commercial papain and papaya l a t e x 2 3 4 F i g u r e 4 8 . P a p a i n - c a t a l y z e d h y d r o l y s i s of carbobenzoxyglycine p - n i t r o p h e n y l e s t e r at pH 6 . 8 and 22<>C. The r e a c t i o n mixture contained 0 . 0 2 M sodium phosphate b u f f e r 1 mM EDTA, 0 . 3 5 mM c y s t e i n e , 6 . 7 % (v/v) a c e t o n i t r i l e and 3 . 3 x 1 0 " T papain. I n i t i a l s u b s t r a t e c o n c e n t r a t i o n as shown 2 3 7 F i g u r e 4 9 . Schematic diagram of the system used f o r the enzyme k i n e t i c s experiments 2 3 8 F i g u r e 50. I d e a l i z e d progress r e a c t i o n curve (A«0o vs. time) f o r the p a p a i n - c a t a l y z e d h y d r o l y s i s of carbobenzoxyglycine p - n i t r o p h e n y l e s t e r 2 3 9 F i g u r e 51. Lineweaver-Burk p l o t f o r the papain a c t i v i t y at d i f f e r e n t c o n c e n t r a t i o n s of t e t r a t h i o n a t e 2 4 6 - x i x -PAGE F i g u r e 52. I n h i b i t i o n e f f e c t of t e t r a t h i o n a t e (TT) i n the presence of three d i f f e r e n t i n i t i a l s u b s t r a t e c o n c e n t r a t i o n s a t a constant TT c o n c e n t r a t i o n (1.06 x 10-J M) 247 F i g u r e 53. E f f e c t of carbobenzoxyglycine p - n i t r o p h e n y l e s t e r c o n c e n t r a t i o n on the second order r a t e constant of i n a c t i v a t i o n of t e t r a t h i o n a t e on papain. (Mean±S.D., n=3). The r e g r e s s i o n l i n e i s shown 249 F i g u r e 54. Change i n the c a t a l y t i c a c t i v i t y of papain as a r e s u l t of the r e a c t i o n with t e t r a t h i o n a t e . A c t i v i t y was measured with carbobenzoxyglycine p - n i t r o p h e n y l e s t e r as the s u b s t r a t e 250 Fi g u r e 55. E f f e c t of the molar r a t i o of t e t r a t h i o n a t e to papain on the c a t a l y t i c a c t i v i t y of papain a t two r e a c t i o n s times. A c t i v i t y was measured with carbobenzoxyglycine p - n i t r o p h e n y l e s t e r as the s u b s t r a t e 251 F i g u r e 56. Progress of i n a c t i v a t i o n of papain by d i f f e r e n t l e v e l s of t e t r a t h i o n a t e (TT). The value of Y was c a l c u l a t e d based on E q . l l when the molar r a t i o of TT to papain was 1:1, f o r the other molar r a t i o s Eq. 12 was used. A c t i v i t y was measured with carbobenzoxyglycine p - n i t r o p h e n y l e s t e r as the s u b s t r a t e 252 -xx-LIST OF APPENDIX PAGE Appendix 1 . L i s t i n g of the IBM-BASIC v e r s i o n of the computer program used by S i e g e l et a l . (1980) to determine secondary s t r u c t u r e f r a c t i o n s from CD s p e c t r a 275 Appendix 2. L i s t i n g of the IBM-BASIC computer program which u t i l i z e s the simplex a l g o r i t h m of Morgan and Deming (1974) used f o r computational o p t i m i z a t i o n of the c o n d i t i o n s to measure the p r o t e o l y t i c a c t i v i t y of papain 277 Appendix 3. L i s t i n g of the IBM-BASIC computer program used to generate the data ( g r i d ) f o r p l o t t i n g the response s u r f a c e of the e f f e c t of 0-mercaptoethanol and t e t r a t h i o n a t e on the p r e c i p i t a t i o n of papain 280 - x x i -ACKNOWLEDGEMENTS I s i n c e r e l y wish to thank Dr. S. Nakai f o r s u g g e s t i n g t h i s p r o j e c t , f o r many h e l p f u l comments and s u g g e s t i o n s , f o r answering my many q u e s t i o n s , f o r p a t i e n c e , and - by being such an expert i n h i s f i e l d - f o r s e r v i n g as a r o l e model...I hope t h a t someday I may know so much ! I would a l s o l i k e to thank p r o f e s s o r s B. J . Skura, J . Vanderstoep, L. E. Hart and Dr. E. Li-Chan f o r t h e i r i n t e r e s t and p a i n s t a k i n g task of checking the t e c h n i c a l aspects and o v e r a l l s t r u c t u r e of t h i s t h e s i s . In my view, t h e i r sharp remarks have c o n s i d e r a b l y improved the q u a l i t y of t h i s r e p o r t . I am a l s o i n debt to Ms. A. Smith and Ms. S. Weintraub f o r k i n d l y h e l p i n g with the formidable task of p r o o f r e a d i n g t h i s t h e s i s . F i n a l l y , I must thank my best f r i e n d , my wife Cecy, whose constant support and encouragement helped to make t h i s t h e s i s p o s s i b l e and helped me keep my s a n i t y d u r i n g the w r i t i n g of t h i s work . F i n a n c i a l support from the Consejo Nacional de C i e n c i a y T e c n o l o g i a (CONACYT) (Mexico) i n the form of a Graduate S c h o l a r s h i p i s g r a t e f u l l y acknowledged. - x x i i -GENERAL INTRODUCTION Fo o d - r e l a t e d enzymes can be c l a s s i f i e d i n two major groups: those enzymes which are added to food and those which occur n a t u r a l l y i n foods (Beck and S c o t t , 1974). Although many enzymes have been i s o l a t e d , there are l e s s than 20 i n d u s t r i a l enzymes on a s c a l e that have had s i g n i f i c a n t impact i n e i t h e r the enzyme i n d u s t r y or the food i n d u s t r y . Most i n d u s t r i a l enzymes are u s u a l l y not h i g h l y p u r i f i e d and r e l a t i v e l y cheap, with a c o s t per kg of < US$10.00 (Hepner and Male, 1983). These i n d u s t r i a l enzymes can be c l a s s i f i e d i n three c a t e g o r i e s : p r o t e o l y t i c , a m y l o l y t i c and other enzymes. The p r o t e o l y t i c enzymes account f o r over 50% of the market and a m y l o l y t i c enzymes fo r 33% (Hepner and Male, 1983). I n d u s t r i a l p r o t e o l y t i c enzymes are d e r i v e d from a wide range of sources and may have w i d e l y d i f f e r e n t pH and temperature optima and s p e c i f i c i t i e s . They are a l s o one of the few groups of i n d u s t r i a l enzymes obtained from animal, p l a n t and m i c r o b i a l sources (Cheetham, 1985). Among the p r o t e o l y t i c enzymes, p l a n t proteases are the most widely used i n the food i n d u s t r y . These enzymes are s u l f h y d r y l or t h i o l proteases such as papain, f i c i n and bromelain, with papain being most commonly used ( L i e n e r , 1974). -1-C h i l l - p r o o f i n g of beer u s i n g papain, a process patented i n 1911- r e p r e s e n t s the e a r l i e s t i n d u s t r i a l a p p l i c a t i o n of commercial papain and s t i l l r e p r e s e n t s the s i n g l e most s i g n i f i c a n t market f o r t h i s enzyme ( B r o c k l e h u r s t et a l . , 1981). The second l a r g e s t use of papain i s i n meat t e n d e r i z a t i o n ( L i e n e r , 1974). Commercial papain has a l s o been used i n b i s c u i t products and f o r the p r e p a r a t i o n of p r o t e i n h y d r o l y z a t e s ( B r o c k l e h u r s t et a l . , 1981). Many other uses of papain f o r food a p p l i c a t i o n s have been c i t e d but most of them have not been commercialized. Enzymes, u n l i k e many other substances, are r e c o g n i z e d and s o l d by t h e i r a c t i v i t y r a t h e r than t h e i r weight, so the s t a b i l i t y of the enzyme d u r i n g i t s i s o l a t i o n , p u r i f i c a t i o n , f o r m u l a t i o n and storage i s of prime importance (Cheetham, 1985). I t i s w e l l documented t h a t d u r i n g the manufacturing process of commercial papain a s i g n i f i c a n t l o s s of p r o t e o l y t i c a c t i v i t y (PA) occurs. In a d d i t i o n , commercial papain l o s e s i t s PA d u r i n g storage, e s p e c i a l l y when i t i s i n s o l u t i o n ( L i e n e r , 1974). As a s u l f h y d r y l enzyme the major causes of PA l o s s e s i n papain are i r r e v e r s i b l e o x i d a t i o n of i t s e s s e n t i a l c y s t e i n e r e s i d u e and a u t o l y s i s . In order to prevent l o s s e s of PA i n the commercial p r o c e s s i n g of papaya l a t e x the a d d i t i o n of reducing agents (e.g. s u l f i t e s ) p r i o r to d r y i n g i s u s u a l l y done. -2-There were two o b j e c t i v e s of the f i r s t p a r t of t h i s t h e s i s r e s e a r c h . The f i r s t o b j e c t i v e was to evaluate a new method to s t a b i l i z e the PA of papaya l a t e x . T h i s new method c o n s i s t e d of the a d d i t i o n of sodium t e t r a t h i o n a t e to achieve c o n t r o l l e d o x i d a t i o n or b l o c k i n g of the s u l f h y d r y l groups of the proteases of papaya l a t e x . Due to the f a c t t h a t sodium t e t r a t h i o n a t e i s p r e s e n t l y more expensive than s u l f i t e s , the second o b j e c t i v e was to develop a chemical method to s y n t h e s i z e sodium t e t r a t h i o n a t e at a c o s t t h a t w i l l make t h i s new method of p r o t e c t i o n of the PA of papaya l a t e x more e c o n o m i c a l l y a t t r a c t i v e . Since the r e a c t i o n between t e t r a t h i o n a t e and the enzymes i n papaya l a t e x was b a s i c a l l y a chemical m o d i f i c a t i o n of the p r o t e i n s , the second p a r t of t h i s study examined the e f f e c t of sodium t e t r a t h i o n a t e on s e l e c t e d physicochemical p r o p e r t i e s of the pure enzyme papain. In a d d i t i o n , k i n e t i c s of the r e a c t i o n between papain and t e t r a t h i o n a t e are r e p o r t e d . -3-CHAPTER 1. P r o t e c t i o n of the p r o t e o l y t i c a c t i v i t y of papaya l a t e x and crude papain by t e t r a t h i o n a t e . LITERATURE REVIEW A. DEFINITIONS The m a t e r i a l termed "papain", i n i n d u s t r i a l usage, i s not a pure form of the enzyme (E.C. 3.4.22.2), but a p a r t i a l l y p u r i f i e d p r e p a r a t i o n of the l a t e x of papaya ( C a r i c i a papaya). Papaya l a t e x c o n t a i n s , i n a d d i t i o n to the pure enzyme papain (E.C. 3.4.22.2), other c y s t e i n e p roteases, c e l l u l a s e , lysozyme, glutamine c y c l o t r a n s f e r a s e s and low molecular weight t h i o l s ( C a y g i l l , 1979). S u r p r i s i n g l y , pure papain i s not the major component of papaya l a t e x (Goodenough and Owen, 1987). The i n d i v i d u a l enzyme papain (E.C. 3.4.22.2) i s the most thoroughly c h a r a c t e r i z e d of the c y s t e i n e p r o t e a s e s . I t s s t r u c t u r e has been determined by X-ray d i f f r a c t i o n s t u d i e s and i t s p h y s i c o - c h e m i c a l c h a r a c t e r i s t i c s and mechanism of a c t i o n are s t i l l the s u b j e c t of i n t e n s i v e study i n many l a b o r a t o r i e s ( B r o c k l e h u r s t et a l . , 1981). In order to a v o i d c o n f u s i o n , the term "pure papain" w i l l be used i n t h i s paper to d e s c r i b e the i n d i v i d u a l p r o t e o l y t i c enzyme (E.C. 3.4.22.2); "crude papain" w i l l be used to i n d i c a t e the d r y papaya l a t e x without f u r t h e r p u r i f i c a t i o n ; and "commercial papain" w i l l be used to name the product r e s u l t i n g from p a r t i a l p u r i f i c a t i o n of crude papain. -4-B. PRODUCTION OF PAPAYA LATEX The p r o d u c t i o n of papaya l a t e x has changed l i t t l e i n r e c e n t decades. The major c o s t i s the establishment and maintenance of the p l a n t a t i o n ( C a y g i l l , 1979). The p r o d u c t i o n and p r o c e s s i n g of papaya l a t e x has been d i s c u s s e d i n d e t a i l by Jones and Mercier (1974), C a y g i l l (1979) and more r e c e n t l y by P o u l t e r and C a y g i l l (1985). 1. Agronomic F a c t o r s P o u l t e r and C a y g i l l (1985) covered the main agronomic f a c t o r s t h at i n f l u e n c e a papaya p l a n t a t i o n . An average d i u r n a l temperature range of 31 to 33°C with a r e g u l a r r a i n y season l a s t i n g from s i x to e i g h t months, followed by d r y weather appears to be i d e a l . A s l i g h t l y a c i d i c s o i l (pH 6.0 to 6.5) i s p r e f e r a b l e , and near the equator i n A f r i c a , a l t i t u d e s of about 1000 m are co n s i d e r e d f a v o r a b l e f o r papaya c u l t i v a t i o n f o r l a t e x ( P o u l t e r and C a y g i l l , 1985). There i s l i t t l e i n f o r m a t i o n about the best v a r i e t i e s f o r papain y i e l d , although Las s o u d i e r e (1969) mentioned Red Panama, F l o r i d e , Richbourg and Ineac 329 as among the best v a r i e t i e s . These c u l t i v a r s were re p o r t e d to have l a t e x y i e l d s s e v e r a l times t h a t of Solo, the v a r i e t y favored f o r f r u i t p r o d u c t i o n i n Hawaii (P o u l t e r and C a y g i l l , 1985). -5-2. Papaya l a t e x h a r v e s t i n g Latex i s harvested by t a p p i n g unripe f r u i t about four months a f t e r s e t t i n g and about s i x weeks before r i p e n i n g . Tapping i n v o l v e s the c u t t i n g of l o n g i t u d i n a l i n c i s i o n s on the s u r f a c e of the green unripe f r u i t u s i n g a sharp blade made of s t a i n l e s s s t e e l , such as a r a z o r blade ( P o u l t e r and C a y g i l l , 1985; B r o c k l e h u r s t et a l . , 1981). The l a t e x i s found i n the l a c i f e r o u s ducts o n l y 1-2 mm below the s u r f a c e . Cuts must not be deep, i n order to a v o i d i n f e c t i o n s of the f r u i t and contamination of the l a t e x with f r u i t j u i c e (CONAFRUT, 1973). The l a t e x flows f o r o n l y a few minutes and d r i p s i n t o cups ( B r o c k l e h u r s t et a l . , 1981), or i n t o c o l l e c t i o n d e v i c e s , resembling i n v e r t e d umbrellas attached around the trunk of the papaya t r e e (Poulter and C a y g i l l , 1985). A d d i t i o n a l l a t e x coagulates on the f r u i t and i s subsequently scraped o f f . Although B r o c k l e h u r s t et a l . (1981) mentioned t h a t t h i s coagulated l a t e x was a second grade m a t e r i a l , with a lower enzyme content, Madrigal et a l . (1980) re p o r t e d no d i f f e r e n c e i n p r o t e o l y t i c a c t i v i t y between the coagulated and the f i r s t l a t e x . The f r u i t should be wiped with a c l o t h soaked with s u l f i t e s or a s u i t a b l e f u n g i c i d e to prevent i n f e c t i o n of unprotected cuts (Salunke and Desai, 1984). F r u i t may be tapped s e v e r a l times, at approximately 4-day i n t e r v a l s , u n t i l r i p e n i n g . A f t e r the f i n a l t a p p i n g , the f r u i t - 6 -should be removed from the t r e e ( P o u l t e r and C a y g i l l - 1985). 3. Y i e l d s of papaya l a t e x The y i e l d s and q u a l i t y of the l a t e x depend on many f a c t o r s i n c l u d i n g f r u i t sex (Madrigal et a l . , 1980), s i z e of the f r u i t ( C a s t r o , 1981), s o i l c h a r a c t e r i s t i c s (Becker, 1958), temperature and season (Ca s t r o , 1981). Y i e l d s of f r e s h l a t e x are f r e q u e n t l y u n r e l i a b l e guides to y i e l d of p r o t e o l y t i c a c t i v i t y (Madrigal et a l . , 1980). F r u i t s from the same t r e e give d i f f e r e n t enzyme c o n c e n t r a t i o n s i n the l a t e x (Madrigal et a l . , 1980). Y i e l d s i n c a r e f u l l y managed p l a n t a t i o n s can be as high as 1,200 Kg of l a t e x / h a per year (Lassoudiere, 1969), though y i e l d s are o f t e n a p p r e c i a b l y lower (Lewis and Woodward, 1948). Krishnamurty et a l . (1960) and Madrigal et a l . (1980) re p o r t e d f r e s h l a t e x y i e l d s of 3-5 g / f r u i t / d a y (approximately 0.17% of the f r u i t weight). However, Castro (1981) re p o r t e d a much lower p r o d u c t i o n of 0.22-0.38 g / f r u i t / d a y under d i f f e r e n t c l i m a t i c c o n d i t i o n s . Kimmel and Smith (1957) i n d i c a t e d t h a t the average annual harvest of d r i e d l a t e x was approximately 100 g per t r e e . Shanmugavelu et a l . (1976) re p o r t e d t h a t the y i e l d s of papaya l a t e x were inc r e a s e d four f o l d over the c o n t r o l s with the use of the l a t e x s t i m u l a n t compound Ethephon. -7-C. PRODUCTION OF CRUDE AND COMMERCIAL PAPAIN 1. Drying Papaya l a t e x c o n t a i n s approximately 15-20% t o t a l s o l i d s and must be d r i e d without d e l a y i n order to inc r e a s e i t s s t a b i l i t y ( P o u l t e r and C a y g i l l , 1985). The moisture content of crude papain (dry papaya l a t e x ) i s u s u a l l y about 5-10% ( O r t i z et a l . , 1980; Vaidya et a l . , 1977). The e a r l i e s t commercial methods of d r y i n g papaya l a t e x were sun d r y i n g or k i l n d r y i n g . The l a t e x was t h i n l y spread on t r a y s or on c o t t o n c l o t h s and d r i e d by exposure to the sun or by open f i r e i n an oven ( P o u l t e r and C a y g i l l , 1985). Sun d r y i n g causes browning of the papaya l a t e x r e s u l t i n g i n a c o n s i d e r a b l e l o s s of enzymatic a c t i v i t y . The f i n a l d r y l a t e x ( i . e . crude papain) i s f r e q u e n t l y malodorous, as a r e s u l t of m i c r o b i a l growth (Salunke and Desai, 1984). Exposure to u l t r a v i o l e t r a d i a t i o n i n s u n l i g h t reduces p r o t e o l y t i c a c t i v i t y , and thus these methods are not recommended (P o u l t e r and C a y g i l l , 1985; Jones and Me r c i e r , 1974). Becker (1958) recommended a d r y i n g shed i n which l a t e x can be d r i e d at 55°C by i n d i r e c t heat. Cabinet d r y e r s have been used to prepare crude papain as a white g r a n u l a t e d powder of higher a c t i v i t y than t h a t obtained by sun or k i l n d r y i n g ( P o u l t e r and C a y g i l l , 1985; O r t i z et a l . , 1980). A summary of the d i f f e r e n t c o n d i t i o n s used f o r oven d r y i n g of papaya l a t e x i s shown i n Table 1. -8-Table 1. C o n d i t i o n s r e p o r t e d f o r oven d r y i n g of papaya l a t e x . Drying C o n d i t i o n s Temperature Time Load Reference*- (°C) (hr) (g/m«) A d d i t i v e s (1) 55 24 2,083 no (2) 40-80 N.R.B N.R. no (3) 55 3-4 944 0.5% PMB C (4) 55 6 1,600 1.0% SMBD+ 0.2% EDTA *Reference: (1) Becker (1958) (2) Castro (1981) (3) Krishnamurty et a l . (1960) (4) O r t i z et a l . (1980) ^.R. = Not rep o r t e d i n the r e f e r e n c e ^MB = Potassium m e t a b i s u l f i t e DSMB = Sodium m e t a b i s u l f i t e -9-Drying under vacuum (55<>c and 28 i n vacuum) minimizes p r o t e o l y t i c a c t i v i t y l o s s e s as compared to sun or oven d r y i n g (Krishnamurty et a l . , 1960). The patented Boudart process (Boudart, 1968; 1970; 1972) i n v o l v e s f i l t r a t i o n and c e n t r i f u g a t i o n of the f r e s h l y c o l l e c t e d l a t e x to remove sm a l l e r i n s o l u b l e i m p u r i t i e s . The l a t e x i s concentrated under vacuum at low temperature to 25% (w/v) s o l i d s or more. The l i q u i d passes to a c o n v e n t i o n a l low c a p a c i t y spray d r i e r . T h i s process produces a f i n e white to o f f - w h i t e powder with high p r o t e o l y t i c a c t i v i t y (Jones and Me r c i e r , 1974) . 2. R e f i n i n g Crude papain i s u s u a l l y exported and r e f i n e d i n the primary importing country ( B r o c k l e h u r s t et a l . , 1981). R e f i n i n g c o n s i s t s of d i s s o l v i n g the s o l u b l e c o n s t i t u e n t s , i n c l u d i n g the enzymes from the l a t e x , and p r e c i p i t a t i n g the enzymes with agents (e.g. ammonium s u l f a t e ) t h a t do not i n a c t i v a t e them ( C a y g i l l , 1979). The main o b j e c t i v e of r e f i n i n g i s to produce a s t a b l e , r e a d i l y s o l u b l e m a t e r i a l , with a low m i c r o b i a l count and s u b s t a n t i a l l y f r e e from the gummy substances and the o f f e n s i v e f l a v o r found i n crude papain (EDC, 1985). In the United S t a t e s imported crude papain i s p u r i f i e d by numerous methods, which g e n e r a l l y i n v o l v e f i l t r a t i o n , s o l v e n t or chemical p r e c i p i t a t i o n and/or spray d r y i n g , to y i e l d commercial -10-p u r i f i e d enzyme p r e p a r a t i o n s of v a r i o u s grades. These grades are u s u a l l y s t a n d a r d i z e d by adding l a c t o s e , sucrose, or s t a r c h as a d i l u e n t , to give commercial papain (Leung, 1980; M i l e s , 1985). These p u r i f i e d p r e p a r a t i o n s , o f t e n c a l l e d "papain", are not pure papain. Pure papain i s not commercially a v a i l a b l e on a l a r g e s c a l e , and i s used mainly f o r r e s e a r c h purposes (Leung, 1980). 3. Grades and p r i c e s of crude and commercial papain U n t i l the mid 1950s when the trade was dominated by s u p p l i e s from S r i Lanka, three grades of crude papain were known: (1) f i n e white powder form prepared by a s p e c i f i c p rocess, (2) oven-dried white crumb, and (3) s u n - d r i e d dark crumb (Salunke and Desai, 1984). Since 1970, other grades of papain, r e s u l t i n g from new p r o c e s s i n g techniques, have a r r i v e d on the market. As a r e s u l t , crude papain can be r e c l a s s i f i e d i n t o three groups (Salunke and Desai, 1984): 1. Crude papain: ranging from f i r s t grade white to second grade brown. 2. Crude papain i n f l a k e or powder form sometimes r e f e r r e d to as s e m i r e f i n e d . 3. S p r a y - d r i e d crude papain of higher a c t i v i t y , i n powder form. Table 2 presents p r i c e s , obtained from d i f f e r e n t sources, of crude and commercial papain. P r o t e o l y t i c a c t i v i t y determines the p r i c e of commercial papain. A d i r e c t comparison of the p r i c e s of commercial papain i s sometimes d i f f i c u l t because of the d i f f e r e n t -11-Table 2. P r i c e s of crude, commercial and pure papain. Type of P r i c e papain Grade (USD/kg) Source* Crude Brown No. 2 3. 60 - 5.20 (1) Crude White No.2 5. 20 - 8.60 (1) Crude A l 3. 00 - 8.50 (2) Crude Type I 103 .40 (3) Crude Type II 148 .50 (3) Commercial Papain 16,00 24 .25 (4) Commercial Papain 30,000 44 .09 (4) Pure 2X C r y s t a l l i z e d 115 .55* (3) "•Sources: (1) Salunke and Desai (1984) (2) Becker (1958) (3) Sigma Chemical Co. L t d (1988) (4) M i l e s L a b o r a t o r i e s (1987) B P r i c e f o r pure papain i n USD/g -12-methods used by enzyme marketing companies to measure the a c t i v i t y of the products. The p r i c e of the pure enzyme papain i s a l s o i n c l u d e d i n Table 2. P r i c e s of commercial papain e x h i b i t a c y c l i c a l b ehavior, r i s i n g to a higher f i g u r e and f a l l i n g , a f u l l c y c l e t a k i n g about f i v e y e a r s . There i s a l s o an o v e r a l l tendency f o r exports and p r i c e s to r i s e s l o w l y over time (Flynn, 1975). A more d e t a i l e d d i s c u s s i o n on the i n t e r n a t i o n a l trade of crude and commercial papain can be found i n F l y n n (1975). D. LOSSES OF PROTEOLYTIC ACTIVITY IN PAPAYA LATEX DUE TO DRYING AND DURING STORAGE OF CRUDE AND COMMERCIAL PAPAIN The p r o t e o l y t i c a c t i v i t y (PA) of commercial papain determines i t s commercial value i n a wide range of a p p l i c a t i o n s ( M i l e s , 1985). Thus any l o s s e s of PA i n crude papain b r i n g about a d i r e c t economic l o s s to the producer. Papaya l a t e x and crude papain are very unstable products, l o s i n g PA and hence commercial value, i n r e l a t i v e l y s h o r t p e r i o d s of time (Castro, 1981; CONAFRUT, 1973). F a c t o r s i n v o l v e d i n the l o s s e s d u r i n g storage and subsequent d r y i n g of the papaya l a t e x i n c l u d e : the a c t i v i t y of l a t e x protease i n h i b i t o r s such as v i t a m i n C and i s o t h i o c y a n a t e s ; m i c r o b i a l d e g r a d a t i o n of the enzymes; a u t o p r o t e o l y s i s ; i r r e v e r s i b l e o x i d a t i o n of c y s t e i n e r e s i d u e s ; and l i g h t s e n s i t i v i t y of the h i s t i d i n e r e s i d u e i n papain, which i s e s s e n t i a l f o r a c t i v i t y ( O r t i z et a l . , 1980; B r o c k l e h u r s t et -13-a l . , 1981). There i s l i m i t e d i n f o r m a t i o n a v a i l a b l e about the l o s s e s of PA d u r i n g storage and d r y i n g of papaya l a t e x , and du r i n g storage of crude papain. Exposure to s u n l i g h t (CONAFRUT, 1973; Salunke and Desai, 1984) and extended storage p r i o r to d r y i n g (CONAFRUT, 1973; Becker, 1958) are not recommended, but no q u a n t i t a t i v e data are r e p o r t e d . O r t i z et a l . , (1980) i n v e s t i g a t e d the e f f e c t s of storage p r i o r to d r y i n g on the PA of f r e s h papaya l a t e x . They r e p o r t e d a maximum l o s s of 20% of the PA a f t e r storage f o r 2 to 24 hr under t r o p i c a l c o n d i t i o n s . Most of the decrease i n PA occurred i n the f i r s t 6 hr of s t o r a g e . Under commercial p r a c t i c e , the minimum d e l a y a f t e r tapping and before d r y i n g i s l i k e l y to be about 2 hr ( O r t i z et a l . , 1980), but o f t e n i s 6-8 hr (Becker, 1958). Storage under s u n l i g h t causes s l i g h t l y g r e a t e r l o s s e s than storage i n the dark ( O r t i z et a l . , 1980). An assay of four samples of f r e s h l a t e x (4 hr o l d at time of assay) i n comparison with crude papain from the same l a t e x , i n d i c a t e d the PA l o s s due to oven d r y i n g at 55°C was 7+2% ( O r t i z et a l . , 1980). Another r e p o r t (CONAFRUT, 1973) mentions t h a t a c t i v i t y l o s s d u r i n g oven d r y i n g could be as high as 20%. Sun-dried crude papain was found to have 20-32% l e s s milk c l o t t i n g a c t i v i t y as compared with vacuum-dried crude papain (Krishnamurty et a l . , 1960). Castro (1981) rep o r t e d t h a t the d i f f e r e n c e of PA between sun- d r i e d and oven-dried crude papain was o n l y i n the order of 10%. The same author found t h a t the l o s s of PA due to storage of crude papain over a p e r i o d -14-of 75 days was 30% and 25% at room and r e f r i g e r a t e d temperature, r e s p e c t i v e l y . Commercial papain i s much more s t a b l e than crude papain. According to one manufacturer ( M i l e s , 1985), the l o s s e s i n PA of commercial papain i n s e a l e d c o n t a i n e r s , s t o r e d under c o o l , d r y c o n d i t i o n s are normally l e s s than 10% over one year. Storage l i f e can be extended by s t o r i n g under r e f r i g e r a t i o n a t 5°C. Dhawalikar and Pandit (1982) r e p o r t e d t h a t the milk c l o t t i n g a c t i v i t y of a "papain c o n c e n t r a t e " (most l i k e l y commercial papain) s t o r e d f o r 100 days at 25 or 37QC, decreased by 16%. Most of the l o s s occurred w i t h i n the f i r s t seven weeks. E. PROCESS TO IMPROVE THE STABILITY OF CRUDE AND COMMERCIAL PAPAIN Crude papain has r e l a t i v e l y low s t a b i l i t y . I t must be s t o r e d at low temperatures to a v o i d l o s s e s of enzymatic a c t i v i t y . Crude papain has been shown to l o s e much of i t s a c t i v i t y a f t e r storage f o r o n l y a few months (CONAFRUT, 1973). Many i n v e s t i g a t o r s have s t u d i e d the problem of the high b a c t e r i a l contamination and low s t a b i l i t y of papaya l a t e x and crude papain. Various methods e i t h e r to improve the p r o c e s s i n g ( i . e . t a p p i n g , d r y i n g and r e f i n i n g ) , or to c h e m i c a l l y s t a b i l i z e papain have been suggested (Jones and M e r c i e r , 1974 ) . -15-1. Improvement of tapping and c o l l e c t i n g procedures Tapping and c o l l e c t i n g procedures have a d i r e c t e f f e c t on the q u a l i t y of the crude papain. Poor tapping and c o l l e c t i n g procedures s t i l l p r e v a i l d e s p i t e the wide a v a i l a b i l i t y of i n f o r m a t i o n on the best techniques (Salunke and Desai, 1984). Common p r a c t i c e s i n the tapping and c o l l e c t i n g procedures t h a t should be avoided are (Salunke and Desai, 1984): (1) tapping by i n c i s i o n s which are too deep, thereby a l l o w i n g j u i c e s and s t a r c h from the f r u i t pulp to contaminate the l a t e x ; (2) tapping at times other than the morning or on overcast days, which r e s u l t s i n q u a l i t y r e d u c t i o n of the l a t e x ; (3) tapping too soon before m a t u r i t y , or too long a f t e r m a t u r i t y ; (4) l e a v i n g the l a t e x too long i n the sun, where i t can l o s e enzyme a c t i v i t y and c o l l e c t f o r e i g n matter such as i n s e c t s and dust. 2. The Boudart Process The Boudart process, a l r e a d y d e s c r i b e d , produces a commercial papain with low b a c t e r i a l contamination, good s o l u b i l i t y and high p r o t e o l y t i c a c t i v i t y . Only mechanical methods of p u r i f i c a t i o n , such as f i l t r a t i o n and c e n t r i f u g a t i o n are used (Baines and B r o c k l e h u r s t , 1979; C a y g i l l , 1979; Jones and M e r c i e r , 1974). The need of modern equipment, somewhat s i m i l a r to t h a t of a milk d r y i n g p l a n t (Jones and M e r c i e r , 1974), l i m i t s i t s a p p l i c a b i l i t y to o n l y l a r g e s c a l e o p e r a t i o n s . I t i s i n a p p r o p r i a t e to many c o u n t r i e s where papain would be considered -16-a by-product of the f r u i t on an intermediate s c a l e ( O r t i z et a l . , 1980; CONAFRUT, 1973). 3. A d d i t i o n of reducing agents The a d d i t i o n of reducing agents, such as sodium b i s u l f i t e or m e t a b i s u l f i t e , i n combination with a c h e l a t i n g compound ( i . e . EDTA) before d r y i n g , has been shown to decrease the l o s s e s of p r o t e o l y t i c a c t i v i t y due to d r y i n g by 20% as compared to an untreated sample ( O r t i z et a l . , 1980). Krishnamurty et a l . (1960) found t h a t a d d i t i o n of 0.5% m e t a b i s u l f i t e to the l a t e x before d r y i n g c o n s i d e r a b l y improved the a c t i v i t y of crude papain ( i . e . d r y l a t e x ) when prepared by sun d r y i n g , but o n l y s l i g h t l y when prepared by vacuum d r y i n g . Jones and Mercier (1974) mentioned t h a t chemical s t a b i l i z a t i o n of l a t e x b r i n g s improvement i n r e t e n t i o n of p r o t e o l y t i c a c t i v i t y , but the treatment of f r e s h l a t e x should be done i n areas where i t i s easy to export and import chemicals and b i o c h e m i c a l products. T h i s i s not g e n e r a l l y the case i n papaya growing d i s t r i c t s . Commercial papain u s u a l l y c o n t a i n s a s t a b i l i z i n g agent such as sodium or potassium m e t a b i s u l f i t e (Perez and Lopez-Munguia, 1985). Numerous p r e p a r a t i o n s to s t a b i l i z e commercial papain s o l u t i o n s have been d e s c r i b e d i n the patent l i t e r a t u r e . C a y g i l l (1979) r e p o r t e d more than ten patents on t h i s s u b j e c t . A t y p i c a l composition of a p r e p a r a t i o n to c h i l l - p r o o f beer i n c l u d e s the f o l l o w i n g (Rommele, 1978): 20-60% sucrose, 1.5-3% sodium m e t a b i s u l f i t e and 1-35% papain. -17-F. SODIUM TETRATHIONATE AS A STABILIZING AGENT OF SULFHYDRYL PROTEASES Sodium t e t r a t h i o n a t e , a symmetrical d i s u l f i d e , has been shown to r e v e r s i b l y o x i d i z e t h i o l s q u a n t i t a t i v e l y to t h e i r c orresponding d i s u l f i d e s . The o x i d a t i o n i s promptly reversed by the a d d i t i o n of a reducing agent such as c y s t e i n e , 0-mercaptoethanol or d i t h i o t h r e i t o l (Means and Feeney, 1971; P i h l and Lange, 1962). 1. Mechanism of r e a c t i o n The r e a c t i o n of t e t r a t h i o n a t e with c y s t e i n e proteases can be seen as a c o n t r o l l e d o x i d a t i o n ; i t proceeds a c c o r d i n g to the f o l l o w i n g scheme: E-SH + R-S-S-R <= = = = = = =» E-S-S-R + R-SH (1) where E-SH r e f e r s to the a c t i v e s u l f h y d r y l enzyme such as papain, bromelain or f i c i n ; R-S-S-R i s the symmetrical d i s u l f i d e i n a c t i v a t o r ( i . e t e t r a t h i o n a t e ) ; E-S-S-R i s the r e v e r s i b l y i n a c t i v a t e d enzyme i n the form of a mixed d i s u l f i d e ; and R-SH i s a low molecular weight t h i o l (Means and Feeney, 1971). -18-2. R e v e r s i b l e i n a c t i v a t i o n Upon r e a c t i o n of p r o t e o l y t i c enzyme with t e t r a t h i o n a t e , the protease becomes r e v e r s i b l y i n a c t i v e . Thus, the process of a u t o l y s i s i s i n h i b i t e d . T e t r a t h i o n a t e a l s o p r o t e c t s the a c t i v e c y s t e i n e r e s i d u e i n c y s t e i n e proteases from i r r e v e r s i b l e o x i d a t i o n (Englund et a l . , 1968). A u t o l y s i s and i r r e v e r s i b l e o x i d a t i o n are the major causes of i r r e v e r s i b l e l o s s e s of enzymatic a c t i v i t y of c y s t e i n e proteases i n g e n e r a l , and of papain i n p a r t i c u l a r . I t can be expected t h a t the a d d i t i o n of sodium t e t r a t h i o n a t e to papaya l a t e x w i l l i n c r e a s e i t s s t a b i l i t y , and minimize l o s s of p r o t e o l y t i c a c t i v i t y d u r i n g d r y i n g and stor a g e . T e t r a t h i o n a t e has been used with great success to prevent of a u t o l y s i s of c y s t e i n e proteases d u r i n g chromatographic p u r i f i c a t i o n or s e p a r a t i o n o f : stem bromelain (Takahashi et a l . , 1973); f i c i n (Englund et a l . , 1968); c a l o t r o p i n , a s u l f h y d r y l protease i s o l a t e d from the l a t e x of the madar p l a n t ( C a l o t r o p i s  qigantea) (Pal et a l . , 1984); a s u l f h y d r y l protease from s p r o u t i n g potato tubers (Kitamura and Maruyama, 1986) and more r e c e n t l y sodom apple protease (Aworth, 1987). Use of t e t r a t h i o n a t e r e s u l t s i n higher y i e l d s of enzymes and an incr e a s e d q u a l i t y of s e p a r a t i o n . Sodium t e t r a t h i o n a t e a l s o has an a n t i m i c r o b i a l e f f e c t a g a i n s t some b a c t e r i a (Palumbo and A l f o r d , 1970). I t i s p o s s i b l e t h a t a d d i t i o n of t e t r a t h i o n a t e to papaya l a t e x w i l l decrease the -19-m i c r o b i a l load of the l a t e x . The a d d i t i o n of another d i s u l f i d e , 2,21-dipyridyl d i s u l f i d e (2PDS) to f r e s h papaya l a t e x i n order to i n c r e a s e i t s s t a b i l i t y has been r e p o r t e d ( B r o c k l e h u r s t et a l . , 1985). The r e v e r s i b l e i n a c t i v a t i o n of papain, with t e t r a t h i o n a t e , was used by Kim Kam et a l . (US Patent 3,818,106 (1974)) to prepare an enzyme f o r m u l a t i o n f o r i n j e c t i o n ante mortem i n t o animals as a meat t e n d e r i z e r . P r o t e o l y s i s occurred without causing severe p h y s i o l o g i c a l r e a c t i o n s i n the i n j e c t e d animals, which might otherwise r e s u l t i n condemnation of the c a r c a s s e s by governmental i n s p e c t o r s . In the Proten process (developed by S w i f t and Company L i m i t e d ) , a concentrated s o l u t i o n of t e t r a t h i o n a t e - i n a c t i v a t e d papain i s i n j e c t e d i n t o the j u g u l a r v e i n , ten to t h i r t y minutes before s l a u g h t e r . C o n t i n u i n g g l y c o l y s i s d e p l e t e s oxygen from the muscle of the f r e s h l y s l a u g h t e r e d c a r c a s s , r e s u l t i n g i n an accumulation of f r e e t h i o l s and other reducing agents. Under these c o n d i t i o n s , the o x i d i z e d papain may become r e a c t i v e . The c o n v e r s i o n i s slow i n c h i l l e d meat, but i s completed r a p i d l y on warming ( D r a n s f i e l d and E t h e r i n g t o n , 1981). T h i s process has been f u l l y approved by the Meat and P o u l t r y I n s p e c t i o n Program of the US Department of A g r i c u l t u r e (Smith et a l . , 1973). Accor d i n g to Bradley et a l . (1987) i n the United Kingdom, some hundred thousand c a t t l e are t r e a t e d a n n u a l l y by the Proten process i n 21 a b b a t t o i r s . R e c e n t l y i t was confirmed (Bradley et a l . , 1987) that the -20-Proten process does not cause b e h a v i o r a l or other c l i n i c a l a b n o r m a l i t i e s i n c a t t l e f o l l o w i n g treatment. Carcasses were examined by gross v i s u a l assessment. I t was concluded t h a t the Proten process d i d not cause d e t e c t a b l e h e p a t o c e l l u l a r or r e n a l damage. In another study, the Proten process s i g n i f i c a n t l y i n c r e a s e d the tenderness and o v e r a l l s a t i s f a c t i o n r a t i n g s of b u l l o c k s t e a k s , i n comparison to steaks from untreated b u l l o c k s (Smith et a l . , 1973) G. CHEMICAL PROPERTIES OF TETRATHIONATE Compounds of the composition H2S*06 have been known f o r a long time i n the form of s a l t s t h at are r a t h e r unstable i n aqueous s o l u t i o n . T h i s holds f o r the ions S J O G " 1 , StOs'2, S S0 6" J and SsOs'% which have been designated as p o l y t h i o n i c a c i d (Schmidt, 1972). Although s a l t s of these compounds are u s u a l l y designated as p o l y t h i o n a t e s , Na2S30s and Na 2S40 s are known as sodium t r i t h i o n a t e and t e t r a t h i o n a t e , r e s p e c t i v e l y . The proper names of s u l f a n e d i s u l f o n a t e s , f o r example monosulfane d i s u l f o n a t e f o r t r i t h i o n a t e , and d i s u l f a n e d i s u l f o n a t e f o r t e t r a t h i o n a t e , should r e p l a c e the e s t a b l i s h e d and customary o l d e r names (Schmidt, 1972). According to Schmidt (1972), the olde r l i t e r a t u r e on these p o l y t h i o n i c a c i d s i s overwhelming. The problems connected with t h e i r chemical nature, r e a c t i o n s , formation, and decomposition were f o r a long time one of the great -21-p u z z l e s i n i n o r g a n i c chemistry. The same author i n d i c a t e s t h a t many of the o l d p u b l i c a t i o n s are f r e q u e n t l y c o n t r a d i c t o r y , which shows t h a t no r e a l understanding e x i s t e d on the nature of the compounds. 1. Some p r o p e r t i e s of t e t r a t h i o n a t e Sodium and potassium t e t r a t h i o n a t e are c o l o r l e s s , with p l a t e l i k e or p r i s m a t i c c r y s t a l s (Feher, 1963). T e t r a t h i o n a t e i s r e a d i l y s o l u b l e i n water up to 12.6% and 23.2% at 0 and 20°C, r e s p e c t i v e l y . They are i n s o l u b l e i n absolute a l c o h o l (Feher, 1963). Aqueous s o l u t i o n s of the s a l t s decompose i n t o t r i t h i o n a t e and s u l f u r i f heated (Haff, 1970). The pure dry m a t e r i a l i s s t a b l e f o r a long time without change, but decomposes i f i m p u r i t i e s ( i . e . t h i o s u l f a t e s or s u l f i t e s ) are present (Feher, 1963). T e t r a t h i o n a t e i s s t a b l e i n c o l d concentrated a c i d (Schmidt, 1972). Fordos and G e l i s (1842) re p o r t e d t h a t the d i l u t e d aqueous s o l u t i o n c o u l d be b o i l e d without decomposition, but the concentrated s o l u t i o n decomposed i n t o s u l f u r , s u l f u r d i o x i d e and s u l f u r i c a c i d . T e t r a t h i o n a t e i s very unstable i n a l k a l i s o l u t i o n , decomposing i n t o s u l f a t e and s u l f i t e . D i l u t e aqueous s o l u t i o n s of t e t r a t h i o n a t e decompose with time at room temperature (Schmidt, 1972) . -22-2. S t r u c t u r e of t e t r a t h i o n a t e Using X-ray c r y s t a l l o g r a p h y f o r s t r u c t u r e d e t e r m i n a t i o n , Foss (1960) r e p o r t e d that t e t r a t h i o n a t e ions c o n s i s t of two d i s t o r t e d S203~2 t e t r a h e d r a j o i n e d by a c o v a l e n t bond: Two types of s u l f u r - s u l f u r bonds occur i n t h i s compound, between two d i v a l e n t s u l f u r atoms i n the middle of the c h a i n and between one d i v a l e n t and one s u l f o n a t e s u l f u r atom i n the ends. 3. Uses T e t r a t h i o n a t e has been r e p o r t e d to be used as a fermentation a i d i n the p r o d u c t i o n of p e n i c i l l i n (Peterson, 1959). I t has a l s o been r e p o r t e d to a c t a g a i n s t cyanide p o i s o n i n g (SSrbo, 1972), as i t r e a c t s spontaneously with cyanide, the l a t t e r being converted to t h i o c y a n a t e . Although an a n t i d o t e e f f e c t has been e s t a b l i s h e d , t e t r a t h i o n a t e i s i n f e r i o r to t h i o s u l f a t e i n t h i s r e s p e c t (Sorbo, 1972). Since t e t r a t h i o n a t e has b a c t e r i c i d a l a c t i o n i t has been used, to a l i m i t e d extent, f o r medical f o r m u l a t i o n s , e s p e c i a l l y l o t i o n s and creams (Sorbo, 1972) . -23-T e t r a t h i o n a t e , produced In. s i t u - i s widely used i n a s e l e c t i v e enrichment medium ( T e t r a t h i o n a t e broth) f o r Salmonella (Poelma et a l . , 1984). Calcium t e t r a t h i o n a t e showed promising r e s u l t s as a f u n g i c i d e a g a i n s t powdery mildew of cucumbers (Golyshin et a l . , 1967). An organic d e r i v a t i v e of t e t r a t h i o n a t e , b i s ( d i i s o b u t y l a m i n e ) t e t r a t h i o n a t e , has been used as a f u n g i c i d e f o r powdery mildew (Abylgaziev, 1967), and hydrazine t e t r a t h i o n a t e has been shown to be u s e f u l f o r combating a p a r a s i t i c fungus (Erysiphe graminis) of cucumber (Sanin et a l . , 1966). T e t r a t h i o n a t e was a l s o used to produce immunoglobulin p r e p a r a t i o n s f o r intravenous use with decreased anti-complementary a c t i v i t y (Yoshida et a l . , 1980; Masuyasu and Tomibe, 1979). Damodaran (1986) repo r t e d a simple method of removal of n u c l e i c a c i d s from yeast n u c l e o p r o t e i n complexes by s u l f i t o l y s i s . T h i s method i n v o l v e d the treatment of yeast n u c l e o p r o t e i n s with sodium s u l f i t e followed by sodium t e t r a t h i o n a t e . T h i s treatment caused d e s t a b i l i z a t i o n and d i s s o c i a t i o n of the n u c l e o p r o t e i n complexes. Subsequent p r e c i p i t a t i o n of p r o t e i n s at pH 4.2 r e s u l t e d i n a p r o t e i n p r e p a r a t i o n with low l e v e l s of n u c l e i c a c i d s (Damodaran, 1986). In the area of biotechnology, t e t r a t h i o n a t e was used to regenerate d i s u l f i d e bonds i n a g e n e t i c a l l y engineered m i c r o b i a l rennet (Hayenga et a l . , 1982). -24-4. T o x i c i t y The t o x i c a c t i o n of t e t r a t h i o n a t e i s due to the f a c t t h at i t r a p i d l y o x i d i z e s , in. v i v o , s u l f h y d r y l compounds to d i s u l f i d e s , i t s e l f being reduced to t h i o s u l f a t e (Sorbo, 1972; Gilman et a l . , 1946a) . T e t r a t h i o n a t e has been found to produce a st r o n g nephrotoxic a c t i o n causing complete a n u r i a i n dogs w i t h i n 30 to 60 min (Gilman et a l . , 1946a). The l e t h a l dose of t e t r a t h i o n a t e when i n j e c t e d i n t r a v e n o u s l y ( i . v . ) i n t o r a b b i t s was 100 mg/kg of body weight. P a t h o l o g i c a l examination of the kidneys r e v e a l e d n e c r o s i s of the c e l l s of the proximal r e n a l tubes (Gilman et a l . , 1946a). An impairment of kidney f u n c t i o n has a l s o been demonstrated i n human p a t i e n t s who r e c e i v e d r e l a t i v e l y l a r g e doses of t e t r a t h i o n a t e (Sorbo, 1972). No LDso value has been repo r t e d f o r sodium t e t r a t h i o n a t e . For b i s ( d i i s o b u t y l a m i n e ) t e t r a t h i o n a t e LDso values obtained by intramuscular i n j e c t i o n were r e p o r t e d as 6,750 mg/kg f o r r a t s , and 2,169 mg/kg f o r mice. C l o n i c spasms and death i n the f i r s t day with i n h i b i t i o n of b r e a t h i n g were found to occur i n both s p e c i e s (Abylgaziev, 1967). Comparison of the t o x i c i t i e s of t e t r a t h i o n a t e and b i s u l f i t e , a compound commonly used as a p r o t e c t i n g agent of the PA of papaya l a t e x , suggest t h a t t e t r a t h i o n a t e i s not as t o x i c as b i s u l f i t e ; the i . v . LDso f o r b i s u l f i t e f o r r a b b i t i s 65 mg/kg (FAO, 1974), -25-compared to an approximate value of 100 mg/kg f o r t e t r a t h i o n a t e . Although LD B 0 and l e t h a l doses are expressions of the t o x i c i t y of a chemical compound, a more r e a l i s t i c procedure to assess the p o t e n t i a l r i s k of a food a d d i t i v e must i n v o l v e a comparison of the most probable consumption l e v e l to v a r i o u s expressions of t o x i c i t y (e.g. LDso or l e t h a l dose values) (Vanderstoep, 1988). In order to estimate the probable d a i l y intake of t e t r a t h i o n a t e by man, the f o l l o w i n g assumptions were made: 1) Commercial papain i s the s o l e source of t e t r a t h i o n a t e i n the d i e t ; 2) For the c a l c u l a t i o n of the d a i l y intake of t e t r a t h i o n a t e o n l y the two major food uses of commercial papain, meat t e n d e r i z a t i o n and c h i l l p r o o f i n g of beer, are c o n s i d e r e d ; 3) The per c a p i t a consumption of beer and red meat were taken as 217.5 mL/day and 195.5 g/day, r e s p e c t i v e l y ( S t a t i s t i c s Canada, 1987); 4) A l l beer was assumed to be c h i l l p r o o f e d at a l e v e l of 8 ppm (8 mg/L) (Reed, 1966) using a commercial product t h a t had 16% of commercial papain (Perez and Lopez Munguia, 1985), and a l e v e l of t e t r a t h i o n a t e i n the commercial papain was taken as 1%; 5) Only 10% of the meat was t e n d e r i z e d with papain. The l e v e l of t e n d e r i z e r added to meat was taken as 12 g/kg meat (U n d e r k o f l e r , 1972). The c o n c e n t r a t i o n of commercial papain i n the t e n d e r i z e r was assumed to be 2% ( U n d e r k o f l e r , 1972), and the l e v e l of t e t r a t h i o n a t e i n the papain was again -26-taken as 1%. Taking these assumptions i n t o c o n s i d e r a t i o n we have: -Consumption of t e t r a t h i o n a t e due to beer i n t a k e : 0.217 L/day x 8 mg/L x 0.16 x 0.01 = 0.0028 mg/day -Consumption of t e t r a t h i o n a t e due to meat i n t a k e : 0.02 kg/day x 0.012 kg/kg x 0.02 x 0.01 = 0.048 mg/day T o t a l consumption of t e t r a t h i o n a t e = 51 \lq/ day Using the i . v . l e t h a l dose of 100 mg/kg f o r r a b b i t s as being a p p l i c a b l e to man a l e t h a l dose of t e t r a t h i o n a t e f o r the average man i s 7,000 mg (70 kg x 100 mg/kg). The intake of 51 Hg/day i s 0.00073% of the assumed l e t h a l dose. T h i s would suggest that the use of t e t r a t h i o n a t e as an a d d i t i v e i n commercial papain does not r e p r e s e n t a h e a l t h hazard. Although t e t r a t h i o n a t e i s not a food a d d i t i v e , i t i s expected to be found i n those products where s u l f u r d i o x i d e (SO*) i s used, s i n c e S0 2 i s very l i k e l y to be o x i d i z e d to t e t r a t h i o n a t e and to other compounds by r e a c t i n g with food c o n s t i t u e n t s (Eckohoff and Okos, 1986). H. PREPARATION OF TETRATHIONATE Since t e t r a t h i o n a t e i s the f i r s t o x i d a t i o n product of t h i o s u l f a t e , most p r e p a r a t i o n methods are through an o x i d a t i o n r e a c t i o n of t h i o s u l f a t e . Table 3 r e p o r t s the standard e l e c t r o d e p o t e n t i a l of some -27-Table 3. Standard e l e c t r o d e p o t e n t i a l (E°) f o r some chemical rea c t i o n s * ' 8 . H a lf r e a c t i o n E<>, v S«CV S + 2e" 2SjO,-a +0.170 la + 2e" 21" +0.535 H»0 2 + 2H* + 2e- 2H20 +1.776 Fe*' + e- Fe f l +0.771 Cu" + e" Cu» +0.167 VCV 1 + 2H* + e" VO*2 + H»0 +0.361 *-Half r e a c t i o n s are w r i t t e n as r e d u c t i o n B S o u r c e : F r i t z and Schenk (1972) -28-o x i d i z i n g agents that have been used to s y n t h e s i z e t e t r a t h i o n a t e from t h i o s u l f a t e . The standard e l e c t r o d e p o t e n t i a l f o r the o x i d a t i o n of t h i o s u l f a t e to t e t r a t h i o n a t e i s o n l y -0.17 V which i n d i c a t e s weak oxidants should be used to s y n t h e s i z e t e t r a t h i o n a t e , i n order to prevent f u r t h e r o x i d a t i o n of t h i o s u l f a t e to higher valences ( i . e . s u l f a t e s ) . Some chemical r e a c t i o n s i n the s y n t h e s i s of t e t r a t h i o n a t e are shown i n Table 4. These r e a c t i o n s are d i s c u s s e d i n d e t a i l i n the f o l l o w i n g s e c t i o n . 1. Iodine o x i d a t i o n O x i d a t i o n of a t h i o s u l f a t e with a c i d i c i o d i n e y i e l d s t e t r a t h i o n a t e q u a n t i t a t i v e l y (Schmidt, 1972). According to Awtrey and Connick (1951) the mechanism i s as f o l l o w s : I-I + :SSOr 2 > I:SS0 3- + I" (2) SS0 3:" 2 + I:SS0 3 > 0 3SS:SS0r 2 + I" (3) I t should be noted t h a t t r a n s f e r of the two e l e c t r o n s occurs i n the second step, when a second t h i o s u l f a t e ion a t t a c k s the i o d i n e t h i o s u l f a t e i n t e r m e d i a t e . The two e l e c t r o n s , bonding i o d i n e to s u l f u r i n the in t e r m e d i a t e , are t r a n s f e r r e d to i o d i d e as i t i s d i s p l a c e d from the intermediate by the second t h i o s u l f a t e . T h i s i s a t y p i c a l example of a bi m o l e c u l a r n u c l e o p h i l i c displacement r e a c t i o n (S N2) ( F r i t z and Schenk, 1972). T h i s r e a c t i o n i s e x t e n s i v e l y used i n a n a l y t i c a l chemistry f o r o x i d a t i o n - r e d u c t i o n -29-Table 4 . Chemical r e a c t i o n s i n the s y n t h e s i s of t e t r a t h i o n a t e . Oxidant Reaction Iodine 2Na 2S 20i + I 2 • 2NaI + Na»S40-Hydrogen Peroxide 2Na 2S 20 3 + H 20 2 * Na 2S«0 6 + 2NaOH F e r r i c c h l o r i d e 2Na2S20- + 2FeCli » Na2S«0< + 2Fe C l 2 + 2NaCl Cu p r i c s u l f a t e 3Na 2S 20j + 2CuSO« » Na-SiOs + 2Na2S04 + Cu 2S 20 3 Sodium vanadate*- 2S 20r J + V O l - i » S«0--J + V O J " 1 * T h i s r e a c t i o n i s only f o r the chemical s p e c i e s i n v o l v e d i n the o x i d a t i o n - r e d u c t i o n -30-t i t r a t i o n . Gilman et a l . (1946a) r e p o r t e d i n d e t a i l the method of p r e p a r a t i o n of sodium t e t r a t h i o n a t e with i o d i n e . They r e p o r t e d t h a t the t e t r a t h i o n a t e obtained was 98.8 to 102% pure, with an o v e r a l l y i e l d of 65% of the t h e o r e t i c a l y i e l d . A s i m i l a r i o d i n e method was repor t e d by Feher (1963). 2. O x i d a t i o n with other compounds O x i d i z i n g agents other than i o d i n e a l s o produce t e t r a t h i o n a t e from t h i o s u l f a t e . M e l l o r (1930), i n h i s c l a s s i c a l book, mentioned many of the r e a c t i o n s , r e p o r t e d u n t i l t h a t time, to s y n t h e s i z e t e t r a t h i o n a t e . Since many of the o r i g i n a l r e f e r e n c e s are from the beginning of t h i s c e n t u r y i t was imp o s s i b l e , i n many cases, to review the o r i g i n a l papers. (a) O x i d a t i o n with hydrogen peroxide Nabl (1900) i n a s h o r t " c o r r e c t i o n " i n d i c a t e d t h at hydrogen peroxide o x i d i z e d t h i o s u l f a t e to t e t r a t h i o n a t e provided that the sodium hydroxide was n e u t r a l i z e d as i t was formed. Otherwise the hydroxide decomposed the t e t r a t h i o n a t e i n t o t h i o s u l f a t e , s u l f a t e and s u l f i t e . T h i s " c o r r e c t i o n " was very s u p e r f i c i a l and no d e t a i l e d procedure was r e p o r t e d . Abel (1907) r e p o r t e d that the second order r a t e constant f o r the r e a c t i o n of hydrogen peroxide with t h i o s u l f a t e was 1.53. No u n i t s were i n d i c a t e d . Assuming the standard u n i t s (M"'sec"M f o r the aforementioned constant, i t can be c a l c u l a t e d t h a t i n approximately 35 min, the r e a c t i o n should reach 90% completion. -31-Since hydrogen peroxide i s a very s t r o n g o x i d i z i n g agent, care has to be taken to c o n t r o l the r e a c t i o n , i n order to av o i d o x i d a t i o n of the t h i o s u l f a t e to s u l f i t e or s u l f a t e . (b) O x i d a t i o n with metals s a l t s Fordos and G e l i s (1842) repo r t e d the s y n t h e s i s of t e t r a t h i o n a t e using f e r r i c c h l o r i d e as the o x i d i z i n g agent. More r e c e n t l y , the same o x i d a t i o n r e a c t i o n was re p o r t e d to be ra t h e r slow, and to be c a t a l y s e d by copper s a l t s (Lar and Singh, 1956). The r e d u c t i o n of c u p r i c to cuprous s a l t s can be used to o x i d i z e t h i o s u l f a t e to t e t r a t h i o n a t e ( Z e l t n o f f , 1867; Raschig, 1920). The standard p o t e n t i a l s f o r f e r r i c and c u p r i c o x i d a t i o n , shown i n Table 3, suggest t h a t both ions are mil d o x i d i z i n g agents. (c) O x i d a t i o n with vanadate Gowda et a l . (1955) developed a method to estimate t h i o s u l f a t e with sodium vanadate. In t h i s method, sodium t h i o s u l f a t e was q u a n t i t a t i v e l y o x i d i z e d to t e t r a t h i o n a t e i n f i v e minutes a t room temperature with excess sodium vanadate. The r e a c t i o n occurred i n a medium c o n t a i n i n g s u l f u r i c or a c e t i c a c i d and a small amount of copper s u l f a t e as a c a t a l y s t . F u r t h e r o x i d a t i o n d i d not occur even when the mixture was heated to b o i l i n g . A f t e r the r e a c t i o n was complete, the excess vanadate was t i t r a t e d with a standard s o l u t i o n of f e r r o u s ammonium s u l f a t e . Since q u a n t i t a t i v e o x i d a t i o n of t h i o s u l f a t e to t e t r a t h i o n a t e -32-Since q u a n t i t a t i v e o x i d a t i o n of t h i o s u l f a t e to t e t r a t h i o n a t e was r e p o r t e d , i t i s p o s s i b l e to use t h i s r e a c t i o n to s y n t h e s i z e t e t r a t h i o n a t e . 3. Other methods of s y n t h e s i s of t e t r a t h i o n a t e . The r e a c t i o n of s u l f u r y l c h l o r i d e with t h i o s u l f a t e s r e s u l t s i n p r a c t i c a l l y q u a n t i t a t i v e y i e l d s of pure t e t r a t h i o n a t e and s u l f u r d i o x i d e (Schmidt, 1972). T e t r a t h i o n a t e can a l s o be prepared by the r e a c t i o n of s u l f u r c h l o r i d e with the r a t h e r complicated mixture t h a t i s formed when s u l f u r d i o x i d e i s d i s s o l v e d i n water (Feher, 1963). T h i s r e a c t i o n mixture i s c a l l e d " s u l f u r o u s a c i d " by t r a d i t i o n , without having a d e f i n i t i v e composition (Schmidt, 1972). T h i s method of p r e p a r a t i o n i s somewhat d i f f i c u l t to c a r r y out and although good y i e l d s and p u r i t y are r e p o r t e d f o r the t e t r a t h i o n a t e obtained, i t s a p p l i c a t i o n i s l i m i t e d to l a b o r a t o r y s c a l e p r o d u c t i o n . Since the s u l f u r bacterium E c t o t h i o r h o d o s p i r a shaposhnikovi has been r e p o r t e d to o x i d i z e t h i o s u l f a t e to t e t r a t h i o n a t e , i t i s p o s s i b l e to use t h i s bacterium to s y n t h e s i z e t e t r a t h i o n a t e (Gogotava and Vainshteim, 1981). Pure c u l t u r e s of T h i o s p i r a accumulate t e t r a t h i o n a t e when grown on a medium c o n t a i n i n g o r g a n i c compounds (Dubinins and S e l v i c h , 1983) . -33-Another r e p o r t i n d i c a t e d t h a t an a u t o t r o p h i c , a c i d o p h i l i c T h i o b a c i l l u s metabolized t h i o s u l f a t e to t e t r a t h i o n a t e d u r i n g growth and c o u l d not r e o x i d i z e t h i s product (Reynolds et a l . , 1981). -34-MATERIALS AND METHODS A. MATERIALS Since papaya l a t e x i s commercially u n a v a i l a b l e , crude papain, Type I (Sigma Chemical Co. St. L o u i s , MO) was used i n s t e a d . Sodium t e t r a t h i o n a t e was obtained from ICN Pharmaceuticals, Inc. ( L i f e Science Group, P l a i n v i e w , NY) and sodium m e t a b i s u l f i t e , anhydrous powder, was purchased from Matheson Coleman & B e l l Manufacturing Chemists, Norwood, OH. Cysteine h y d r o c h l o r i d e and EDTA were from Sigma Chemical Co. St. L o u i s , MO. Casein, a c c o r d i n g to Hammersten was from BDH Chemicals, Vancouver B.C. Glass d i s t i l l e d d e i o n i z e d water was used i n the p r e p a r a t i o n of a l l s o l u t i o n s and b u f f e r s . A l l other chemicals were of a n a l y t i c a l grade. A l l pH-values were measured using a F i s h e r model 420 pH-meter ( F i s h e r S c i e n t i f i c Co., P i t t s b u r g h , PA). B. REHYDRATION OF CRUDE PAPAIN In order to simulate a system s i m i l a r to f r e s h papaya l a t e x , crude papain was rehydrated with d i s t i l l e d water to give a 20% s o l u t i o n (w/v), a s o l i d r a t i o s i m i l a r to that of f r e s h papaya l a t e x ( O r t i z et a l . , 1980). The r e h y d r a t i o n was as f o l l o w s : crude papain was mixed with c o l d d i s t i l l e d water and l e f t at 4°C f o r 2 hr with slow a g i t a t i o n . T h i s rehydrated crude papain w i l l be r e f e r r e d to as papaya l a t e x . -35-C. DRYING CHARACTERISTICS OF PAPAYA LATEX The e f f e c t of temperature (55, 80 and 100°C), d r y i n g load (1,190, 2,381, and 4,792 g/ra* of d r y i n g a r e a ) , and a d d i t i o n of s u l f i t e or t e t r a t h i o n a t e (both at 1% (w/v)), on the d r y i n g r a t e of papaya l a t e x was i n v e s t i g a t e d . An e l e c t r i c a l l y heated, mechanical c o n v e c t i o n , f o r c e d h o r i z o n t a l a i r - f l o w t r a y d r i e r was used (Blue M Stabil-Therm Oven, Model OV-490a-2, Blue M E l e c t r i c Company, Blue I s l a n d , I L ) . The samples were d r i e d i n aluminum di s h e s ( i . d . 2.3 cm and 0.5 cm deep). The oven was preheated f o r a t l e a s t one hour at the d e f i n e d temperature before the s t a r t of the d r y i n g experiments. In order to o b t a i n the d r y i n g curves, samples were withdrawn from the oven at v a r i o u s time i n t e r v a l s , and the moisture content of a l l samples was determined as the weight l o s s a f t e r 12 hr a t 80°C under vacuum. D. DETERMINATION OF INFLUENTIAL FACTORS ON THE LOSSES OF PROTEOLYTIC ACTIVITY OF PAPAYA LATEX The f r a c t i o n a l f a c t o r i a l d e s ign L IT (3") of Taguchi (1957) was used to determine the f a c t o r s which may s i g n i f i c a n t l y a f f e c t the l o s s e s of p r o t e o l y t i c a c t i v i t y of papaya l a t e x due to d r y i n g . By u s i n g a f r a c t i o n a l f a c t o r i a l d e s ign i t was p o s s i b l e to determine i f t e t r a t h i o n a t e p r o t e c t e d the PA of papaya l a t e x d u r i n g d r y i n g . P r o t e c t i o n of the PA provided by t e t r a t h i o n a t e was compared to -36-that produced by sodium metabisulfite, a compound commonly used in the commercial drying of papaya latex. The factors evaluated, together with their assigned upper and lower l i m i t s are shown in Table 5 . The scheme used is presented in F i g . 1. The pre-drying additive, sodium metabisulfite or sodium tetrathionate, was added d i r e c t l y to the latex. The treated or control latex was stored at room temperature (20-22°C) prior to subjecting i t to drying under the conditions s p e c i f i e d by the f r a c t i o n a l f a c t o r i a l design. After the storage period the samples were dried in the e l e c t r i c a l l y heated, forced a i r oven, at the temperature spe c i f i e d by the f r a c t i o n a l f a c t o r i a l design. Drying was continued u n t i l the latex hardened and crumbled r e a d i l y when pressed by the fingers. This usually occurred when the residual moisture was 6+2%. Dried samples were sealed in polyethylene bags. The sealed bags were placed inside amber glass jars with dessicant ( d r i e r i t e ) and stored at -20°C u n t i l PA determination (within three weeks). Preliminary experiments showed that no losses of PA occurred during storage of crude papain at -20°C. PA determination was carried out using the method reported below. The values of PA from the 27 drying treatments (in duplicate) were analyzed using a Taguchi's f a c t o r i a l analysis of variance computer program written in IBM-Basic (Arteaga, 1986). -37-Table 5. F a c t o r s , and t h e i r a s s i g n e d l e v e l s i n v e s t i g a t e d f o r t h e i r p o s s i b l e i n f l u e n c e on the l o s s e s of p r o t e o l y t i c a c t i v i t y of papaya l a t e x . L e v e l F a c t o r 1 2 3 P r e d r y i n g treatment* TT MBS CON Drying temperature, °C 55 70 100 Storage p r i o r to d r y i n g , h 2 12 24 Drying l o a d , g/m* 1,190 2,381 4,762 Type of storage p r i o r to d r y i n g 1 1 D D/L L * P r e d r y i n g treatments: TT = a d d i t i o n of 1% sodium t e t r a t h i o n a t e . MBS = a d d i t i o n of 1% sodium m e t a b i s u l f i t e . CON = no a d d i t i v e s ( c o n t r o l treatment). BType of storage p r i o r to d r y i n g : D = storage under dark c o n d i t i o n s . D/L = f i r s t h a l f of the storage time under dark and other h a l f under l i g h t c o n d i t i o n s . L = storage under l i g h t c o n d i t i o n s . -38-Treatment prior to drying Drying temperature Storage time prior to drying Drying load Type of storage prior to drying F i g u r e 1. Scheme used i n the Taguchi L21 f r a c t i o n a l f a c t o r i a l experiment ( 3 1 1 ) . - 3 9 -E. EFFECT OF DIFFERENT TYPES OF DRYING AND ADDITIVES ON THE LOSSES OF PROTEOLYTIC ACTIVITY OF PAPAYA LATEX The e f f e c t of three types of d r y i n g , namely, sun d r y i n g , vacuum d r y i n g and oven ( a i r ) d r y i n g , on the l o s s e s of PA of papaya l a t e x , with or without a d d i t i v e s was determined using a 3 x 3 f u l l f a c t o r i a l experimental d e s i g n . The two f a c t o r s , and the corresponding l e v e l s were: type of d r y i n g (sun, vacuum, and a i r (oven) dr y i n g ) and treatment p r i o r to d r y i n g ( a d d i t i o n of 1% m e t a b i s u l f i t e , or t e t r a t h i o n a t e , and no a d d i t i o n of a d d i t i v e s ) . The a d d i t i v e s were added d i r e c t l y to the l a t e x . A l l samples were d r i e d i n aluminum d i s h e s with a d r y i n g load of 1,190 g/m*. Each treatment was done i n t r i p l i c a t e . Sun d r y i n g (20-25°C) was c a r r i e d out f o r 6 hr. A f t e r t h i s p e r i o d , the moisture content of the samples was s t i l l high (30%), so the samples were vacuum d r i e d f o r 3 hr at 55°C, 27 inches of vacuum, to a moisture content of 6+2%. Oven d r y i n g was done a t 55<>C f o r a p e r i o d of 1.5 hr, to reach a f i n a l moisture content of 6±2%. Vacuum d r y i n g was c a r r i e d out at 55<>C with a vacuum of 27 inches, f o r 5 hr; the f i n a l moisture of the product was a l s o 6±2%. Dried samples were s e a l e d i n po l y e t h y l e n e bags, and the sea l e d bags were placed i n s i d e amber g l a s s j a r s with d e s s i c a n t . The j a r s were s t o r e d at -20°C u n t i l PA det e r m i n a t i o n ( w i t h i n three weeks). PA de t e r m i n a t i o n was c a r r i e d out using the method rep o r t e d below. -40-F. LOSSES OF THE PROTEOLYTIC ACTIVITY OF CRUDE PAPAIN DURING STORAGE Papaya l a t e x with 1% m e t a b i s u l f i t e or 1% t e t r a t h i o n a t e was oven d r i e d at 55°C for 1.5 hr. The r e s u l t i n g crude papain was s e a l e d i n p o l y e t h y l e n e bags, and the s e a l e d bags were placed i n t r a n s p a r e n t g l a s s j a r s and s t o r e d at room temperature. At 1 wk i n t e r v a l s f o r up to 13 wk the PA was measured i n d u p l i c a t e , u s i n g the method re p o r t e d below. A c o n t r o l sample ( i . e . crude papain with no a d d i t i v e s added) was s t o r e d and analysed i n the same manner. G. PROTEOLYTIC ACTIVITY ASSAY The method of Hanada et a l . (1978) with m o d i f i c a t i o n s was used. T h i s method i s based on the q u a n t i t a t i o n by u.v. spectrophotometry of t r i c h l o r o a c e t i c a c i d (TCA)-soluble peptides f o l l o w i n g c a s e i n h y d r o l y s i s . The crude papain samples were f i n e l y ground with a p e s t l e and mortar. Approximately 5 mg of powder was t r a n s f e r r e d to a 25 mL v o l u m e t r i c f l a s k and the f l a s k was made to volume with f r e s h l y prepared a c t i v a t i n g b u f f e r (40 mM c y s t e i n e HCl and 20 mM EDTA i n 0.05 M phosphate b u f f e r , pH 6.8). T h i s s o l u t i o n was incubated i n a water bath at 40°C f o r 15 min. An a l i q u o t of 0.5 mL of the a c t i v a t e d enzyme s o l u t i o n was added to 5 mL of 1% Hammarsten-type c a s e i n s o l u t i o n i n 0.05 M T r i s - H C l b u f f e r (pH 8), p r e e q u i l i b r a t e d a t 40°C. A f t e r a d d i t i o n of the enzyme s o l u t i o n the t e s t tube -41-contents were immediately mixed with a vortex mixer and incubated at 40°C f o r 10 min. Then 5 mL of 0.44 M TCA s o l u t i o n was added to the mixture and the contents of the t e s t tube were shaken v i g o r o u s l y . The t e s t tube was placed again i n the water bath at 40°C f o r 30-40 min to l e t the p r e c i p i t a t e d p r o t e i n f u l l y c o a g u l a t e . Samples were c e n t r i f u g e d (10,000 xg, 15 min, 4<>C S o r v a l l R2-B) and f i l t e r e d under vacuum through Whatman No.42 f i l t e r paper. A l l f i l t r a t e s were completely c l e a r . The absorbance of the f i l t r a t e was measured at 280 nm with a Cary 210 spectrophotometer (Varian A s s o c i a t e s Inc., Palo A l t o , CA). P r o t e o l y t i c a c t i v i t i e s were expressed as i n t e r n a t i o n a l u n i t s i . e Jimols of t y r o s i n e l i b e r a t e d min"1mg"1 of sample, under assay c o n d i t i o n s . A standard curve prepared with L - t y r o s i n e plus c a s e i n s o l u t i o n , under the c o n d i t i o n s f o r assay was used to convert absorbance to Hmols of t y r o s i n e . The 1% c a s e i n s o l u t i o n was prepared as d e s c r i b e d i n the Food Chemical Codex (FCC I I I , 1981), with the m o d i f i c a t i o n that Hammarsten-type c a s e i n was d i s s o l v e d i n 0.05 M T r i s - H C l b u f f e r (pH 8.0) i n s t e a d of i n p h o s p h a t e - c i t r a t e b u f f e r , pH 6.8. The use of the T r i s b u f f e r enabled the storage of the c a s e i n s o l u t i o n f o r p e r i o d s of more than one week (Arnon, 1970). Each sample e v a l u a t i o n c o n s i s t e d of four tubes; two incubated a c t i v e enzyme/substrate mixtures and two s u b s t r a t e mixtures to which a c t i v e enzyme was added a f t e r the TCA s o l u t i o n . -42-H. PREPARATION OF SODIUM TETRATHIONATE I. Iodine o x i d a t i o n The method r e p o r t e d by Gilman et a l . (1946a) was used without m o d i f i c a t i o n . T e t r a t h i o n a t e was prepared by the dropwise a d d i t i o n of a concentrated s o l u t i o n of sodium t h i o s u l f a t e (250 g of NaiSaOj^SHjO i n 250 mL of water) to an i c e - c o o l e d a l c o h o l i c s o l u t i o n of i o d i n e (127 g of I* and 50 g of Nal i n 500 .mL of a b s o l u t e e t h a n o l ) . The r e a c t i o n mixture was v i g o r o u s l y s t i r r e d and maintained below 20°C d u r i n g the a d d i t i o n of t h i o s u l f a t e . P r e c i p i t a t i o n of t e t r a t h i o n a t e began when approximately h a l f of the t h i o s u l f a t e had been i n t r o d u c e d . A f t e r the r e a c t i o n was completed, one l i t e r of a b s olute ethanol and 500 mL of anhydrous e t h y l ether were added to the r e a c t i o n mixture, i n order to separate t e t r a t h i o n a t e . The mixture was l e f t at 4°C f o r approximately 12 hr. The p r e c i p i t a t e was c o l l e c t e d on a buchner funnel and washed with sma l l p o r t i o n s of a b s o l u t e ethanol to remove excess i o d i n e . The p r e c i p i t a t e was then d r i e d at room temperature, i n vacuo, over d r i e r i t e . The s o l i d was s t o r e d at 4°C i n amber b o t t l e s u n t i l a n a l y s e d . 2. Hydrogen peroxide o x i d a t i o n A f t e r a s e r i e s of p r e l i m i n a r y experiments, the f i n a l method adopted was as f o l l o w s : 16 g of Na 2Sj0 3»5H 20 was d i s s o l v e d i n 50 mL of d i s t i l l e d water. To t h i s s o l u t i o n was added dropwise, 50 mL -43-of a 3% s o l u t i o n of hydrogen peroxide a t an approximate flow r a t e of 1.5-2.0 mL/min. The s o l u t i o n was mixed with a magnetic s t i r r e r and the pH was c o n t i n u o u s l y monitored with the use of a pH-meter. With each drop of hydrogen peroxide s o l u t i o n added, i t was necessary to add one or two drops of e i t h e r 0.10 N HaSOi or 0.50 M a c e t i c a c i d , to maintain the pH near n e u t r a l i t y (pH 6-8). Two r e a c t i o n temperatures were t e s t e d : 10 and 20°C. A f t e r a l l the hydrogen peroxide was added, the r e a c t i o n mixture was allowed to stand f o r 20 min. C r y s t a l l i z a t i o n with ethanol and ether, d r y i n g and storage were c a r r i e d out as d e s c r i b e d f o r the i o d i n e o x i d a t i o n method. 3. F e r r i c o x i d a t i o n The f i n a l technique used was as f o l l o w s : 6 g of f e r r i c c h l o r i d e (FeCl-»6H 20) was d i s s o l v e d i n 25 mL of d i s t i l l e d water. Upon d i s s o l u t i o n of the f e r r i c c h l o r i d e , the pH of the mixture dropped to approximately pH 3.5. One s e t of experiments was done without pH adjustment and i n the other the pH was adjus t e d with 0.1 N NaOH to approximately pH 7. To t h i s s o l u t i o n was added dropwise 20 mL of a s o l u t i o n of t h i o s u l f a t e (5 g of Na2S203»5HaO i n 20 mL of d i s t i l l e d water) at a flow r a t e of 1.5-2.0 mL/min. Continuous a g i t a t i o n was c a r r i e d out by using a magnetic s t i r r e r . Copper s a l t s have been r e p o r t e d to a c t as c a t a l y s t s i n the o x i d a t i o n of t h i o s u l f a t e with f e r r i c ions (Lar and Singh, 1956). Thus, i n a s e r i e s of experiments, 1 mL of a 10% s o l u t i o n of -44-c u p r i c a c e t a t e was added to the r e a c t i o n mixture j u s t before a d d i t i o n of the t h i o s u l f a t e s o l u t i o n . A f t e r the t h i o s u l f a t e was added, the r e a c t i o n mixture was allowed t o stand f o r one hour. C r y s t a l l i z a t i o n with ethanol and ether, d r y i n g and storage were c a r r i e d out as d e s c r i b e d f o r the io d i n e o x i d a t i o n method. 4. Cu p r i c o x i d a t i o n The f i n a l method used was as f o l l o w s : 5 g of c u p r i c s u l f a t e (CuS0«»5Hj0) was d i s s o l v e d i n 25 mL of d i s t i l l e d water. A drop i n pH to about 3.5 was observed upon d i s s o l u t i o n of the s a l t . In some experiments, as i n the f e r r i c o x i d a t i o n , the pH was adj u s t e d with 0.1 N NaOH to pH 7. To t h i s s o l u t i o n was added dropwise 20 mL of a t h i o s u l f a t e s o l u t i o n (7.5 g of Na2S 20 3»5H20 i n 20 mL of d i s t i l l e d water). The s o l u t i o n was a g i t a t e d with a magnetic s t i r r e r . A f t e r the t h i o s u l f a t e was added, the r e a c t i o n mixture was allowed to stand f o r one hour. C r y s t a l l i z a t i o n with ethanol and ether, d r y i n g , and storage were c a r r i e d out as d e s c r i b e d f o r the io d i n e o x i d a t i o n method. 5. Vanadate o x i d a t i o n The technique r e p o r t e d by Gowda et a l . (1955), f o r e s t i m a t i o n of t h i o s u l f a t e v i a r e a c t i o n with vanadate was modified f o r p r e p a r a t i o n of t e t r a t h i o n a t e from t h i o s u l f a t e . To a s o l u t i o n of sodium vanadate (1.5 g NaV0 3»2H20 d i s s o l v e d -45-i n 10 mL d i s t i l l e d water) were added 5 mL of a 1% s o l u t i o n of c u p r i c a c e t a t e and 0.7 mL of 7 M s u l f u r i c a c i d , followed by the dropwise a d d i t i o n of 10 mL of a sodium t h i o s u l f a t e s o l u t i o n (10 g NajS203»5H20 i n 10 mL of water) at a flow r a t e of 1-1.5 mL/min. According to Gowda et a l . (1955) the r e a c t i o n between vanadate and t h i o s u l f a t e i s completed i n f i v e minutes a t 28°C. The mixture was allowed to stand f o r 10 min a f t e r the t h i o s u l f a t e s o l u t i o n was added. C r y s t a l l i z a t i o n with ethanol and et h e r , d r y i n g , and storage was c a r r i e d out as d e s c r i b e d f o r the i o d i n e o x i d a t i o n method. I. DETERMINATION OF PURITY AND YIELD Many methods have been r e p o r t e d f o r the de t e r m i n a t i o n of p o l y t h i o n a t e s ( f o r a review see W i l l i a m s , 1979). An HPLC method has been r e p o r t e d by Chapman and Beard (1973). As mentioned by Wil l i a m s (1979), d e t e r m i n a t i o n of i n d i v i d u a l pure p o l y t h i o n a t e s i s r e l a t i v e l y easy but a n a l y s i s of a mixture i s more d i f f i c u l t . A f t e r the s o l v e n t c r y s t a l l i z a t i o n and d r y i n g of the t e t r a t h i o n a t e - c o n t a i n i n g p r e c i p i t a t e , i t s weight was recorded. The % t e t r a t h i o n a t e i n the s o l i d was determined by the f o l l o w i n g two methods. 1. I o d a t e - i o d i n e t i t r a t i o n T h i s method was f i r s t r e p o r t e d by Gilman et a l . (1946a,b) and -46-i s a r e l a t i v e l y simple and s t r a i g h t f o r w a r d procedure. The method i s an i n d i r e c t i o d i n e t i t r a t i o n ; the general form f o r t h i s type of r e a c t i o n s i s ( F r i t z and Schenk, 1972): Aox + I"(excess) ^ ABBD + 12 (4) I 2 + 2SjCV 2 > 21" + S«CV 2 (5) where Aox and ABBD are the o x i d i z e d and reduced s t a t e s of A, r e s p e c t i v e l y . The i o d i n e formed i n r e a c t i o n (4) i s e q u i v a l e n t to the amount of Aox i n the sample to be analysed, and i s t i t r a t e d with standard t h i o s u l f a t e with s t a r c h as an i n d i c a t o r ( r e a c t i o n 5) . The a n a l y s i s of t e t r a t h i o n a t e with iodate i s based on the r e a c t i o n i n which iodate (I0 3) q u a n t i t a t i v e l y o x i d i z e s t e t r a t h i o n a t e i n the presence of HC1 to s u l f a t e ( r e a c t i o n 6). The unreacted iodate r e a c t s with an added excess of io d i d e to give f r e e i o d i n e ( r e a c t i o n 7), which was then t i t r a t e d with 0.10 N or 0.05 N t h i o s u l f a t e ( r e a c t i o n 8). 2SAOS-2 + 7I0 3- + 2H+ + 7C1" > 7IC1 + 8SOr 2 + H20 (6) IOr + 51- + 6H* > 3I 2 + 3HzO (7) I» + 2S2O3-2 > 21- + S<CV2 (8) For the d e t e r m i n a t i o n of t e t r a t h i o n a t e 25 mL of 0.1 N KI0 3 was added to 25 mg of the s o l i d , followed by 5 mL of 2 N HC1. A f t e r s t a n d i n g a minimum of 5 min, 5 mL of 10% KI were added to convert IC1 and unreacted ICV to f r e e i o d i n e which was then t i t r a t e d with 0.1 N t h i o s u l f a t e . A blank was run p a r a l l e l to the samples. -47-Since t h i o s u l f a t e was a l s o o x i d i z e d with iodate to s u l f a t e - i t was necessary to determine i n the same sample the amount of t h i o s u l f a t e . For the d e t e r m i n a t i o n of t h i o s u l f a t e a s i m i l a r i n d i r e c t i o d i n e method was used. Both ions- t e t r a t h i o n a t e and t h i o s u l f a t e , were o x i d i z e d by iodate to s u l f a t e . Only t h i o s u l f a t e was o x i d i z e d , however, by i o d i n e (Gilman et a l . , 1946b). In the a n a l y s i s of t h i o s u l f a t e 25 mg of the s o l i d was d i s s o l v e d i n 5-10 mL of water. One drop of p h e n o l p h t h a l e i n was added and the s o l u t i o n was made a l k a l i n e (pink c o l o r ) with 0.1 N NaOH. To t h i s s o l u t i o n 25 mL of 0.1 N KI0 3, 5 mL of 10% KI and 5 mL of 2 N HC1 were added i n the order named. Under a l k a l i n e c o n d i t i o n s , iodate r e a c t s with i o d i d e to produce i o d i n e ( r e a c t i o n 9) , without r e a c t i n g with the t h i o s u l f a t e present. Upon a d d i t i o n of the a c i d , the i o d i n e formed o x i d i z e d t h i o s u l f a t e ( r e a c t i o n 10) . The excess i o d i n e was then t i t r a t e d with 0.1 N t h i o s u l f a t e ( r e a c t i o n 11). A blank was run p a r a l l e l to the samples. IOr + I" * I2 (9) S-03 + I- (excess) -» 21" + S<Or2 (10) S 20 3 + I i r 21- + S«Os-2 (11) The f o l l o w i n g equations were used to c a l c u l a t e the amount of t h i o s u l f a t e and t e t r a t h i o n a t e i n the sample: -48-Iodate r e a c t i o n mg Na 2S 20 3= DELTA * N * 158 (12) where DELTA = (mL t i t r a t i o n blank - mL t i t r a t i o n sample) N = n o r m a l i t y of t h i o s u l f a t e Iodine r e a c t i o n mg Na 2S 20 3= DELTA * N * (158/8) (13) where DELTA = (mL t i t r a t i o n blank- mL t i t r a t i o n sample) N = n o r m a l i t y of t h i o s u l f a t e mg Na 2S«Ot = mg Na 2S 20 3 ( i o d i n e ) - mg Na 2 S 20 3 (iodate) (14) 2. A l k a l i n e c y a n o l y s i s The second method used f o r the d e t e r m i n a t i o n of t e t r a t h i o n a t e and t h i o s u l f a t e was the c o l o r i m e t r i c method r e p o r t e d by Nor and Tabatabai (1975). This method i n v o l v e s a l k a l i n e c y a n o l y s i s of t h i o s u l f a t e i n the presence of c u p r i c ions or a l k a l i n e c y a n o l y s i s of 57% of t e t r a t h i o n a t e i n the absence of c u p r i c i o n s . C o l o r i m e t r i c d e t e r m i n a t i o n of the t h i o c y a n a t e i s based on the formation of a f e r r i c t h i o c y a n a t e complex. The r e a c t i o n s i n t h i s case were: S 2 0 r 2 + CN" + Cu" SCV 1 + CNS* (15) S«0t-2 + CN" + HsO • S 20 3" 2 + SOr 2 + 2HCN + CNS* (16) CNS* + F e ° • Fe-CNS complex (17) For the d e t e r m i n a t i o n of t h i o s u l f a t e + t e t r a t h i o n a t e the method was as f o l l o w s (Nor and Tabatabai, 1975): an a l i q u o t (1-2 mL) of sample c o n t a i n i n g 25-200 ug of S as t e t r a t h i o n a t e and t h i o s u l f a t e was placed i n a 25 mL v o l u m e t r i c f l a s k . One m i l l i l i t e r of 0.1 M KCN was added, and the f l a s k was s w i r l e d to -49-mix the co n t e n t s . A f t e r 15 min 2 mL of 0.033 M CuCl 2 and 1 mL of a s o l u t i o n of f e r r i c n i t r a t e - n i t r i c a c i d s o l u t i o n (0.25 M Fe(NO-) 3»9H 20:3.1 M HN0-) was added. The s o l u t i o n was made to volume with d i s t i l l e d water, and the f l a s k was i n v e r t e d s e v e r a l times to mix the co n t e n t s . A f t e r two minutes, the absorbance at 460 nm of the reddish-brown c o l o r f e r r i c t h i o c y a n a t e complex was measured. For the d e t e r m i n a t i o n of onl y t e t r a t h i o n a t e the procedure d e s c r i b e d above was repeated with the omission of the a d d i t i o n of 2 mL of 0.033 M CuCl 2. The t e t r a t h i o n a t e and t h i o s u l f a t e S or t e t r a t h i o n a t e S content of the a l i q u o t was c a l c u l a t e d by r e f e r r i n g to a c a l i b r a t i o n curve based on the r e s u l t s obtained with standards c o n t a i n i n g 0, 50, 100, 150, 200 and 250 jUg of t h i o s u l f a t e S. 3. M e l t i n g p o i n t d e t e r m i n a t i o n The m e l t i n g p o i n t of the t e t r a t h i o n a t e - s o l i d s obtained was determined using a Perkin-Elmer DSC-2 System (The Perkin-Elmer C o r p o r a t i o n , Norwalk, CT). Samples of the s o l i d (5-7 mg) were weighed i n t o t a r e d aluminum pans, and the pans were s e a l e d . The pans were held a t 20<>C f o r 10 min, then heated to 200<>C at a r a t e of 10°C/min. An empty pan was used as a r e f e r e n c e . The thermograms were a u t o m a t i c a l l y recorded and the mel t i n g p o i n t was taken as the maximum temperature of the endothermic peak. -50-4. C a l c u l a t i o n of p u r i t y and y i e l d P u r i t y and y i e l d were c a l c u l a t e d u s i n g the f o l l o w i n g formulas: P u r i t y (%) = mg of t e t r a t h i o n a t e i n sample X 100 (18) mg sample Y i e l d (%) = q p r e c i p i t a t e X p u r i t y X 100 (19) t h e o r e t i c a l y i e l d g NajS«0 6 #2H aO T h e o r e t i c a l y i e l d s were c a l c u l a t e d on the b a s i s of the s t o i c h i o m e t r i c r e a c t i o n s r e p o r t e d i n Table 4. J . COST EVALUATION In order to determine the most c o s t - e f f i c i e n t method f o r s y n t h e s i s of t e t r a t h i o n a t e , the c o s t of the t e t r a t h i o n a t e obtained using the d i f f e r e n t methods was c a l c u l a t e d . The c a l c u l a t i o n s were based on the p r i c e s of the p a r t i c u l a r chemicals used i n the s y n t h e s i s and the y i e l d obtained f o r each method. The p r i c e s of the chemicals were those from Sigma Chemical Company (1988). K. INACTIVATION/ACTIVATION EFFICIENCY OF THE SYNTHESIZED TETRATHIONATES Each t e t r a t h i o n a t e p r e p a r a t i o n was t e s t e d f o r i t s a b i l i t y to r e v e r s i b l y i n a c t i v a t e papaya l a t e x . For t h i s d e t e r m i n a t i o n crude papain was rehydrated with water to give a 20% (w/v) s o l u t i o n as p r e v i o u s l y d e s c r i b e d i n s e c t i o n B. One m i l l i l i t e r of t h i s papaya -51-l a t e x was t r a n s f e r r e d to a 25 mL v o l u m e t r i c f l a s k and 1 mL of a s o l u t i o n of the t e t r a t h i o n a t e to be t e s t e d (0.5 mg/mL i n 0.05 M phosphate b u f f e r pH 6.8) was added. A f t e r g e n t l y shaking the f l a s k to mix the conten t s , the mixture was incubated at room temperature f o r 15 min. The s o l u t i o n was made to volume with 0.05 M phosphate b u f f e r , pH 6.8, 0.02 M EDTA ( i n a c t i v a t i o n ) , or with the same b u f f e r c o n t a i n i n g 0.05 M c y s t e i n e ( r e a c t i v a t i o n ) . A f t e r 10 min a t room temperature, d u p l i c a t e 1 mL a l i q u o t s were used to determine the PA, using the method r e p o r t e d i n s e c t i o n G. A c o n t r o l sample, to which no t e t r a t h i o n a t e was added, was run p a r a l l e l to each experiment. I n a c t i v a t i o n was taken as the r a t i o of the d i f f e r e n c e between the PA of a c o n t r o l sample (no t e t r a t h i o n a t e added) and the PA of the t e t r a t h i o n a t e t r e a t e d sample, to the PA of the c o n t r o l sample. R e a c t i v a t i o n was c a l c u l a t e d as the r a t i o of the PA a c t i v i t y of the t e t r a t h i o n a t e t r e a t e d samples d i l u t e d i n the c y s t e i n e c o n t a i n i n g b u f f e r to the PA of the c o n t r o l sample (no t e t r a t h i o n a t e ) d i l u t e d with the same b u f f e r . The i n a c t i v a t i o n e f f i c i e n c y of the s o l i d was expressed as the percentage i n a c t i v a t i o n of a sample of pure t e t r a t h i o n a t e . S i m i l a r l y , the r e a c t i v a t i o n e f f i c i e n c y of the s o l i d was taken as the percentage r e a c t i v a t i o n of a sample of pure t e t r a t h i o n a t e . -52-RESULTS AND DISCUSSION A. DRYING RATES OF PAPAYA LATEX The e f f e c t s of temperature and d r y i n g load on the d r y i n g r a t e s of papaya l a t e x , without a d d i t i v e s , are shown i n F i g . 2 and 3. As expected, the s h o r t e s t time f o r r e a c h i n g a constant weight was needed at the h i g h e s t d r y i n g temperature (100°C), or the lowest d r y i n g load (1,190 g/m*). At 55<>C, with a d r y i n g load of 2,381 g/m*, i t took approximately 150 min (2.5 hr) to reach a constant weight (moisture content of 6+2%). O r t i z et a l . (1980) re p o r t e d t h a t f r e s h papaya l a t e x r e q u i r e d approximately 240 min (4 hr) to d r y a t 55<>C. The d i f f e r e n c e i n d r y i n g time i s l i k e l y due to the d i f f e r e n t type of oven used. The s h o r t e r d r y i n g time f o r rehydrated crude papain, however, co u l d i n d i c a t e t h a t d r y i n g of papaya l a t e x caused changes i n some of the components of f r e s h papaya l a t e x , with the r e s u l t t h a t the r e h y d r a t a t i o n water added to the crude papain was not as t i g h t l y bound as the o r i g i n a l water i n the f r e s h papaya l a t e x . The e f f e c t of a d d i t i o n of sodium m e t a b i s u l f i t e or sodium t e t r a t h i o n a t e on the d r y i n g r a t e s , at 55°C, of papaya l a t e x i s shown i n F i g . 4. A d d i t i o n of these compounds at a 1% l e v e l , d i d not change the d r y i n g r a t e compared to a c o n t r o l papaya l a t e x . S i m i l a r r e s u l t s were found f o r the other temperatures. -53-F i g u r e 2. The e f f e c t of three d i f f e r e n t d r y i n g temperatures on the d r y i n g r a t e of papaya l a t e x at a d r y i n g load of 2,381 g/m-. Each p o i n t r e p r e s e n t s the mean of three d e t e r m i n a t i o n s . In a l l case the c o e f f i c i e n t of v a r i a t i o n was l e s s than 5%. -54-Figure 3. The e f f e c t of drying load on the drying rate of papaya latex at a drying temperature of 8 0 ° C . Each point represents the mean of three determinations. In a l l cases the c o e f f i c i e n t of variation was less than 5%. - 5 5 -100-, 75 - O • 8 8 8 S 50 -§ o e Drying Temperature 55°C o Control o 1* Metabisulfite A 1* Tetrathionate 25 -T— i — r I i i — i — i — I — i — i — i — i — | — i — i — i — i — j — i — i — i — i — | 50 100 150 200 250 Drying Time (min) F i g u r e 4 . The e f f e c t of the a d d i t i o n of 1% t e t r a t h i o n a t e or m e t a b i s u l f i t e on the d r y i n g r a t e of papaya l a t e x at 55 °C and d r y i n g load of 2,381 g/m«. Each p o i n t r e p r e s e n t s the mean of three d e t e r m i n a t i o n s . In a l l cases the c o e f f i c i e n t of v a r i a t i o n was l e s s than 5%. -56-B. DETERMINATION OF INFLUENTIAL FACTORS ON THE LOSS OF PROTEOLYTIC ACTIVITY DUE TO DRYING OF PAPAYA LATEX By using the f r a c t i o n a l f a c t o r i a l design L 2 7 ( 3 1 3 ) of Taguchi (1957), i t was p o s s i b l e to determine which f a c t o r s a f f e c t e d the l o s s e s of PA d u r i n g d r y i n g of papaya l a t e x . The f a c t o r s examined were a d d i t i o n of a d d i t i v e s ( m e t a b i s u l f i t e or t e t r a t h i o n a t e ) , type ( l i g h t or dark c o n d i t i o n s ) and l e n g t h of storage p r i o r to d r y i n g , d r y i n g temperature, and d r y i n g load, as w e l l as p o s s i b l e i n t e r a c t i o n s among the f a c t o r s . Data c o l l e c t e d from the 27 d r y i n g treatments, f o l l o w i n g the f r a c t i o n a l experimental design L 2 7 ( 3 1 3 ) of Taguchi (1957), were analyzed by a n a l y s i s of v a r i a n c e to determine the s i g n i f i c a n c e of the f a c t o r s and the p o s s i b l e i n t e r a c t i o n s on the p r o t e o l y t i c a c t i v i t y l o s s e s of papaya l a t e x d u r i n g d r y i n g . Table 6 shows the a n a l y s i s of v a r i a n c e of the three l e v e l f a c t o r i a l d e s i g n . The f o l l o w i n g main e f f e c t s were computed to be h i g h l y s i g n i f i c a n t (p<0.01): treatment p r i o r to d r y i n g , d r y i n g temperature and, d r y i n g l o a d . The type of storage p r i o r to d r y i n g ( l i g h t or dark c o n d i t i o n s ) was found to have a s i g n i f i c a n t e f f e c t at p<0.05. The i n t e r a c t i o n (treatment p r i o r to d r y i n g ) x ( d r y i n g temperature) was h i g h l y s i g n i f i c a n t (p<0.01). The other i n t e r a c t i o n s were non s i g n i f i c a n t at p=0.05. The e f f e c t curves i n F i g . 5 i l l u s t r a t e the f a c t t h a t a s i g n i f i c a n t l y higher mean PA r e t e n t i o n was achieved with the a d d i t i o n of 1% t e t r a t h i o n a t e than with the a d d i t i o n of m e t a b i s u l f i t e a t the same l e v e l , at a d r y i n g temperature of 55°C. -57-Table 6. A n a l y s i s of v a r i a n c e (Taguchi's L 2 7 3 1 3 ) of p r o t e o l y t i c a c t i v i t y values of papaya l a t e x obtained from 27 d r y i n g experiments. Source of v a r i a t i o n DF Mean Square F-value P r e d r y i n g treatment (PT) 2 730.91 13.64" Drying temperature (DT) 2 9959.86 185.87" Storage p r i o r to d r y i n g 2 146.49 2.73 n.s. Drying load 2 421.51 7.87" Type of storage p r i o r t o d r y i n g 2 270.96 5.06* PT X DT 4 311.17 5.81" Errors- 12 53. 59 T o t a l 26 ' s i g n i f i c a n t a t p < 0.05 " s i g n i f i c a n t at p < 0.01 n.s. not s i g n i f i c a n t at p > 0.05 • *-The sums of square values f o r the i n t e r a c t i o n s t h a t do not appear i n the ANOVA t a b l e were very low and, t h e r e f o r e , were i n c o r p o r a t e d i n t o the e r r o r sums of squares. -58-Figure 5. E f f e c t curve for the interaction between drying temperature and treatment prior to drying on the pr o t e o l y t i c a c t i v i t y retained by crude papain. (Mean+Confidence l i m i t s calculated at p<0.05). - 5 9 -Although the mean value of the PA a f t e r d r y i n g a t 70<>C was s l i g h t l y higher when the l a t e x was t r e a t e d with t e t r a t h i o n a t e than the corresponding value with the treatment of m e t a b i s u l f i t e , o v e r l a p p i n g of the confidence l i m i t s (p<0.05) i n d i c a t e d no s i g n i f i c a n t d i f f e r e n c e between the two treatments. Duncans m u l t i p l e range t e s t confirmed the above c o n c l u s i o n . At a d r y i n g temperature of 100°C n e i t h e r treatment p r o t e c t e d the PA of papaya l a t e x . Commercial d r y i n g of papaya l a t e x i s u s u a l l y done a t 50-55°C ( O r t i z et a l . , 1980). Since t e t r a t h i o n a t e has been found to be s i g n i f i c a n t l y s u p e r i o r to m e t a b i s u l f i t e as a p r o t e c t i n g agent of the PA of papaya l a t e x at 55°C, i t can be concluded t h a t t e t r a t h i o n a t e has p o t e n t i a l commercial a p p l i c a t i o n as an agent f o r p r o t e c t i n g the PA of papaya l a t e x . The e f f e c t curves i n F i g . 6 show th a t h i g h e s t mean r e t e n t i o n of the PA of papaya l a t e x occurred when the d r y i n g load was at the minimum l e v e l t e s t e d (1,190 g/m* of d r y i n g area) f o r the c o n t r o l papaya l a t e x and the l a t e x t r e a t e d with e i t h e r a d d i t i v e . Overlapping of the confidence l i m i t s (not shown i n the graph), however, i n d i c a t e d no s i g n i f i c a n t d i f f e r e n c e s between the r e t e n t i o n of PA at the d i f f e r e n t d r y i n g loads f o r the papaya l a t e x t r e a t e d with t e t r a t h i o n a t e or m e t a b i s u l f i t e . -60-100-i Q) C 0) C£ >s o < o CD -+-» o CL Treatment o 156 tetrathionate o 158 metabisulfite A Control 80 60 40 -20 1,190 2,381 Drying load ( g / m 2 ) 4,762 Figure 6. E f f e c t curve for the interaction between drying load and treatment prior to drying on the proteolytic a c t i v i t y retained by crude papain. Only the confidence l i m i t s (at p<0.05) for the latex treated with tetrathionate are shown. - 6 1 -C. EFFECT OF DIFFERENT TYPES OF DRYING AND ADDITIVES ON LOSS OF PROTEOLYTIC ACTIVITY OF PAPAYA LATEX As p r e v i o u s l y r e p o r t e d (Krishnamurty et a l . , 1960), sun d r y i n g caused higher l o s s e s of PA i n papaya l a t e x as compared to the other d r y i n g methods (Table 7). For sun d r y i n g the a d d i t i o n of t e t r a t h i o n a t e or m e t a b i s u l f i t e d i d not s i g n i f i c a n t l y decrease the l o s s of PA as compared to an untreated l a t e x . I t i s thought t h a t a major cause of l o s s of PA d u r i n g sun d r y i n g i s the e f f e c t of UV r a d i a t i o n on a h i s t i d i n e r e s i d u e e s s e n t i a l f o r PA i n papain ( B r o c k l e h u r s t et a l . , 1981). Since n e i t h e r t e t r a t h i o n a t e nor m e t a b i s u l f i t e p r o t e c t t h i s h i s t i d i n e r e s i d u e from UV r a d i a t i o n , a major decrease i n the l o s s e s of PA i s not l i k e l y to occur when t e t r a t h i o n a t e or m e t a b i s u l f i t e i s added. As expected, vacuum d r y i n g produced the l e a s t l o s s of PA i n papaya l a t e x (Table 7). Although o x i d a t i o n was a l r e a d y prevented with the use of hypobaric c o n d i t i o n s i n t h i s d r y i n g method, the a d d i t i o n of t e t r a t h i o n a t e or m e t a b i s u l f i t e to the rehydrated papaya l a t e x caused s i g n i f i c a n t improvement i n the PA r e t e n t i o n . In the case of f r e s h papaya l a t e x Krishnamurty et a l . (1960) re p o r t e d t h a t a d d i t i o n of m e t a b i s u l f i t e d i d not i n c r e a s e the r e t e n t i o n of PA d u r i n g vacuum d r y i n g as compared with untreated l a t e x . -62-Table 7. Mean r e t e n t i o n of the p r o t e o l y t i c a c t i v i t y * - (PA) of the crude papain r e s u l t i n g from papaya l a t e x t r e a t e d with 1% t e t r a t h i o n a t e or 1% m e t a b i s u l f i t e and d r i e d using d i f f e r e n t methods 3' 0. Type of d r y i n g 1 Treatment Sun Vacuum Oven C o n t r o l * 61.3 + 2.63- 78.7+3.5 1 65.6+3.5 1 M e t a b i s u l f i t e 64.4 + 3.2 1 86.6+3.0 2 84.0 + 3.9 2 T e t r a t h i o n a t e 63.7+3.I 1 96.2+4.0 2 100.0+3.8 3 *-PA expressed as % of the o r i g i n a l PA present i n the l a t e x before d r y i n g B E a c h value i s the mean of three d e t e r m i n a t i o n s . Mean +S.D °Means i n the same column which are not followed by the same number are s i g n i f i c a n t l y d i f f e r e n t a t p<0.05 l e v e l ^Drying c o n d i t i o n s are r e p o r t e d i n M a t e r i a l s and Methods "Latex with no added a d d i t i v e s -63-D. EFFECT OF ADDITIVES ON LOSS OF PROTEOLYTIC ACTIVITY OF CRUDE PAPAIN DURING STORAGE A d d i t i o n of 1% t e t r a t h i o n a t e to papaya l a t e x p r o t e c t e d the PA of the r e s u l t i n g crude papain d u r i n g storage at room temperature. T h i s p r o t e c t i o n was g r e a t e r than that provided by the same l e v e l of m e t a b i s u l f i t e ( F i g . 7). The a d d i t i o n of m e t a b i s u l f i t e a l s o decreased the l o s s e s of PA of crude papain, but to a l e s s e r extent than t e t r a t h i o n a t e . R e l a t i v e l y high l o s s e s of PA occurred d u r i n g storage of the c o n t r o l sample; almost 40% of the o r i g i n a l PA was l o s t i n 13 wk (91 days) ( F i g . 7). C a s t ro (1981) r e p o r t e d l o s s e s of PA of 30% f o r crude papain s t o r e d 75 days at room temperature. Since crude papain i s u s u a l l y exported from the producing to the r e f i n i n g c o u n t r i e s (Leung, 1980; F l y n n , 1975), i t i s very l i k e l y t h a t i t i s s t o r e d f o r r e l a t i v e l y long p e r i o d s with consequent l o s s e s of PA. By adding t e t r a t h i o n a t e to the l a t e x before d r y i n g , i t i s p o s s i b l e to minimize the l o s s e s i n PA, thus, i n c r e a s i n g the s t a b i l i t y of the crude papain. For the c o n t r o l l a t e x (no a d d i t i v e s added), the decrease i n PA with time f o l l o w e d a n o n - l i n e a r r e l a t i o n s h i p ( F i g . 7). The e m p i r i c a l r e a c t i o n order of the PA l o s s over time f o r t h i s l a t e x , determined u s i n g the l i n e a r i z a t i o n procedure of Durance et a l . (1986), was found to be 1.5. Since l o s s of PA of crude papain are due to a number of d i f f e r e n t f a c t o r s ( i . e . o x i d a t i o n , m i c r o b i a l d e g r a d a t i o n , a u t o d i g e s t i o n , i n t e r a c t i o n of p r o t e i n with the carbohydrates present i n the l a t e x , e t c . ) i t i s -64-110-i "O c CD LY. 100-90-80 o < 70H o 8 6 C ^ O 0_ 50 0 1 Treatment O Control • \% Metabisulfite A 1* Tetrathionate ~i 1 1 1 r 3 4 5 6 7 i 1 1 1 1 1 8 9 10 11 12 13 Storage Time (weeks) Figure 7. Change in the p r o t e o l y t i c a c t i v i t y of crude papain with or without additives during storage at room temperature. Each point is mean of duplicates determinations. - 6 5 -expected that the o v e r a l l k i n e t i c s o£ degradation are complex. For the samples of crude papain with e i t h e r a d d i t i v e , the decrease i n PA was l i n e a r over the storage p e r i o d analysed, which i n d i c a t e d a zero order r e a c t i o n . Although no o b j e c t i v e measure of the c o l o r of the crude papain was done, no change i n t h i s parameter was observed d u r i n g the storage time t e s t e d f o r a l l samples. E. COMPARISON OF DIFFERENT METHODS TO SYNTHESIZE TETRATHIONATE Y i e l d s and p u r i t y of t e t r a t h i o n a t e s y n t h e s i z e d by the d i f f e r e n t methods used are shown i n Table 8. Two methods, the i o d i n e - i o d a t e r e a c t i o n and the a l k a l i n e c y a n o l y s i s , were used to determine % t e t r a t h i o n a t e i n the prepared samples. A n a l y s i s of v a r i a n c e of the r e s u l t s of both methods showed no s i g n i f i c a n t d i f f e r e n c e between them. As expected, i o d i n e o x i d a t i o n gave t e t r a t h i o n a t e of high p u r i t y with good y i e l d (65 % ) . The s o l i d obtained was p r a c t i c a l l y pure t e t r a t h i o n a t e . D i f f e r e n t c o n d i t i o n s were t e s t e d i n the hydrogen peroxide o x i d a t i o n method. Decreasing the temperature from 22 to 10<>C inc r e a s e d the amount of t e t r a t h i o n a t e obtained. A d d i t i o n of copper s a l t s had the same e f f e c t . Copper s a l t s have been r e p o r t e d to a c t as c a t a l y s t s i n the o x i d a t i o n of t h i o s u l f a t e with f e r r i c ions (Lar and Singh, 1956). Two d i f f e r e n t a c i d s were used to n e u t r a l i z e the sodium hydroxide t h a t was formed -66-Table 8. Yields*- and p u r i t y of t e t r a t h i o n a t e s l n t h e s i z e d by the d i f f e r e n t methods. C o n d i t i o n s Temp. Y i e l d * P u r i t y c Method pH (°C) (%) (%) Iod ine N, ,D.D 4 65 98 Peroxide™ 6, .8 10 35 50 Peroxide™ 6. .8 22 41 60 Peroxide** 6, .8 10 40 60 Peroxide 1 1' . a 6. .8 10 41 68 Peroxide* -' . o 6. .8 10 43 70 F e r r i c 3, ,0 22 15 15 F e r r i c 6, . 8 22 18 20 F e r r i c 0 6. .8 10 25 17 Cupr i c 3. .0 22 30 28 Cupr i c 6. .8 22 30 30 Cupr i c 6. . 8 10 35 20 Vanadate™' a N. ,D.D 22 40 69 Vanadate*"' . a N. ,D.D 22 35 88 ^-Values were c a l c u l a t e d u s i n g the mean values of d u p l i c a t e d e t e r m i n a t i o n s B Y i e l d expressed as the % of t h e o r e t i c a l y i e l d °Purity expessed as the % t e t r a t h i o n a t e i n p r e c i p i t a t e measured using the i o d a t e - i o d i n e r e a c t i o n DN.D.= Not determinated " S u l f u r i c a c i d used ""Acetic a c i d used a C u p r i c s u l f a t e was added as a c a t a l y s t - 6 7 -d u r i n g the r e a c t i o n of peroxide with t h i o s u l f a t e . I t was found t h a t the weak a c i d ( a c e t i c ) gave s l i g h t l y higher y i e l d s of t e t r a t h i o n a t e than the s t r o n g a c i d ( h y d r o c h l o r i c ) . Under the c o n d i t i o n s t e s t e d , f e r r i c s u l f a t e d i d not give good y i e l d s of t e t r a t h i o n a t e . I t i s p o s s i b l e t h a t the p r e c i p i t a t e obtained was mainly unreacted t h i o s u l f a t e and f e r r o u s s a l t s . Somewhat higher y i e l d s were obtained using c u p r i c c h l o r i d e as the o x i d i z i n g agent, however, they were s m a l l e r than the y i e l d s f o r the peroxide or vanadate o x i d a t i o n . The vanadate o x i d a t i o n method r e s u l t e d i n s i m i l a r y i e l d s to the peroxide o x i d a t i o n . Gowda et a l . (1955) repo r t e d t h a t a c e t i c a c i d was p r e f e r r e d to s u l f u r i c a c i d i n the d e t e r m i n a t i o n of t h i o s u l f a t e with vanadate. In these experiments use of s u l f u r i c a c i d produced higher y i e l d s of t e t r a t h i o n a t e . O v e r a l l o n l y the peroxide and vanadate methods gave adequate y i e l d s , which were s t i l l lower than the y i e l d obtained with the i o d i n e method. I t i s p o s s i b l e t h a t with f u r t h e r r e s e a r c h , the y i e l d s of these methods can be i n c r e a s e d to make them a more economical a l t e r n a t i v e f o r the s y n t h e s i s of t e t r a t h i o n a t e . F. COST EVALUATION OF THE METHODS OF TETRATHIONATE SYNTHESIS To determine the most c o s t e f f e c t i v e method of s y n t h e s i s of t e t r a t h i o n a t e a c o s t e v a l u a t i o n of each method was performed. The p r i c e of the chemicals used f o r the s y n t h e s i s of t e t r a t h i o n a t e -68-by the d i f f e r e n t methods, together with the p r i c e s of sodium m e t a b i s u l f i t e and sodium t e t r a t h i o n a t e are shown i n Table 9. Sodium t e t r a t h i o n a t e i s roughly 24 times more expensive per gram than m e t a b i s u l f i t e , a t c u r r e n t commercial p r i c e s . Since p u r i t y has a major e f f e c t on the p r i c e of a chemical compound, i n order to s t a n d a r d i z e c o s t , the grade of the chemicals r e p o r t e d i n Table 9 i s ACS Grade (American Chemical S o c i e t y Grade). Taking i n t o c o n s i d e r a t i o n the c o s t of the d i f f e r e n t chemicals, with or without s o l v e n t s , the c o s t of s y n t h e s i s of sodium t e t r a t h i o n a t e using the d i f f e r e n t methods was c a l c u l a t e d ( F i g . 8a and 8b). T h i s a n a l y s i s i n d i c a t e d t h a t the t e t r a t h i o n a t e obtained with the c u p r i c o x i d a t i o n method had the h i g h e s t c a l c u l a t e d p r i c e . In the case of the i o d i n e o x i d a t i o n , the c o s t of the i o d i n e represented a high p r o p o r t i o n (24.3%) of the t o t a l c o s t of t e t r a t h i o n a t e obtained ( F i g . 9). For the peroxide method, the c o s t of the oxidant represented o n l y 3.7% of the t o t a l c o s t . Ethanol and e t h y l ether c o n t r i b u t e d to a major p r o p o r t i o n of the t o t a l c o s t of the t e t r a t h i o n a t e ( F i g . 9). Although, i n these experiments, the volumes and p r o p o r t i o n s of the s o l v e n t s were kept cons t a n t , i t could be p o s s i b l e to decrease the t o t a l c o s t of t e t r a t h i o n a t e by e i t h e r d e c r e a s i n g the volumes of s o l v e n t used or by u s i n g l e s s expensive s o l v e n t s to c r y s t a l l i z e the t e t r a t h i o n a t e . The hydrogen peroxide method f o r t e t r a t h i o n a t e s y n t h e s i s was 18% l e s s expensive than the i o d i n e o x i d a t i o n ( s o l v e n t s -69-Table 9. Commercial p r i c e of t e t r a t h i o n a t e , m e t a b i s u l f i t e and chemicals used to s y n t h e s i z e t e t r a t h i o n a t e . P r i c e * Compound Formula (USD/g) Sodium t e t r a t h i o n a t e Na 2S«0«»2H 20 0.600* Sodium m e t a b i s u l f i t e Na-SiOs 0.025° Sodium t h i o s u l f a t e NajS-Oj»5H20 0.013 Iodine I i 0.141 Sodium i o d i d e Nal 0.082 Hydrogen peroxide 1* H 20 2 0.117 F e r r i c c h l o r i d e FeCl,»6H 20 0.006 C u p r i c s u l f a t e CuSO-»5H-,0 0.024 Sodium vanadate NaV0i»2H 20 0.060 * P r i c e s a c c o r d i n g to Sigma Chemical Comp. except when i n d i c a t e d B P r i c e from A l d r i c h Chemical Comp. a P r i c e from F i s h e r S c i e n t i f i c D P r i c e per g of H 20 2 based on a c o n c e n t r a t i o n of 30% H 20 2 i n the commercial s o l u t i o n -70-2.00 1.80 -Iodine Peroxide Cupric Ferric Vanadate Method F i g u r e 8a. The c o s t of s y n t h e s i s of t e t r a t h i o n a t e f o r the d i f f e r e n t methods e v a l u a t e d . Cost i n c l u d i n g that of the s o l v e n t s . Note: The experimental y i e l d s obtained were used for the c a l c u l a t i o n of the c o s t . - 7 1 -0.40 Iodine Peroxide Cupric Method i r Ferric Vanadate Figure 8b. The cost of synthesis tetrathionate for the d i f f e r e n t , methods evaluated. Cost without including that of the solvents. Note: The experimental yields obtained were used for the ca l c u l a t i o n of the c o s t . - 7 2 -F i g u r e 9. D i s t r i b u t i o n o f t h e c o s t o f s y n t h e s i s of t e t r a t h i o n a t e u s i n g t h e i o d i n e (A) and t h e h y d r o g e n p e r o x i d e (B) m e t h o d s . i n c l u d e d ) , while the vanadate o x i d a t i o n method was 16% l e s s expensive than the i o d i n e method ( s o l v e n t s i n c l u d e d ) . G. MELTING POINT DETERMINATION Determination of the a b s o l u t e p u r i t y of m a t e r i a l s by d i f f e r e n t i a l scanning c a l o r i m e t r y (DSC) has been an accepted technique i n the pharmaceutical and chemical i n d u s t r i e s s i n c e the development of DSC i n the e a r l y 1960s (Brennan et a l . , 1984). The m e l t i n g p o i n t of a commercial sample of sodium t e t r a t h i o n a t e (102.0±5% pure as measured by i o d i n e - i o d a t e t i t r a t i o n ) , determined by DSC, was 115±2°C (mean of three d e t e r m i n a t i o n s ) . The thermogram of the commercial t e t r a t h i o n a t e gave, as expected, on l y one endothermic peak, with a temperature range between 110-120<>C ( F i g . 10). T e t r a t h i o n a t e s y n t h e s i z e d by the i o d i n e method (98% pure as measured by i o d i n e - i o d a t e t i t r a t i o n ) gave very s i m i l a r r e s u l t s ( F i g . 11). When samples of t e t r a t h i o n a t e s y n t h e s i z e d by the other methods were su b j e c t e d to DSC a n a l y s i s , the c h a r a c t e r i s t i c endothermic peak of commercial t e t r a t h i o n a t e was not observed. Instead, an endothermic peak at a lower temperature appeared ( F i g . 12-15), except f o r t e t r a t h i o n a t e s y n t h e s i z e d by the vanadate method us i n g a c e t i c a c i d ( F i g . 16). Even when the sample contained 50% t e t r a t h i o n a t e (measured by the i o d i n e - i o d a t e t i t r a t i o n ) , the c h a r a c t e r i s t i c peak of t e t r a t h i o n a t e d i d not appear. Since m e l t i n g p o i n t d e t e r m i n a t i o n i s very s e n s i t i v e to -74-Figure 10. Typical DSC thermogram of a commercial tetrathionate sample from ICN Pharmaceutical, Inc. Tetrathionate content (%) determined using the iodate-iodine reaction (Mean±S.D n = 3)= 102.0+.5% -75-F i g u r e 11. T y p i c a l DSC thermogram o£ t e t r a t h i o n a t e s y n t h e s i z e d by the i o d i n e r e a c t i o n . T e t r a t h i o n a t e content (%) determined u s i n g the i o d a t e - i o d i n e r e a c t i o n (Mean+S.D n=3)= 98.31±6% -77-F i g u r e 12. T y p i c a l DSC thermogram of t e t r a t h i o n a t e s y n t h e s i z e d by the peroxide method. T e t r a t h i o n a t e content (%) determined u s i n g the i o d a t e - i o d i n e r e a c t i o n (Mean+S.D n=3)= 37.9+6% -79-F i g u r e 13. T y p i c a l DSC thermogram of t e t r a t h i o n a t e s y n t h e s i z e d by the c u p r i c method. T e t r a t h i o n a t e content (%) determined using the i o d a t e - i o d i n e r e a c t i o n (Mean+S.D n=3)= 20.4+8% -81-15.0 CUS04 PH-6 0.0 TEMPERATURE <C> DSC F i g u r e 14. T y p i c a l DSC thermogram of t e t r a t h i o n a t e s y n t h e s i z e d by the f e r r i c method. T e t r a t h i o n a t e content (%) determined using the i o d a t e - i o d i n e r e a c t i o n (Mean+S.D n=3)= 18.3+5% -83-F i g u r e 15. T y p i c a l DSC thermogram of t e t r a t h i o n a t e s y n t h e s i z e d by the vanadate method with h y d r o c h l o r i c a c i d . T e t r a t h i o n a t e content (%) determined u s i n g the i o d a t e - i o d i n e r e a c t i o n (Mean+S.D n=3)= 38.6+7% -85-1 5 . 0 VANADATE HAC 0 . 0 i U . O " '• 7 0 . 0 • 1 5 0 . 0 TEMPERATURE <C> DSC F i g u r e 16. T y p i c a l DSC thermogram of a t e t r a t h i o n a t e s y n t h e s i z e d by the vanadate method with a c e t i c a c i d . T e t r a t h i o n a t e content (%) determined using the i o d a t e - i o d i n e r e a c t i o n (Mean+S.D n=3)= 35.0+7% -87-8.00 CO CO A Q Q Z U LU cn x < u V A N A D A T E H C L WT» 11. 45 mg SCAN RATE, 20.00 d PEAK FROMi 64.22 TOi 118.08 ONSETi 05.24 CAL/GRAMi 10.47 4.00 + 3O0 50.00 MAXi 104.44 90.00 110.00 1307 T E M P E R A T U R E C O 150.00 DSC the p u r i t y of the compound, i t was thought that i m p u r i t i e s present i n the s o l i d s ( i . e . t h i o s u l f a t e , s u l f i t e , s u l f a t e s and/or other s a l t s ) i n t e r f e r e d with the m e l t i n g p o i n t d e t e r m i n a t i o n . A sample of pure t h i o s u l f a t e run on the DSC gave a m e l t i n g p o i n t of 55±3°C ( F i g . 17) (mean of three d e t e r m i n a t i o n s ) , s i m i l a r to p r e v i o u s l y r e p o r t e d l i t e r a t u r e values (Weast, 1987). I t i s i n t e r e s t i n g to note t h a t the endothermic peak of pure t h i o s u l f a t e occurred i n the same temperature range (50-55<>C) as the peak of the s o l i d s obtained with the other methods of t e t r a t h i o n a t e s y n t h e s i s which contained 30-50% t e t r a t h i o n a t e . In order to determine the e f f e c t of i m p u r i t i e s on the m e l t i n g p o i n t of t e t r a t h i o n a t e , pure t e t r a t h i o n a t e was mixed with d i f f e r e n t p r o p o r t i o n s of t h i o s u l f a t e . The r e s u l t s i n d i c a t e d t h a t even s m a l l amounts of t h i o s u l f a t e s (10% w/w) had a dramatic e f f e c t on the thermogram of t e t r a t h i o n a t e . When t h i o s u l f a t e was mixed at l e v e l s of 35-40% (w/w) or higher with pure t e t r a t h i o n a t e the sharp endothermic peak at 110-115°C disappeared ( F i g . 18). A d d i t i o n of t h i o s u l f a t e to t e t r a t h i o n a t e decreased the area of the endothermic peak at 110-115°C and i n c r e a s e d the area of the endothermic peak at 50-55°C ( F i g . 19). A decrease i n the m e l t i n g p o i n t of t e t r a t h i o n a t e a l s o occurred with the a d d i t i o n of t h i o s u l f a t e ( F i g . 20). These r e s u l t s c o n f i r m the f a c t t h a t the d e t e r m i n a t i o n of p u r i t y by DSC becomes u n r e l i a b l e as the p r o p o r t i o n of contaminants i n c r e a s e s . A s i m i l a r e f f e c t , where i m p u r i t i e s i n the compound cause -89-F i g u r e 17. T y p i c a l DSC thermograms of pure t h i o s u l f a t e from F i s h e r S c i e n t i f i c Comp. -90-I U5 20.0 T-A a a u CO - J < u THIOSULPHATE WTi 4.50 mg SCAN RATEi 20.00 deg/min MAXi S 3 . 3 3 10. u t 'PEAK FROM. 47 ONSETi 49. 13 CAL/GRAMi. 513, 34 ' . ' n 1 ' i ^ — +— ~ « — : ~ 4 ; -20.0 . " " " 110.0 " 230 .0 TEMPERATURE CO D S C F i g u r e 18. T y p i c a l DSC thermogram of a mixture of pure t h i o s u l f a t e and t e t r a t h i o n a t e at a 1:1 (w/w) r a t i o . -92-10.0 130.0 2 2 0 . 0 ' TEMPERATURE <C> DSC 60.00 - i % Thiosulphate in Mixture (w/w) Figure 19. Effect of addition of thiosulphate on the area of two endothermic peaks of tetrathionate: Endothermic peak with temperature range 47-66°C (solid l i n e ) ; endothermic peak with temperature range 104-137<>C (broken l i n e ) . Mean + S.D (n = 3). -94-120.00 - i 110.00 H O o o Q_ 100.00 H cr> 90.00 H c 80.00 H 70.00 10 20 30 40 % Thiosulphate in Mixture (w/w) Figure 2 0 . E f f e c t of addition of thiosulfate on the melting point of tetrathionate. - 9 5 -dramatic changes i n the thermogram, was r e p o r t e d f o r many compounds (Brennan et a l . , 1984). H. INACTIVATION AND ACTIVATION EFFICIENCY OF THE SYNTHESIZED TETRATHIONATE As expected, f o r most samples, the higher the p r o p o r t i o n of t e t r a t h i o n a t e i n the sample, the higher the i n a c t i v a t i o n e f f i c i e n c y (IE) of the s o l i d (Table 10). T e t r a t h i o n a t e s y n t h e s i z e d by the i o d i n e method gave 100% IE, which i n d i c a t e s t h a t i t i n a c t i v a t e d the PA of papaya l a t e x to the same extent as a commercial sample of t e t r a t h i o n a t e . In the case of the s o l i d prepared by the f e r r i c o x i d a t i o n method, the IE was higher than expected, c o n s i d e r i n g i t s low t e t r a t h i o n a t e content. T h i s r e s u l t suggests t h a t other compounds present i n the s o l i d (e.g. f e r r o u s s a l t s ) may a l s o have an i n a c t i v a t i o n e f f e c t on the PA of papaya l a t e x . In most cases the a c t i v a t i o n e f f i c i e n c y (AE) was n e a r l y 100%, with the e x c e p t i o n of the s o l i d prepared with the f e r r i c method. Since the p r o t e o l y t i c enzymes i n papaya l a t e x are i n a c t i v a t e d by metal ions (Fukal et a l . , 1984), and t h i s i n a c t i v a t i o n i s not reversed by a d d i t i o n of c y s t e i n e , these r e s u l t s suggest that t h i s s o l i d contained a l a r g e amount of f e r r o u s s a l t s which r e a c t with the proteases of papaya l a t e x . T h i s e x p l a n a t i o n i s supported by the f a c t t h a t i n c r e a s i n g the c o n c e n t r a t i o n of EDTA i n the b u f f e r i n order to c h e l a t e more metal ions, i n c r e a s e d the a c t i v a t i o n e f f i c i e n c y (Table 10). -96-Table 10. I n a c t i v a t i o n (IE) and a c t i v a t i o n (AE) e f f i c i e n c y * of t e t r a t h i o n a t e s s y n t h e s i z e d u s i n g d i f f e r e n t methods. E f f i c i e n c y * Method of % t e t r a t h i o n a t e i n IE AE s y n t h e s i s sample 0 (%) (%) C o n t r o l 0 100 100 100 Iodine 100 100 100 Peroxide 43 40 95 F e r r i c 23 38 60 F e r r i c 23 18= 100 C u p r i c 30 23 85 Vanadate 42 34 100 *The d e f i n i t i o n s of i n a c t i v a t i o n (IE) and a c t i v a t i o n e f f i c i e n c y (AE) are giv e n i n the Methods s e c t i o n *Values were c a l c u l a t e d u s i n g the mean values of d u p l i c a t e d e t e r m i n a t i o n s f o r both i n a c t i v a t i o n and a c t i v a t i o n e f f i c i e n c i e s c % t e t r a t h i o n a t e r e p o r t e d i s the mean value of d u p l i c a t e d e t e r m i n a t i o n s u s i n g the i o d a t e - i o d i n e r e a c t i o n ^Commercial sample from ICN Pharmaceuticals, Inc. BThe c o n c e n t r a t i o n of EDTA i n the b u f f e r was inc r e a s e d to 50 mM -97-CONCLUSION The main o b j e c t i v e of t h i s study was to determine i f a d d i t i o n of t e t r a t h i o n a t e to papaya l a t e x would p r o t e c t i t s p r o t e o l y t i c a c t i v i t y (PA) d u r i n g d r y i n g , as w e l l as d u r i n g storage of crude papain. The r e s u l t s obtained i n d i c a t e that a d d i t i o n of t e t r a t h i o n a t e , at a 1% l e v e l , to papaya l a t e x c ompletely i n h i b i t e d the l o s s e s of PA when the d r y i n g temperature was low (50-55<>C). Sinc e , i n commercial p r a c t i c e , 55<>C i s the commonly used d r y i n g temperature f o r papaya l a t e x , a d d i t i o n of t e t r a t h i o n a t e has p o t e n t i a l i n d u s t r i a l a p p l i c a t i o n . A d d i t i o n of 1% m e t a b i s u l f i t e a l s o p r o t e c t e d the PA of papaya l a t e x d u r i n g oven d r y i n g , however, i t s p r o t e c t i v e e f f e c t was s i g n i f i c a n t l y lower than t h a t one given by t e t r a t h i o n a t e . A f r a c t i o n a l f a c t o r i a l experimental design showed t h a t the best c o n d i t i o n s (minimum PA l o s s e s ) f o r d r y i n g papaya l a t e x were: a d d i t i o n of 1% t e t r a t h i o n a t e ; a d r y i n g temperature of 55<>C; a d r y i n g load of 1,190 g/m2, and i f storage p r i o r to d r y i n g was necessary, i t had to be under dark c o n d i t i o n s . The e f f e c t s of three d i f f e r e n t d r y i n g methods (sun, oven, and vacuum) on the PA of papaya l a t e x were a l s o i n v e s t i g a t e d . As r e p o r t e d by other r e s e a r c h e r s , vacuum d r y i n g produced the s m a l l e s t PA l o s s e s . In t h i s case a d d i t i o n of t e t r a t h i o n a t e or m e t a b i s u l f i t e , both a t a 1% l e v e l , was found to be e q u a l l y e f f e c t i v e i n p r o t e c t i n g the PA of papaya l a t e x . The g r e a t e s t PA l o s s e s occurred d u r i n g sun d r y i n g , and f o r t h i s d r y i n g method, n e i t h e r t e t r a t h i o n a t e nor m e t a b i s u l f i t e minimized the PA l o s s e s i n papaya l a t e x as compared with an untreated l a t e x . The second p a r t of t h i s study was aimed a t i n v e s t i g a t i n g d i f f e r e n t chemical methods f o r s y n t h e s i s of t e t r a t h i o n a t e and to compare the y i e l d s and c o s t s of the t e t r a t h i o n a t e o b tained. T e t r a t h i o n a t e y i e l d s (as % of the t h e o r e t i c a l y i e l d ) ranged from 65% f o r the i o d i n e r e a c t i o n to 15% f o r the f e r r i c o x i d a t i o n method. O v e r a l l , o n l y the peroxide and vanadate methods gave y i e l d s comparable to the one obtained by the io d i n e method. A c o s t a n a l y s i s i n d i c a t e d t h a t the s o l v e n t s needed f o r c r y s t a l l i z i n g t e t r a t h i o n a t e c o n t r i b u t e d s i g n i f i c a n t l y to the o v e r a l l c o s t of the compound. Both the peroxide and vanadate methods were e a s i e r to performed on a l a b o r a t o r y s c a l e , and both methods gave t e t r a t h i o n a t e at about 20% lower c o s t s than the i o d i n e method. In order to c h a r a c t e r i z e the t e t r a t h i o n a t e obtained from the d i f f e r e n t s y n t h e s i s methods, DSC was used to determine the me l t i n g p o i n t of the s o l i d s . The m e l t i n g p o i n t of pure t e t r a t h i o n a t e was found to be 115<>C. However, due to the high p r o p o r t i o n of compounds other than t e t r a t h i o n a t e i n the s o l i d s , the m e l t i n g behavior of the s o l i d s was completely d i f f e r e n t than the one found f o r pure t e t r a t h i o n a t e . - 9 9 -CHAPTER 2. Chemical m o d i f i c a t i o n of papain by t e t r a t h i o n a t e . LITERATURE REVIEW A. CHEMICAL MODIFICATION OF PROTEINS M o d i f i c a t i o n of a p r o t e i n u s u a l l y r e f e r s to p h y s i c a l , chemical, or enzymatic treatments changing i t s conformation, i t s s t r u c t u r e , and consequently i t s physicochemical and f u n c t i o n a l p r o p e r t i e s (Chobert et a l . , 1988); Chemical m o d i f i c a t i o n i s g e n e r a l l y r e f e r r e d to as the i n t e n t i o n a l a l t e r a t i o n of a p r o t e i n s t r u c t u r e , or conformation, by chemical agents (Means and Feeney, 1971). In g e n e r a l , i t i n v o l v e s d e r i v a t i z a t i o n , by s p e c i f i c reagents, of some r e a c t i v e s i d e c h a i n groups i n the p r o t e i n molecule, such as charged a n i o n i c and c a t l o n i c groups, h y d r o x y l , amide and t h i o l r e s i d u e s . The amino a c i d s i d e chains of p r o t e i n s most o f t e n modified are probably the e-amino group of l y s i n e , the s u l f h y d r y l group of c y s t e i n e , or i t s o x i d i z e d product the d i s u l f i d e group of c y s t i n e (Feeney, 1977). Chemical m o d i f i c a t i o n of a p r o t e i n i s a technique widely used i n fundamental s t u d i e s i n v a r i o u s areas of p r o t e i n r e s e a r c h i n c l u d i n g enzymology, immunochemistry, X-ray c r y s t a l l o g r a p h y and p u r i f i c a t i o n of p r o t e i n s . The techniques have advanced so r a p i d l y t h a t almost every j o u r n a l of b i o c h e m i s t r y has an a r t i c l e which r e p o r t s chemical m o d i f i c a t i o n , many of these using s e v e r a l d i f f e r e n t methods f o r d i f f e r e n t s i d e chains (Feeney, 1987). With the present a v a i l a b i l i t y of -100-s p e c i f i c chemical reagents and s o p h i s t i c a t e d a n a l y t i c a l t echniques, chemical m o d i f i c a t i o n has become a powerful t o o l fo r p r o t e i n chemists, i n the study of s t r u c t u r e and f u n c t i o n of b i o l o g i c a l l y a c t i v e p r o t e i n s (Feeney, 1987; Feeney, 1977; Means and Feeney, 1971). Chemical m o d i f i c a t i o n i s r o u t i n e l y used to i n v e s t i g a t e the r o l e s of i n d i v i d u a l amino a c i d chains i n r e l a t i o n to the p h y s i c a l , chemical, and b i o l o g i c a l p r o p e r t i e s of p r o t e i n s and to determine the a c t i v e - s i t e r e s i d u e s i n enzymes. The v e r y powerful methods of i n v i t r o mutagenesis and d i r e c t chemical s y n t h e s i s are a c h i e v i n g r e s u l t s beyond those capable with chemical m o d i f i c a t i o n , p a r t i c u l a r l y f o r the d e t e r m i n a t i o n of the r o l e s of i n d i v i d u a l r e s i d u e s i n p r o t e i n s t r u c t u r e and f u n c t i o n . Yet chemical m o d i f i c a t i o n i s s t i l l a good adjunct f o r these methods (Feeney, 1987). Feeney (1987) i n d i c a t e d t h a t "chemical m o d i f i c a t i o n of p r o t e i n s should continue to serve as an important method i n p r o t e i n chemistry. Although there are now b e t t e r methods f o r a t t a c k i n g some of the problems, these methods s t i l l r e q u i r e chemical procedures f o r many of t h e i r a n a l y s i s and a p p l i c a t i o n s " . T h i s author concluded with a l i s t of 24 areas where chemical m o d i f i c a t i o n should continue to be important i n p r o t e i n r e s e a r c h . -101-B. CHEMICAL MODIFICATION OF FOOD RELATED PROTEINS Commercial a p p l i c a t i o n s of chemical m o d i f i c a t i o n of p r o t e i n s have a long h i s t o r y r e l a t e d to the pharmaceutical, dyeing, and t e x t i l e i n d u s t r i e s (Means and Feeney, 1971). Although the a d d i t i o n of chemicals to food has long been p r a c t i c e d , the i n t e n t i o n a l chemical m o d i f i c a t i o n of food p r o t e i n s i s s t i l l l a r g e l y found on l y i n the patent l i t e r a t u r e and i s p r a c t i c e d to o n l y a l i m i t e d extent. Obvious b a r r i e r s to the chemical m o d i f i c a t i o n of food p r o t e i n s f o r human usage in c l u d e e s t h e t i c , c u l t u r a l , l e g a l , and medical aspects (Feeney, 1977). The three general areas of a p p l i c a t i o n of chemical m o d i f i c a t i o n of food p r o t e i n s are : 1) b l o c k i n g of d e t e r i o r a t i v e r e a c t i o n s ; 2) improvement of p h y s i c a l p r o p e r t i e s , and 3) improvement of n u t r i t i o n a l p r o p e r t i e s (Feeney, 1977). D i f f e r e n t d e t e r i o r a t i v e r e a c t i o n s such as o x i d a t i o n , M a i l l a r d r e a c t i o n s , and c r o s s l i n k i n g a f f e c t food p r o t e i n s d u r i n g p r o c e s s i n g and s t o r a g e . In t h i s case, the general purpose i s to modify the p r o t e i n s to e i t h e r prevent a chemical r e a c t i o n or g r e a t l y r e t a r d i t s r a t e . These o b j e c t i v e s can be accomplished by b l o c k i n g a p r o t e i n group which undergoes a r e a c t i o n , or by changing the c o n d i t i o n s , thus g r e a t l y r e t a r d i n g the d e t e r i o r a t i v e r e a c t i o n (Feeney, 1977). Improvements i n p h y s i c a l p r o p e r t i e s can be obtained by chemical m o d i f i c a t i o n . M o d i f i c a t i o n i n t h i s case i s aimed at -102-changing the gross p h y s i c a l and chemical i n t e r a c t i o n s of the molecule which are important to f u n c t i o n a l p r o p e r t i e s such as foaming and whipping c a p a b i l i t i e s (Feeney, 1977). Improvement of the n u t r i t i o n a l q u a l i t y by chemical m o d i f i c a t i o n may prove to be the most important use from the sta n d p o i n t of s o c i e t y ' s fundamental needs. T h i s o b j e c t i v e might be brought about by i n c r e a s i n g the d i g e s t i b i l i t y of the p r o t e i n , i n a c t i v a t i n g t o x i c or i n h i b i t o r y substances, or a t t a c h i n g e s s e n t i a l n u t r i e n t s to the p r o t e i n s . An example of t h i s approach i s the c o v a l e n t i n c o r p o r a t i o n of e s s e n t i a l amino a c i d s to soy p r o t e i n s by means of the ca r b o d i i m i d e r e a c t i o n (Voutsinas, 1978). Attachment of c o l o r i n g or f l a v o r i n g agents to p r o t e i n s might a l s o improve t h e i r a c c e p t a b i l i t y (Feeney, 1977). C. MODIFICATION OF SULFHYDRYL GROUPS IN PROTEINS The ease and s p e c i f i c i t y with which the s u l f h y d r y l or t h i o l (-SH) group can be modified makes i t a prime t a r g e t f o r experimental study, and presumably accounts f o r i t s widespread involvement i n b i o l o g i c a l phenomena ( B r o c k l e h u r s t , 1979). Proton d i s s o c i a t i o n provides the t h i o l a t e i on ( R S - ) , probably the most powerful n u c l e o p h i l e found i n b i o l o g i c a l m a t e r i a l s ( B r o c k l e h u r s t , 1979). The RS~ ions are over 500 times more n u c l e o p h i l i c than the corresponding oxygen analogue RO~ ( J o c e l y n , 1972). -103-A l a r g e number of reagents t h a t r e a c t r e a d i l y with -SH groups have been d e s c r i b e d ( B r o c k l e h u r s t , 1979; Friedman, 1973; J o c e l y n , 1972; Means and Feeney, 1971). Although the high i n t r i n s i c n u c l e o p h i l i c i t y of the t h i o l a t e ions i s o f t e n s u f f i c i e n t to ensure t h e i r s p e c i f i c m o d i f i c a t i o n by many types of e l e c t r o p h i l i c reagents, t h i s cannot always be guaranteed when the -SH groups r e s i d e i n p r o t e i n s ( B r o c k l e h u r s t , 1979). The number of r e a c t i v e s u l f h y d r y l groups r e p o r t e d f o r p r o t e i n s f r e q u e n t l y d i f f e r s because of the use of d i f f e r e n t reagents. The hydrophobic c h a r a c t e r of s u l f h y d r y l groups, and t h e i r presence, i n many cases, i n hydrophobic environments i n c o n j u n c t i o n with the v a r i e d a b i l i t y of reagents to penetrate hydrophobic r e g i o n s p a r t l y account f o r the d i f f e r e n c e s i n r e a c t i v i t y of -SH (Means and Feeney, 1971). The two r e a c t i o n s most commonly employed to modify the -SH groups i n p r o t e i n s are o x i d a t i o n and a l k y l a t i o n . 1. O x i d a t i o n Perhaps the most b i o l o g i c a l l y i n t e r e s t i n g p r o p e r t y of -SH groups i s t h a t they can be o x i d i z e d . The -SH group i s r e l a t i v e l y e a s i l y o x i d i z e d by a wide v a r i e t y of o x i d i z i n g agents, but the m a j o r i t y of these substance ( i . e . HaOa, I a) are not regarded as s p e c i f i c reagents f o r -SH groups. Since s u l f u r has valences ranging from -2 to +6, s e v e r a l types of o x i d a t i o n products are p o s s i b l e . Table 11 shows the most common o x i d a t i o n products of -SH groups. The most e a s i l y formed product i s the d i s u l f i d e -104-Table 11. O x i d a t i o n products of s u l f h y d r y l groups. Formula Name S u l f u r valence R-SH s u l f h y d r y l -1 R-SOH s u l f e n i c -1 R-S-S-R d i s u l f i d e 0 R-SO2H s u l f i n i c + 3 R - S O 3 H s u l f o n i c + 5 Source: Friedman (1973) -105-(-S-S-) ( J o c e l y n , 1972). D i s u l f i d e s are much l e s s a c t i v e than -SH groups and may f u n c t i o n to s t a b i l i z e p r o t e i n s t r u c t u r e . However v a r i o u s reagents can c l e a v e them c o n v e r t i n g them back to -SH groups or other d e r i v a t i v e s (Friedman- 1973; J o c e l y n , 1972). The most s p e c i f i c reagents f o r o x i d i z i n g s u l f h y d r y l groups to d i s u l f i d e s are probably d i s u l f i d e s themselves. T h e i r s p e c i f i c i t y f o r -SH groups may be c o n s i d e r e d a b s o l u t e f o r p r a c t i c a l purposes ( B r o c k l e h u r s t , 1979; Dixon and Webb, 1964). T h i s r e a c t i o n between s u l f h y d r y l groups with d i s u l f i d e s i s commonly known as t h i o l d i s u l f i d e interchange ( J o c e l y n , 1972) . (a) M o d i f i c a t i o n by aromatic d i s u l f i d e s The compound 5 , 5 1 - d i t h i o b i s ( 2 - n i t r o b e n z o i c a c i d ) (DTNB) r e a c t s with f r e e -SH groups of p r o t e i n s , forming t h i o n i t r o b e n z o a t e - p r o t e i n and l i b e r a t i n g one mole of t h i o n i t r o b e n z o a t e anion f o r each -SH group ( B r o c k l e h u r s t , 1979; Means and Feeney, 1971). Although i t s main a p p l i c a t i o n has been i n the measurement of -SH groups of p r o t e i n s (Habeeb, 1972). DTNB has a l s o been used to modify -SH groups i n many p r o t e i n s , such as s t r e p t o c o c c a l d i h y d r o f o l a t e reductase (Warwick and F r e i s h e i m , 1975) and c y t o l y s i n , a t h i o l - a c t i v a t e d e x o t o x i n produced by C l o s t r i d i u m p e r f r i n g e n s type A (Iwamoto et a l . , 1987). In some cases the m o d i f i c a t i o n i s r e v e r s i b l e by treatment with G-mercaptoethanol. However, i n some p r o t e i n s , a -106-secondary change takes place d u r i n g storage t h a t e l i m i n a t e s the a b i l i t y to be r e a c t i v a t e d . T h i s secondary i n a c t i v a t i o n appears to r e s u l t from d i s u l f i d e interchange with unreacted s u l f h y d r y l groups i n the p a r t i a l l y m o d ified enzyme (Means and Feeney, 1971). Another type of aromatic d i s u l f i d e i s the p y r i d y l d i s u l f i d e such as the symmetrical reagents, 2,2' d i p y r i d y l d i s u l f i d e (2PDS) and 4,4' d i p y r i d y l d i s u l f i d e , which are u s u a l l y r e f e r r e d to as two p r o t o n i c s t a t e e l e c t r o p h i l e s . These reagents have been used to modify -SH groups with low pKa's ( B r o c k l e h u r s t , 1979). T i t r a t i o n with 2PDS at pH 4 and 8 has been shown to permit d e t e c t i o n of chymopapain contaminants i n p r e p a r a t i o n s of papain (Baines and B r o c k l e h u r s t , 1979). (b) M o d i f i c a t i o n by t e t r a t h i o n a t e Sodium t e t r a t h i o n a t e (Na2S»0 6) i s another d i s u l f i d e t h a t has been used to modify s u l f h y d r y l groups of p r o t e i n s . As mentioned before (see CHAPTER 1) t e t r a t h i o n a t e r a p i d l y o x i d i z e s simple t h i o l s to corresponding d i s u l f i d e s . In the case of some s u l f h y d r y l p r o t e i n s , r a t h e r s t a b l e s u l f e n y l t h i o s u l f a t e i n t e r m e d i a t e s have been observed (Means and Feeney, 1971). The r e a c t i o n of t e t r a t h i o n a t e with s u l f h y d r y l p r o t e i n s i s r a p i d l y r e v e r s i b l e upon a d d i t i o n of excess t h i o l (e.g. c y s t e i n e ) ( L i u , 1967). -107-Sodium t e t r a t h i o n a t e has been used i n p r o t e i n chemistry i n three d i f f e r e n t areas: I. As a s t a b i l i z i n g agent of s u l f h y d r y l protease T h i s a p p l i c a t i o n has a l r e a d y been reviewed (see CHAPTER 1). B r i e f l y , sodium t e t r a t h i o n a t e , by r e v e r s i b l y i n a c t i v a t i n g s u l f h y d r y l enzymes, i n c r e a s e s the storage s t a b i l i t y of the p r o t e i n s , m i nimizing a c t i v i t y l o s s e s due to a u t o l y s i s and i r r e v e r s i b l e o x i d a t i o n . I I . As a b l o c k i n g agent of c y s t e i n e r e s i d u e s The r e v e r s i b i l i t y of the r e a c t i o n of t e t r a t h i o n a t e with c y s t e i n e r e s i d u e s has been u t i l i z e d to p r o t e c t (block) c y s t e i n e r e s i d u e s i n p r o t e i n s from m o d i f i c a t i o n , thus p e r m i t t i n g the s e l e c t i v e m o d i f i c a t i o n of l e s s r e a c t i v e r e s i d u e s . The f i r s t paper d e s c r i b i n g the use of t e t r a t h i o n a t e as a b l o c k i n g agent was p u b l i s h e d by L i u (1967). Using t h i s method L i u (1967) demonstrated the presence of a h i s t i d i n e r e s i d u e at the a c t i v e s i t e of s t r e p t o c o c c a l p r o t e i n a s e . A s i m i l a r approach was used by G l e i s n e r and L i e n e r (1973) to demonstrate the presence of a h i s t i d i n e r e s i d u e at the a c t i v e s i t e of f i c i n . Murachi and Okumura (1974) a l s o used t e t r a t h i o n a t e as a b l o c k i n g agent d u r i n g the p h o t o o x i d a t i o n of h i s t i d i n e i n bromelain and papain. The use of r e v e r s i b l e b l o c k i n g of c y s t e i n e r e s i d u e s with t e t r a t h i o n a t e has not been r e s t r i c t e d only to s u l f h y d r y l enzymes. -108-Nakai et a l . (1973) i n v e s t i g a t e d the e f f e c t of h i s t i d i n e m o d i f i c a t i o n of K-casein, with d i f f e r e n t compounds, on the s t a b i l i z i n g a b i l i t y of t h i s p r o t e i n on a 3 i - c a s e i n . In order to prevent m o d i f i c a t i o n of the c y s t e i n e r e s i d u e s of K-casein, sodium t e t r a t h i o n a t e was used as a b l o c k i n g agent. In t h i s case, the amino groups of the p r o t e i n were a l s o blocked by a c y l a t i o n with c i t r a c o n i c anhydride. With the use of these b l o c k i n g r e a c t i o n s , i t was found t h a t the h i s t i d i n e r e s i d u e s of K-casein are not e s s e n t i a l f o r i t s s t a b i l i z i n g a b i l i t y on o t s i - c a s e i n . Rothenbuhler and K i n s e l l a (1986), working on the e f f e c t of d i s u l f i d e r e d u c t i o n and molecular d i s s o c i a t i o n of soy g l y c i n i n i n i t s r a t e of p r o t e o l y s i s , used t e t r a t h i o n a t e to prevent r e o x i d a t i o n of the f r e e SH groups formed f o l l o w i n g r e d u c t i o n of the d i s u l f i d e groups. I I I . As a chemical m o d i f i c a t i o n agent of c y s t e i n e r e s i d u e s Since t e t r a t h i o n a t e r e a c t s with s u l f h y d r y l groups i n a h i g h l y s p e c i f i c way, i t has been widely used to modify them. The r o l e of the s u l f h y d r y l groups i n D-glyceraldehyde-3-phosphate dehydrogenase from r a b b i t muscle was s t u d i e d by P i h l and Lange (1962) u s i n g chemical m o d i f i c a t i o n of the c y s t e i n e r e s i d u e s with d i f f e r e n t reagents, i n c l u d i n g t e t r a t h i o n a t e . T e t r a t h i o n a t e was found to i n h i b i t the enzyme r a p i d l y and completely and the i n h i b i t i o n was promptly reversed by c y s t e i n e . Approximately 3 moles of t e t r a t h i o n a t e were needed -109-to i n h i b i t the enzyme completely. Fur t h e r d eterminations i n d i c a t e d t h a t the b i n d i n g of r a d i o a c t i v e s u l f u r ( 3 S S ) was a s s o c i a t e d with the disappearance of an e q u i v a l e n t number of enzyme -SH groups, thus r u l i n g out the formation of i n t r a - or i n t e r - m o l e c u l a r d i s u l f i d e bonds. The i n h i b i t i o n was found to be due to the f a c t t h a t t e t r a t h i o n a t e r e a c t e d s t o i c h i o m e t r i c a l l y with three p a r t i c u l a r l y r e a c t i v e -SH groups of the enzyme, r e s u l t i n g i n the formation of p r o t e i n s u l f e n y l t h i o s u l f a t e s . Parker and A l l i s o n (1969) working with the same enzyme, but from a d i f f e r e n t source ( p i g muscle), r e p o r t e d s i m i l a r r e s u l t s . They found t h a t t e t r a t h i o n a t e r e a c t e d s t o i c h i o m e t r i c a l l y with the c a t a l y t i c a l l y a c t i v e -SH groups of the enzyme to form s u l f e n y l t h i o s u l f a t e d e r i v a t i v e s . These enzyme d e r i v a t i v e s were found to be s t a b l e at 0<>C but decomposed at higher temperatures. In a more rec e n t a r t i c l e Tsou et a l . (1982), r e p o r t e d t h a t t e t r a t h i o n a t e or i o d o a c e t i c a c i d m o d i f i c a t i o n of the a c t i v e s i t e , c y s t e i n e - 1 4 9 , of D-glyceraldehyde-3-phosphate dehydrogenase caused co n f o r m a t i o n a l changes i n the enzyme. T e t r a t h i o n a t e was a l s o used to modify the -SH groups of the enzyme D-amino a c i d oxidase and of hemoglobin (Neims et a l . , 1966). Chung and Folk (1970) s t u d i e d the mechanism of i n a c t i v a t i o n of guinea p i g l i v e r transglutaminase by t e t r a t h i o n a t e . They re p o r t e d t h a t treatment of t h i s enzyme with 2 moles of t e t r a t h i o n a t e r e s u l t e d i n almost complete l o s s i n enzymic a c t i v i t y , and t h a t t h i s l o s s was accompanied by a -110-concomitant disappearance of four s u l f h y d r y l groups i n the enzyme. The n a t i v e enzyme contained 17-18 s u l f h y d r y l groups per molecule and no d i s u l f i d e bonds. However, i t was found t h a t l e s s than 0.15 moles of " S - t e t r a t h i o n a t e were bound per mole of enzyme. These authors concluded t h a t the enzymatic changes which occurred upon r e a c t i o n of transglutaminase with 2 moles of t e t r a t h i o n a t e were a r e s u l t of i n t r a m o l e c u l a r d i s u l f i d e bond formation and t h a t these c a t a l y t i c changes were a s s o c i a t e d with a s i n g l e d i s u l f i d e bond i n the enzyme molecule. The a c t i v i t i e s of l a c t a t e dehydrogenase, glutamate dehydrogenase, a s p a r t a t e amino t r a n s f e r a s e , (3-galactosidase, N-acetyl-p-D-glucosamidase, l e u c i n e aminopeptidase, Y-g l u t a m y l t r a n s f e r a s e and a l k a l i n e phosphatase i n r e n a l t i s s u e and i n u r i n e of r a t s were decreased by t e t r a t h i o n a t e (Kunovic et a l . , 1981). Pyruvate kinase from human e r y t h r o c y t e s was a l s o modified by t e t r a t h i o n a t e . Although i n h i b i t i o n o c c urred, no changes i n conformation or t h e r m o l a b i l i t y occurred ( V a l e n t i n e and P a g l i a , 1983). Sodium t e t r a t h i o n a t e was found to cause co n f o r m a t i o n a l changes i n the g l u c o s e - t r a n s p o r t i n g p r o t e i n of human e r y t h r o c y t e s (Krupka, 1985). In a rec e n t a r t i c l e , Prasad and Horowitz (1987) i n v e s t i g a t e d the chemical m o d i f i c a t i o n of bovine l i v e r rhodanase with t e t r a t h i o n a t e . Rhodanase c a t a l y s e s the t r a n s f e r of the outer s u l f u r atom of t h i o s u l f a t e to the n u c l e o p h i l i c acceptor -111-s u b s t r a t e , cyanide (Prasad and Horowitz, 1987). T h i s enzyme i s a s i n g l e p o l y p e p t i d e c h a i n c o n t a i n i n g 293 amino a c i d r e s i d u e s of which four are c y s t e i n e r e s i d u e s . One of the c y s t e i n e r e s i d u e s , Cys-247, i s at the a c t i v e s i t e . There are no d i s u l f i d e bonds present i n the enzyme. These r e s e a r c h e r s r e p o r t e d a l i n e a r r e l a t i o n s h i p between l o s s of enzymatic a c t i v i t y and amount of t e t r a t h i o n a t e used f o r the m o d i f i c a t i o n . T e t r a t h i o n a t e m o d i f i c a t i o n of rhodanase could be c o r r e l a t e d with the changes i n i n t r i n s i c f l u o r e s c e n c e or with the b i n d i n g of the a c t i v e s i t e reagent 2- a n i l i n o n a p h t h a l e n e - 8 -s u l f o n i c a c i d . C i r c u l a r d i c h r o i s m s p e c t r a of the p r o t e i n suggested i n c r e a s e d ordered secondary s t r u c t u r e i n the p r o t e i n a f t e r r e a c t i o n with t e t r a t h i o n a t e . The authors suggested t h a t t e t r a t h i o n a t e m o d i f i c a t i o n of rhodanase may proceed through formation of s u l f e n y l t h i o s u l f a t e i n t e r m e d i a t e s at s u l f h y d r y l groups, c l o s e t o , but not i d e n t i c a l , with the a c t i v e s i t e s u l f h y d r y l group, which then can r e a c t f u r t h e r with the a c t i v e - s i t e s u l f h y d r y l group to form d i s u l f i d e b r i d g e s . (c) M o d i f i c a t i o n by Iodobenzoates and m e r c u r i a l s Iodobenzoates have become widely used enzyme i n h i b i t o r s . T h e i r p r i n c i p a l r e a c t i o n with p r o t e i n s i s the o x i d a t i o n of -SH groups to d i s u l f i d e bonds. O x i d a t i o n of other r e s i d u e s has not been dete c t e d but may be p o s s i b l e , e s p e c i a l l y the o x i d a t i o n of methionine r e s i d u e s ( B r o c k l e h u r s t , 1979; Means and Feeney, 1971). T h i s m o d i f i c a t i o n i s u s u a l l y r e v e r s i b l e with the -112-a d d i t i o n of c y s t e i n e or d i t h i o t h r e i t o l (Means and Feeney, 1971). M e r c u r i a l s are widely used to q u a n t i t a t e and determine the e f f e c t of s u b s t i t u t i o n of s u l f h y d r y l groups (Means and Feeney, 1971). The r e a c t i o n of m e r c u r i a l s with p r o t e i n s was thoroughly reviewed by Dixon and Webb (1964). The most commonly used i n o r g a n i c m e r c u r i a l i s mercuric c h l o r i d e . Organic m e r c u r i a l s such as p - c h l o r o m e r c u r i c benzoate are a l s o w i d e l y used (Storey and Wagner, 1986). 2. A l k y l a t i o n Compounds with -SH groups are a l k y l a t e d , under m i l d c o n d i t i o n s , by v a r i o u s s t a b l e and water s o l u b l e halogenated a c i d s or t h e i r s a l t s (R'-Hal), such as i o d o a c e t i c a c i d or iodoacetamide, to g i v e s u l f i d e s (R-S-R') ( J o c e l y n , 1972). L i k e most other r e a c t i o n s of t h i o l s , the r e a c t i v e s p e c i e s i n such a l k y l a t i o n r e a c t i o n s i s the t h i o l a t e anion (RS~). Hence, the r a t e of r e a c t i o n decreases with d e c r e a s i n g pH of the medium, and a l s o , due mainly to pK d i f f e r e n c e s , with the nature of the t h i o l s themselves ( J o c e l y n , 1972). Neighboring groups can e i t h e r enhance or suppress the r e a c t i v i t y of a group. For example, the -SH i n s t r e p t o c o c c a l p r o t e i n a s e i s 50 to 100 times more r e a c t i v e than t h a t of g l u t a t h i o n e ( J o c e l y n , 1972). A s i m i l a r a c t i v a t e d -SH has been observed i n papain (Sluyterman, 1967a) . R e a c t i v i t i e s of -SH groups of p r o t e i n s can be e i t h e r enhanced or suppressed by s u b s t r a t e s , a l l o s t e r i c e f f e c t o r s and other -113-s p e c i f i c a l l y i n t e r a c t i n g s u b s t r a t e s . In the case of both papain and f i c i n , the presence of c e r t a i n s u b s t r a t e s caused a s e v e r a l - f o l d s t i m u l a t i o n of t h e i r r e a c t i v i t y to h a l o a c e t a t e s (Whitaker, 1969). A l l the h a l o a c e t a t e compounds modify -SH groups in an i r r e v e r s i b l e way. N-Ethylmaleimide (NEM) and i t s d e r i v a t i v e s are other w i d e l y used s u l f h y d r y l reagents. NEM has been used to determine the e f f e c t s of -SH m o d i f i c a t i o n and the number of s u l f h y d r y l groups i n a wide v a r i e t y of p r o t e i n s . I t a l s o r e a c t s under c e r t a i n c o n d i t i o n s with amino groups (Means and Feeney, 1971). Long c h a i n a l k y l maleimides are sometimes more e f f e c t i v e reagents than NEM f o r r e a c t i o n s with -SH i n nonpolar environment ( J o c e l y n , 1972) . D. PAPAIN 1. D e f i n i t i o n and i s o l a t i o n . Pure papain (E.C.3.4.22.2) i s a s u l f h y d r y l protease (or p r o t e i n a s e ) i s o l a t e d from C a r i c l a papaya l a t e x ( B r o c k l e h u r s t et a l . , 1981). Papain was f i r s t i s o l a t e d i n c r y s t a l l i n e form from f r e s h papaya l a t e x by B a l l s and co-workers i n 1937 but i s more c o n v e n i e n t l y i s o l a t e d from commercially d r i e d l a t e x by the procedure of Kimmel and Smith (1957), or by the method of Baines and B r o c k l e h u r s t (1979) i f d r i e d papaya l a t e x of high s o l u b i l i t y i s used. -114-Papain prepared u s i n g the mentioned procedures i s u s u a l l y present i n three forms: a c t i v e papain, r e v e r s i b l y o x i d i z e d and i r r e v e r s i b l y o x i d i z e d papain ( B r o c k l e h u r s t et a l . , 1981). A c t i v e papain (50%-80% of the t o t a l enzyme content) c o n t a i n s one f r e e c y s t e i n e group per molecule, t h a t of c y s t e i n e - 2 5 , which i s p a r t of the c a t a l y t i c s i t e of the enzyme. In r e v e r s i b l y o x i d i z e d papain, t h i s c a t a l y t i c s i t e -SH group i s l i n k e d i n a d i s u l f i d e bond with t h a t of a f r e e c y s t e i n e molecule, while i n i r r e v e r s i b l y o x i d i z e d papain, the c a t a l y t i c s i t e -SH group has been o x i d i z e d t o a s u l f i n i c a c i d group ( B r o c k l e h u r s t e t a l . , 1981) . F u l l y a c t i v e papain, c o n t a i n i n g one mole SH per mole of p r o t e i n , may be prepared by a f f i n i t y chromatography on columns of agarose to which G l y - G l y - T y r ( B z l ) - A r g (Blumberg et a l . , 1970), p-aminophenylmercuriacetate (Sluyterman and Widjenes, 1970), or g l u t a t h i o n e - 2 - p y r i d y l d i s u l f i d e were attached ( B r o c k l e h u r s t and L i t t l e , 1970). 2. Physicochemical p r o p e r t i e s and s t r u c t u r e Various p h y s i c a l p r o p e r t i e s of papain and three other s u l f h y d r y l p r o t e a s e s , are l i s t e d i n Table 12. G e n e r a l l y speaking, there does not appear to be any major d i f f e r e n c e i n the s i z e and charge d i s t r i b u t i o n s among the v a r i o u s enzymes. One of the most c h a r a c t e r i s t i c d i f f e r e n c e s i s the absence of carbohydrate i n papain and chymopapain (Arnon, 1970). -115-Table 12. Physicochemical p r o p e r t i e s of some c y s t e i n e p r o t e a s e s . P r o p e r t y Papain F i c i n Stem Bromelain* Chymopapain B Molecular weight 23,000 25,500 20,000 33,200 34,500 17 2- * B 25.00 21.00 19 .00 18 .40 I s o e l e c t r i c p o i n t 8.75 9.00 9 . 55 10.40 No. of amino a c i d s 212 248 179-285 318 % Carbohydrate 0 3.4 1.4-2.1 0 * Stem bromelain c o n s i s t s of v a r i o u s enzymes B at 280 nm Source: B r o c k l e h u r s t et a l . (1981) L i e n e r (1974) -116-The papain molecule i s composed of a s i n g l e p o l y p e p t i d e c h a i n of 212 amino a c i d r e s i d u e s , the sequence of which has been determined. S i x of the seven c y s t e i n e r e s i d u e s are engaged i n d i s u l f i d e bond formation, two being i n the f i r s t h a l f of the c h a i n (Cys-22-Cys-63 and Cys-56-Cys-95) and one i n the second h a l f (Cys-153-Cys-200). The seventh c y s t e i n e r e s i d u e (Cys-25) forms p a r t of the c a t a l y t i c s i t e ( B r o c k l e h u r s t et a l . , 1981). The s t r u c t u r a l conformation i s s t a b i l i z e d by the three d i s u l f i d e b r i d g e s . T h e i r rupture r e s u l t s i n l o s s of b i o l o g i c a l a c t i v i t y , c a t a l y t i c as w e l l as immunological (Shapira and Arnon, 1969). The high i s o l e c t r i c p o i n t (pl= 8.1) of papain suggests t h a t a high p r o p o r t i o n of the charged amino a c i d s are b a s i c . Around 18% of the t o t a l amino a c i d r e s i d u e s i n papain have charged groups, 7.1% a c i d i c ( Asp and Glu) and 11.3% b a s i c (Lys, His and A r g ) , which g i v e s a r a t i o of a c i d i c to b a s i c amino a c i d s of 0.63 (Yada, 1984). The average Bigelow h y d r o p h o b i c i t y f o r papain i s 1,159 c a l r e s i d u e - 1 , which i s s i m i l a r to other proteases such as chymosin, pepsin and t r y p s i n (Yada, 1984). H y d r o p h o b i c i t y values s i m i l a r to other proteases were a l s o found when t h i s parameter was measured with f l u o r e s c e n t probes (Yada, 1984). In i t s secondary s t r u c t u r e , papain has 28% h e l i x , 14% beta sheet, 17% beta t u r n and 41% random s t r u c t u r e (Chang et a l . , 1978). The three dimensional s t r u c t u r e of papain has been determined -117-by X-ray c r y s t a l l o g r a p h y to a r e s o l u t i o n of 0.28 nm. For a review of X-ray s t u d i e s of papain see Drenth et a l . (1971). The papain molecule i s e l l i p s o i d a l with approximate dimensions of 5.0 x 3.7 x 3.7 nm. I t i s a b i n u c l e a r p r o t e i n , c o n s t r u c t e d around two hydrophobic c o r e s . The two lobes c o n t a i n s the same number of amino a c i d r e s i d u e s and are separated by a c l e f t i n which the a c t i v e center r e g i o n l i e s ( B r o c k l e h u r s t et a l . , 1981). 3. S t a b i l i t y C r y s t a l l i n e papain shows a high degree of s t a b i l i t y . As c r y s t a l s i n suspension i n NaCl s o l u t i o n s , i t can be kept a t 4°C f o r months without d e t e c t a b l e l o s s i n a c t i v i t y (Arnon, 1970; B r o c k l e h u r s t et a l . , 1981). Papain i s an enzyme with high t h e r m o s t a b i l i t y ; the papain powder r e s i s t s d r y heat a t 100<>C f o r 3 hours (Arnon, 1970). Papain i n s o l u t i o n a l s o shows a remarkable temperature s t a b i l i t y , which i s pH dependent. The enzyme i s unstable under a c i d i c c o n d i t i o n s . Below pH 2.8 the enzyme s u f f e r s a d r a s t i c decrease i n a c t i v i t y (Arnon, 1970). Papain i s l e s s heat s t a b l e than chymopapain ( S k e l t o n , 1968). Papain i s u n a f f e c t e d by high c o n c e n t r a t i o n s of some d e n a t u r i n g agents such as methanol (up to 70%), dimethyl s u l f o x i d e (up to 20%), and urea (8 M) (Arnon, 1970). However exposure t o 10% t r i c h l o r o a c e t i c a c i d or 6 M guanidine h y d r o c h l o r i d e causes i r r e v e r s i b l e changes i n the s t r u c t u r e and a c t i v i t y of papain (Arnon, 1970). Since papain a c t i v i t y depends on a f r e e -SH group i t i s -118-expected t h a t a l l t h i o l reagents should a c t as i n h i b i t o r s . Papain i s i n a c t i v a t e d i n the presence of a i r and low c o n c e n t r a t i o n s of c y s t e i n e ( B r o c k l e h u r s t et a l . , 1981; Arnon, 1970). 4 . A c t i v i t y Papain i s a protease with broad s i d e c h a i n s p e c i f i c i t y , and w i l l degrade most p r o t e i n s u b s t r a t e s more e x t e n s i v e l y than t r y p s i n , pepsin or chymotrypsin, i n many cases g i v i n g r i s e to f r e e amino a c i d s (Arnon, 1970). Studie s on p a p a i n - c a t a l y z e d cleavage of alpha and beta chains of haemoglobin, o x i d i z e d chains of i n s u l i n and of glucagon i n d i c a t e t h a t peptide bonds of the type A-X-Y, i n which X-Y i s the s c i s s i l e bond and A i s Phe, V a l , l i e or Leu are c l e a v e d p r e f e r e n t i a l l y ( B r o c k l e h u r s t et a l . , 1981). In a d d i t i o n to the h y d r o l y s i s of peptide bonds, papain i s very e f f e c t i v e as an e s t e r a s e or t h i o l e s t e r a s e , and a l s o possesses t r a n s f e r a s e a c t i v i t y . Papain i s capable of c a t a l y z i n g not o n l y t r a n s a m i d a t i o n and t r a n s p e p t i d a t i o n , but t r a n s e s t e r i f i c a t i o n r e a c t i o n s as w e l l (Arnon, 1970). E. CHEMICAL MODIFICATION OF PAPAIN E x p l o r a t i o n of the a c t i v e s i t e of an enzyme by chemical m o d i f i c a t i o n f r e q u e n t l y p r o v i d e s important evidence about i t s p r o p e r t i e s (Lowe, 1976). Since papain i s a s u l f h y d r y l enzyme, -119-i t i s not s u r p r i s i n g t h a t most of the chemical m o d i f i c a t i o n s t u d i e s on t h i s p r o t e i n have concerned the m o d i f i c a t i o n of the e s s e n t i a l c y s t e i n e r e s i d u e (Cys-25). Many of the s t u d i e s may be c o n s i d e r e d more as k i n e t i c than chemical m o d i f i c a t i o n s t u d i e s , s i n c e more emphasis was placed on the k i n e t i c s of the i n a c t i v a t i o n or i n h i b i t i o n . A d i s c u s s i o n on the k i n e t i c s of i r r e v e r s i b l e i n h i b i t i o n ( i . e . i r r e v e r s i b l e m o d i f i c a t i o n ) i s a l s o given i n t h i s l i t e r a t u r e review (see s e c t i o n " I r r e v e r s i b l e i n h i b i t o r s " ) . 1. M o d i f i c a t i o n of Cys-25 M o d i f i c a t i o n of the Cys-25 group of papain has been c a r r i e d out with a v a r i e t y of a l k y l a t i n g agents (e.g. i o d o a c e t a t e , iodoacetamide, c h l o r o a c e t a t e ) . I r r e v e r s i b l e i n a c t i v a t i o n of the enzymes occurs by a l k y l a t i o n but i n most cases no observable changes i n s t r u c t u r e occur (Lowe, 1976; B r o c k l e h u r s t et a l . , 1981). Papain a l k y l a t e d with i o d o a c e t i c a c i d has been r e p o r t e d to have the same immunological i n t e r a c t i o n s with a n t i p a p a i n serum as n a t i v e papain. These r e s u l t s suggest t h a t v e r y l i t t l e , i f any, s t r u c t u r a l change occurred d u r i n g t h i s m o d i f i c a t i o n (Shapira and Arnon, 1969). O x i d a t i o n of the Cys-25 r e s i d u e of papain has been performed with a v a r i e t y of reagents; however, i n many cases o n l y the e f f e c t on the papain a c t i v i t y was r e p o r t e d . Since o x i d a t i o n per se causes l o s s e s i n the a c t i v i t y , i t i s d i f f i c u l t to i n d i c a t e -120-i f changes i n s t r u c t u r e occur upon chemical m o d i f i c a t i o n of papa i n . Reaction of papain with 2-bromo-2 1,4'dimethoxy acetophenone produced a d e r i v a t i v e t h a t was used to prepare modified papain molecules i n which c y s t e i n e - 2 5 was changed i n t o : (1) d ehydro-serine, (2) s e r i n e and (3) g l y c i n e (Clark and Lowe, 1978). S e r i n e - and dehydro-serine papain lacked e s t e r a s e a c t i v i t y , but possessed b i n d i n g p r o p e r t i e s to a f f i n i t y chromatography columns s i m i l a r to those of n a t i v e papain. These r e s u l t s i n d i c a t e t h a t the t e r t i a r y s t r u c t u r e of the n a t i v e enzyme has been preserved (Clark and Lowe, 1978). 2. M o d i f i c a t i o n of other amino a c i d r e s i d u e s Few s t u d i e s aimed at modifying other r e s i d u e s i n papain have been r e p o r t e d . In g e n e r a l , chemical m o d i f i c a t i o n of amino a c i d r e s i d u e s , other than the e s s e n t i a l c y s t e i n e r e s i d u e s , i n s u l f h y d r y l enzymes presents a d i f f i c u l t problem. Since the c y s t e i n e group l o c a t e d at the a c t i v e s i t e i s so r e a c t i v e , most reagents r e a c t with i t i n preference to any other r e s i d u e which might a l s o be a f u n c t i o n a l component of the a c t i v e s i t e ( G l e i s n e r and L i e n e r , 1973). In order to s o l v e t h i s problem, the technique of r e v e r s i b l e b l o c k i n g of the c y s t e i n e r e s i d u e with t e t r a t h i o n a t e has been used with success ( L i u , 1967; G l e i s n e r and L i e n e r , 1973). G l e i s n e r and L e i n e r (1973) modified the h i s t i d i n e r e s i d u e l o c a t e d at the a c t i v e s i t e of f i c i n . A f u l l y a c t i v e d e r i v a t i v e of f i c i n was prepared by -121-r e v e r s i b l y b l o c k i n g i t s a c t i v e t h i o l group with sodium t e t r a t h i o n a t e and by o x i d a t i o n of a methionine r e s i d u e with sodium metaperiodate. Treatment of t h i s a c t i v e d e r i v a t i v e with bromoacetone i n the presence of 2 M urea a t pH 6.5 r e s u l t e d i n i t s complete i n a c t i v a t i o n . The s o l e change i n i t s composition was the l o s s of one of i t s two h i s t i d i n e r e s i d u e s . M o d i f i c a t i o n of h i s t i d i n e r e s i d u e s by p h o t o o x i d a t i o n with methylene blue caused l o s s of the c a s e i n o l y t i c a c t i v i t y of papain and bromelain. D i s c r e p a n c i e s between the r a t e of the l o s s of p r o t e o l y t i c a c t i v i t y and t h a t of h i s t i d i n e r e s i d u e s suggests t h a t f a c t o r s other than o x i d a t i o n of h i s t i d i n e r e s i d u e s were i n v o l v e d i n the mechanism of i n a c t i v a t i o n (Murachi and Okumura, 1974). T e t r a t h i o n a t e was a l s o used i n t h i s experiment as a b l o c k i n g agent f o r the c y s t e i n e groups of papain and f i c i n . S u c c i n y l a t i o n of papain caused, as expected, a dramatic change i n the i s o l e c t r i c p o i n t ( p i ) , from 8.2 to 4.3. In s p i t e of the change i n p i , an a c t i v i t y assay with b e n z o y l - g l y c i n e e t h y l e s t e r demonstrated a l o s s of a c t i v i t y of o n l y 10% (Sluyterman and DeGraff, 1972). One of the most wi d e l y used reagents f o r the m o d i f i c a t i o n of tryptophan r e s i d u e s i s N-bromosuccinimide, which with an i n t a c t enzyme g e n e r a l l y o x i d i z e s the i n d o l e to an oxyindole r i n g and may subsequently brominate i t . I t i s known, however, t h a t t h i s reagent i s capable of modifying c y s t e i n e , methionine, t y r o s i n e and h i s t i d i n e r e s i d u e s (Lowe and Whitworth, 1974). In -122-a d e t a i l e d i n v e s t i g a t i o n , Lowe and Whitworth (1974) s t u d i e d the m o d i f i c a t i o n of tryptophan r e s i d u e s of papain by N-bromosuccinimide. The c y s t e i n e group of papain was p r o t e c t e d f i r s t , as a d i s u l f i d e with mercaptoethanol. The authors r e p o r t e d that a 2 molar e q u i v a l e n t of the reagent N-bromosuccinimide modified tryptophan-69, and a 4 molar e q u i v a l e n t m o d i f i e d tryptophan-69 and -177. The enzymic a c t i v i t y , measured with N - b e n z y l o x y c a r b o n y l g l y c i n e p - n i t r o p h e n y l e s t e r , was not s e r i o u s l y impaired. By c o n t r a s t , p h o t o o x i d a t i o n of tryptophan -177 and a d d i t i o n a l c o n v e r s i o n i n t o formylkynuremine, l e d to complete l o s s of enzymic a c t i v i t y toward N-a-benzoyl-L-arginine e t h y l e s t e r with a p p a r e n t l y o n l y minor changes i n papain conformation ( J o r i and G a l i a z z o , 1971). As mentioned b e f o r e , r e d u c t i o n of a l l the d i s u l f i d e bonds of papain r e s u l t e d i n t o t a l l o s s of a c t i v i t y i n papain. I t should be pointed out that i n order to o b t a i n a complete r e d u c t i o n of the three d i s u l f i d e bonds of papain, i t was necessary to incubate the enzyme with 0.34 M (3-mercaptoethanol p l u s 6 M guanidine h y d r o c h l o r i d e f o r three hours (Shapira and Arnon, 1969). I t was a l s o p o s s i b l e to s e l e c t i v e l y reduce o n l y one d i s u l f i d e bond of papain. In t h i s case, i t was necessary to incubate the p r o t e i n with the red u c i n g agent p l u s 8 M urea; 60% of the o r i g i n a l a c t i v i t y was r e t a i n e d (Shapira and Arnon, 1969). Reacting t h i s reduced papain with mercury caused the formation of an i n t r a m o l e c u l a r mercaptide bond -S-Hg-S-, i n s t e a d of the -123-o r i g i n a l d i s u l f i d e bond -S-S-. T h i s enzyme d e r i v a t i v e was e n z y m a t i c a l l y a c t i v e on a range of s u b s t r a t e s . The i n t e r n a l mercury atom, i n c o n t r a s t to the one found i n the a c t i v e s i t e s u l f h y d r y l , was r e t a i n e d i n the molecule under the c o n d i t i o n s r e q u i r e d f o r a c t i v a t i o n of papain (5 mM c y s t e i n e and 2 mM EDTA). The immunological r e a c t i v i t y of t h i s d e r i v a t i v e with a n t i p a p a i n serum was e s s e n t i a l l y i d e n t i c a l with that of n a t i v e papain (Shapira and Arnon, 1969). Mercury papain, papain with Cys-25 blocked with mercury, i s a commonly used d e r i v a t i v e of the enzyme. I t has been shown to have the same s t r u c t u r e as papain, and s i n c e i t i s r e v e r s i b l y i n a c t i v e , i t i s more s t a b l e d u r i n g storage than the n a t i v e papain molecule (Arnon, 1970). -124-F. ENZYME KINETICS 1. R e a c t i o n r a t e s The mathematical equations t h a t d e s c r i b e the changes i n c o n c e n t r a t i o n of the product or r e a c t a n t d u r i n g a chemical or enzymatic r e a c t i o n a t any time are c a l l e d r e a c t i o n r a t e s . The r e a c t i o n r a t e s most f r e q u e n t l y found i n enzymatic r e a c t i o n s a r e : zero, f i r s t and second order r e a c t i o n r a t e s (Whitaker, 1972). (a) Zero order r e a c t i o n s In a zero order r e a c t i o n the r a t e of disappearance of r e a c t a n t or r a t e of appearance of product i s independent of the c o n c e n t r a t i o n of r e a c t a n t . A monomolecular, i r r e v e r s i b l e zero order r e a c t i o n i s represented as: A - * ¥P (1) where A i s the r e a c t a n t and P i s the product. The r a t e of c o n v e r s i o n i s expressed mathematically f o r a zero order r e a c t i o n as: -dA/dt = k = dP/dt (2) By i n t e g r a t i n g Eq.(2) between the l i m i t s of s u b s t r a t e c o n c e n t r a t i o n a t zero time (A 0) and a t time t (A), the f o l l o w i n g equation i s o b t a i n e d : Ao-A = kt = P (3) The p l o t of l A o l - l A ) or IP] a g a i n s t time g i v e s a s t r a i g h t l i n e with i n t e r c e p t of zero and slope k. Zero order r e a c t i o n s with r e s p e c t to s u b s t r a t e c o n c e n t r a t i o n i n enzymatic r e a c t i o n s are encountered whenever the enzymes are s a t u r a t e d with s u b s t r a t e (Whitaker, 1972). (b) F i r s t order r e a c t i o n s In a f i r s t order r e a c t i o n the r a t e of the r e a c t i o n depends on the r e a c t a n t c o n c e n t r a t i o n and i s expressed mathematically as: -dA/dt = kA = dP/dt (4) I n t e g r a t i n g Eg.(4) between the l i m i t s of r e a c t a n t c o n c e n t r a t i o n of Ao a t time 0, and A a t time t , g i v e s the f o l l o w i n g equation: In A = In A 0 - kt (5) In enzyme-catalyzed r e a c t i o n s , the r a t e of r e a c t i o n i s f i r s t order with r e s p e c t to s u b s t r a t e c o n c e n t r a t i o n when the enzyme i s l e s s than 5% s a t u r a t e d with the s u b s t r a t e (Whitaker, 1972). (c) Second order r e a c t i o n s T h i s type of r e a c t i o n i s c h a r a c t e r i z e d as being dependent upon two molecules, e i t h e r of the same compound or of two d i f f e r e n t compounds. A second order r e a c t i o n can be represented as: A + B "s *P (6) M a t h e m a t i c a l l y the r a t e of r e a c t i o n of a second order r e a c t i o n i s expressed as: -dA/dt = -dB/dt = dP/dt = kAB (7) Depending on the i n i t i a l c o n c e n t r a t i o n of A and B, three types of second order r e a c t i o n s can be found (Whitaker, 1972): -126-I. Type I When the c o n c e n t r a t i o n of A i s equal to the c o n c e n t r a t i o n of B, the r e a c t i o n r a t e can be expressed as: -dA/dt = kA* (8) By i n t e g r a t i n g Eq.(8) under the c o n d i t i o n that a t t=0, [A]=[AoJ or [B]=[B 0] one o b t a i n s : 1/tA] - 1/[A 0] = kt or 1/[B] - l / [ B o ] = kt (9) Eq.(9) i s wid e l y used to determine the second order r a t e constant of the r e a c t i o n of an enzyme with an i r r e v e r s i b l e i n h i b i t o r compound. The second order r a t e c o n s t a n t , k (M _ I s " 1 ) , i s obtained by p l o t t i n g the data a c c o r d i n g t o Eq.(9 ) . I I . Type II When the c o n c e n t r a t i o n s of the r e a c t a n t A and B are s i m i l a r , the r e a c t i o n r a t e i s expressed as Eq.(7). By i n t e g r a t i n g Eq.(7) and e x p r e s s i n g the equation i n terms of i n i t i a l r e a c t a n t c o n c e n t r a t i o n we o b t a i n (Whitaker, 1972): kt = 2.3/( [ B o l - I A o l ) log{ ( [ A o]/[B 0] ) * ( [ B a ] - [ P ] ) / ( [ A A ] - [ P ] ) } (10) Eq.(lO) i s a l s o commonly used to determined the second order r a t e constant of i n a c t i v a t i o n of an enzyme with an i r r e v e r s i b l e i n h i b i t o r (Tonomura e t a l . , 1985). I I I . Type I I I When the c o n c e n t r a t i o n of one of the r e a c t a n t s i s r e l a t i v e l y high i n r e l a t i o n to the other r e a c t a n t , i t can be assumed that the c o n c e n t r a t i o n of the r e a c t a n t present i n high c o n c e n t r a t i o n i s constant over the course of the r e a c t i o n and the second order r a t e equation can be s i m p l i f i e d t o the f i r s t order r e a c t i o n r a t e equation ( E q . ( 5 ) ) . 2. S t a t e s of an enzymatic r e a c t i o n Enzyme c a t a l y z e d r e a c t i o n s f o l l o w the progress curve shown i n F i g . 21. G e n e r a l l y three s t a t e s f o r the o v e r a l l time course of an enzyme r e a c t i o n can be r e c o g n i z e d : Phase I : The pre-steady s t a t e or t r a n s i e n t i n i t i a l phase, a few seconds i n d u r a t i o n , Phase II : A s t e a d y - s t a t e phase, Phase I I I : A n o n l i n e a r phase up to completion. (a) The pre-steady s t a t e The i n i t i a l p a r t of the curve where the v e l o c i t y , or r a t e of the r e a c t i o n , expressed as dP/dt, i s i n c r e a s i n g with time i s c a l l e d the pre-steady s t a t e . A c c o r d i n g t o the Michaelis-Menten theory (see s e c t i o n " E f f e c t of s u b s t r a t e c o n c e n t r a t i o n on the i n i t i a l v e l o c i t y " ) , i t i s the time r e q u i r e d to e s t a b l i s h the e q u i l i b r i u m between the formation and breakdown of the enzyme-s u b s t r a t e complex. Since the formation of the enzyme-substrate complex i s an a d s o r p t i v e phenomena t h a t i s c o n t r o l l e d by d i f f u s i o n , the pre-steady s t a t e can o n l y be s t u d i e d by s p e c i a l techniques such as stopped-flow or p e r t u r b a t i o n methods (Engel, 1984). -128-[Product] phase Reaction time F i g u r e 21. Progress r e a c t i o n curve f o r an i d e a l enzyme r e a c t i o n . -129-(b) The steady s t a t e In t h i s p a r t of the r e a c t i o n a l l the r e a c t a n t s are i n dynamic e q u i l i b r i u m and t h e r e f o r e operate at maximum e f f i c i e n c y . I f the i n i t i a l s u b s t r a t e c o n c e n t r a t i o n i s high enough to s a t u r a t e the enzyme, there w i l l be a p e r i o d i n which the r e a c t i o n r a t e with r e s p e c t to time i s zero order, i . e . dP/dt = k. The l e n g t h of t h i s p e r i o d w i l l depend, among other t h i n g s , on the i n i t i a l s u b s t r a t e c o n c e n t r a t i o n ( F u l l b r o o k , 1983). (c) The n o n l i n e a r s t a t e As the r e a c t i o n proceeds the i n i t i a l r e a c t i o n v e l o c i t y decreases, even i f complete enzyme a c t i v i t y i s r e t a i n e d . T h i s i s due to the r e a c t i o n approaching i t s e q u i l i b r i u m p o i n t and the reverse r e a c t i o n beginning to operate ( F u l l b r o o k , 1983). The i n t e g r a t e d form of the Michaelis-Menten equation can be used to d e s c r i b e the three s t a t e s , assuming t h a t the enzyme maintains i t s f u l l a c t i v i t y d u r i n g the whole course of the r e a c t i o n ( F u l l b r o o k , 1983). 3. Measurement of v e l o c i t y of enzyme c a t a l y z e d r e a c t i o n s Since the r e a c t i o n v e l o c i t y of an enzyme-catalyzed r e a c t i o n i s not constant over the e n t i r e r e a c t i o n time, the r a t e most f r e q u e n t l y determined i s the i n i t i a l r a t e , or i n i t i a l v e l o c i t y of the r e a c t i o n (Ainsworth, 1977). T h i s method i n v o l v e s d e t e r m i n a t i o n of r a t e of the r e a c t i o n as c l o s e to time zero as p o s s i b l e . T h i s method has the f o l l o w i n g advantages: (a) i t i s -130-u n a f f e c t e d by i n s t a b i l i t y of the enzyme, (b) i t i s u n a f f e c t e d by the product c o n c e n t r a t i o n s i n c e t h i s i s zero, and (c) s u b s t r a t e c o n c e n t r a t i o n can be taken as that i n i t i a l l y added to the r e a c t i o n (Whitaker, 1972). (a) E f f e c t of s u b s t r a t e c o n c e n t r a t i o n on the i n i t i a l v e l o c i t y Many f a c t o r s a f f e c t the i n i t i a l v e l o c i t y of an enzyme c a t a l y z e d r e a c t i o n : enzyme and s u b s t r a t e c o n c e n t r a t i o n , pH, temperature, i o n i c s t r e n g t h and the presence of a c t i v a t o r s or i n h i b i t o r s . From a k i n e t i c s p o i n t of view and a t a constant enzyme c o n c e n t r a t i o n , s u b s t r a t e c o n c e n t r a t i o n i s one of the most important f a c t o r s which determine the v e l o c i t y of an enzymatic r e a c t i o n (Dixon and Webb, 1964). In most cases when the i n i t i a l v e l o c i t y i s p l o t t e d a g a i n s t s u b s t r a t e c o n c e n t r a t i o n a s e c t i o n of a r e c t a n g u l a r hyperbola i s obtained, as shown i n F i g . 22. The shape of the curve i s t y p i c a l of a process t h a t depends on a simple d i s s o c i a t i o n ( F u l l b r o o k , 1983). A theory i n v o l v i n g d i s s o c i a t i o n of t h i s type was put forward i n 1913 by M i c h a e l i s and Menten and has been the foundation of the g r e a t e r p a r t of enzyme k i n e t i c s . T h i s t h e o r y assumes t h a t the enzyme (E) forms a complex (ES) with the s u b s t r a t e (S) and t h a t the complex (ES) d i s s o c i a t e s i n t o the f r e e enzyme (E) and the end product (P) of the enzyme r e a c t i o n (Dixon and Webb, 1964). E + S — »ES (11) ES^ »E + P (12) -131-Vmax Figure 22. T y p i c a l graph o£ the i n i t i a l v e l o c i t y of an enzymatic r e a c t i o n as a f u n c t i o n of i n i t i a l s u b s t r a t e c o n c e n t r a t i o n . - 1 3 2 -The d e r i v a t i z a t i o n of the Michaelis-Menten equation can be found i n many biochem i c a l textbooks (e.g. Whitaker, 1972). The Michaelis-Menten equation i s the equation of a r i g h t hyperbola and d e s c r i b e s the data as p l o t t e d i n F i g . 22: v = Vmax [ S ] / (Km +[S]) (13) v = i n i t i a l v e l o c i t y [S ] = i n i t i a l s u b s t r a t e c o n c e n t r a t i o n Vmax = maximum v e l o c i t y Km = M i c h a e l i s constant The M i c h a e l i s constant (Km) can be d e f i n e d as the value of the s u b s t r a t e c o n c e n t r a t i o n t h a t g i v e s an i n i t i a l v e l o c i t y equal to h a l f the maximum v e l o c i t y at t h a t enzyme c o n c e n t r a t i o n . Thus i t i s a measure of the a f f i n i t y of the enzyme f o r the s u b s t r a t e ; the sm a l l e r the value of Km the higher the a f f i n i t y t h a t the enzyme shows f o r the s u b s t r a t e ( F u l l b r o o k , 1983). The Km of enzymes range wi d e l y but f o r most i n d u s t r i a l l y used enzymes i t l i e s i n the range of 10"1 to 10"9 M ( F u l l b r o o k , 1983). Km i s independent of enzyme c o n c e n t r a t i o n and i s a tr u e c h a r a c t e r i s t i c of the enzyme f o r a s p e c i f i c s u b s t r a t e under d e f i n e d c o n d i t i o n s of temperature, pH and i o n i c s t r e n g t h (Dixon and Webb, 1964). (b) Determination of Km and Vmax According to Eq.(13) a t a given i n i t i a l s u b s t r a t e c o n c e n t r a t i o n , two parameters, Km and Vmax, d e f i n e the i n i t i a l v e l o c i t y of an enzymatic r e a c t i o n . In order to c a l c u l a t e Km and Vmax, i n i t i a l v e l o c i t i e s at d i f f e r e n t s u b s t r a t e c o n c e n t r a t i o n s -133-must be found. G. DETERMINATION OP INITIAL VELOCITIES Determination of acc u r a t e i n i t i a l v e l o c i t i e s i s a p r e r e q u i s i t e f o r the c a l c u l a t i o n of accurate Michaelis-Menten parameters of an enzymatic r e a c t i o n (Durance et a l . , 1986). The most common method to estimate i n i t i a l v e l o c i t i e s i s the fi x e d - t i m e assay. In t h i s method the r e a c t i o n i s s t a r t e d and at a predetermined time, a sample i s analyzed f o r product or s u b s t r a t e c o n c e n t r a t i o n . Since i t i s assumed t h a t the i n i t i a l v e l o c i t y i s constant over the p r e d e f i n e d i n i t i a l time, meaning t h a t the r e a c t i o n i s zero order with r e s p e c t to time, the i n i t i a l v e l o c i t y , Vo i s c a l c u l a t e d by: (So- S ) / ( t - t 0 ) = Vo (14) where So i s the i n i t i a l s u b s t r a t e c o n c e n t r a t i o n and S i s the s u b s t r a t e c o n c e n t r a t i o n a t time t . The main advantage of t h i s method i s i t s s i m p l i c i t y . However, estimates of the i n i t i a l v e l o c i t i e s tend to be i n a c c u r a t e even when the t r a c e s are almost s t r a i g h t ( A t k i n s and Nimmo, 1980). When two v a r i a b l e s are mutually c o r r e l a t e d (e.g. r e a c t i o n time and product c o n c e n t r a t i o n ) l i n e a r i z a t i o n through data t r a n s f o r m a t i o n i s q u i t e u s e f u l ( P u j i i and Nakai, 1980). P u j i i and Nakai (1980) developed a procedure f o r data t r a n s f o r m a t i o n f o r l i n e a r i z a t i o n and suggested t h a t t h i s t r a n s f o r m a t i o n c o u l d be used t o f i n d the r e a c t i o n order which best f i t t e d the r e a c t a n t --134-product r e l a t i o n s h i p d u r i n g the course of the r e a c t i o n . Durance et a l . (1986) a p p l i e d t h i s technique, with e x c e l l e n t r e s u l t s , to determine r e a c t i o n orders f o r h y p o t h e t i c a l models and k i n e t i c parameters with reduced standard e r r o r s f o r an enzymatic h y d r o l y s i s . T h i s method has a l s o been a p p l i e d i n s h e l f l i f e s t u d i e s of corn products (Arteaga, 1988). The l i n e a r i z a t i o n method i s as f o l l o w s : f o r a monomolecular, i r r e v e r s i b l e r e a c t i o n the r a t e of disappearance of a s u b s t r a t e can be expressed as: dS/dt = kS" (15) where S i s the s u b s t r a t e c o n c e n t r a t i o n , t i s r e a c t i o n time, n i s the order of the r e a c t i o n , and k i s the r a t e c o n s t a n t . By i n t e g r a t i n g Eq.(15) the f o l l o w i n g equations are obtained (Durance et a l . , 1986): S 1"" = S o 1 - " + ( n - l ) k t f o r n f 1 (16) InS = InSo - kt f o r n = 1 (17) The dependent v a r i a b l e s of these equations ( S 1 - " and InS) are transformed a c c o r d i n g to Durance et a l . (1986) t o : Y B = So 1"" + ( n - l ) k t f o r n ? 1 (18) lnY = InSo - kt f o r n = 1 (19) B values are s e l e c t e d , using the computer program r e p o r t e d by Durance et a l . (1986) such t h a t the c o e f f i c i e n t of d e t e r m i n a t i o n of the l i n e Y B a g a i n s t time i s maximized. The r e a c t i o n order (n) i s then c a l c u l a t e d from: n = 1-B (20) The value of the r a t e constant k i s taken as the slope of the -135-equation f o r the l i n e a r i z e d data c a l c u l a t e d by l i n e a r r e g r e s s i o n . By o b t a i n i n g the d e r i v a t i v e of t h i s l i n e a r i z e d equation, and e q u a l i z i n g i t to zero, the value of the i n i t i a l v e l o c i t y i s obtained. A f t e r i n i t i a l v e l o c i t i e s have been c a l c u l a t e d at d i f f e r e n t i n i t i a l s u b s t r a t e c o n c e n t r a t i o n s , d i f f e r e n t methods can be used to c a l c u l a t e Km and Vmax. For best r e s u l t s , i n i t i a l v e l o c i t i e s should be obtained a t 6 to 10 i n i t i a l s u b s t r a t e c o n c e n t r a t i o n s ranging from 0.1 to 10 Km. A t k i n s and Nimmo (1980) reviewed the present methods f o r e s t i m a t i o n of Michaelis-Menten parameters. The Michaelis-Menten equation (Eq.(13)) may be p l o t t e d i n s e v e r a l d i f f e r e n t ways f o r the det e r m i n a t i o n of Vmax and Km from a s e t of measurements of v e l o c i t y at d i f f e r e n t s u b s t r a t e c o n c e n t r a t i o n s . The most common g r a p h i c a l methods are the Lineweaver-Burk method and the Hofstee method. A d e t a i l e d d i s c u s s i o n of these methods can be found i n many biochemical textbooks. More r e c e n t l y , n o n - l i n e a r r e g r e s s i o n a n a l y s i s ( O e s t r e i c h e r and P i n t o , 1983; Lutz et a l . , 1986) has been used to c a l c u l a t e Km and Vmax more a c c u r a t e l y . H. NITROPHENYL ESTERS AS SUBSTRATES FOR PAPAIN N i t r o p h e n y l e s t e r d e r i v a t i v e s of s p e c i f i c compounds are commonly used s u b s t r a t e s i n enzyme k i n e t i c s experiments (Whitaker, 1972). Since the n i t r o p h e n y l e s t e r i s a good l e a v i n g -136-group and the c o n c e n t r a t i o n of n i t r o p h e n o l l i b e r a t e d can be e a s i l y determined s p e c t r o p h o t o m e t r i c a l l y at d i f f e r e n t pH's, the ra t e of h y d r o l y s i s can be followed c o n t i n u o s l y . R e c e n t l y the c o n s t r u c t i o n of a p - n i t r o p h e n o l a t e s e n s i t i v e e l e c t r o d e and i t s a p p l i c a t i o n to an enzyme assay was re p o r t e d (Katsu et a l . , 1987). Some of the most common a r t i f i c i a l s u b s t r a t e s used i n k i n e t i c s t u d i e s of papain are the n i t r o p h e n y l e s t e r s of carbobenzoxyglycine. Both the pre-steady and steady s t a t e k i n e t i c s f o r the papain c a t a l y z e d h y d r o l y s i s of t h i s n i t r o p h e n y l are w e l l documented (Ascenzi e t a l . , 1987; K i r s c h and Ingelstrom, 1966). 1. Steady s t a t e k i n e t i c s f o r the papain c a t a l y z e d  h y d r o l y s i s of carbobenzoxyglycine K i r s c h and Ingelstrom (1966) s t u d i e d i n d e t a i l the steady s t a t e k i n e t i c s of the p a p a i n - c a t a l y z e d h y d r o l y s i s of e s t e r s of carbobenzoxyglycine. They concluded t h a t the r e a c t i o n of papain with the p - n i t r o p h e n y l e s t e r p r o v i d e s a ve r y s e n s i t i v e and convenient assay f o r enzyme a c t i v i t y . The r e a c t i o n i s f a s t , t a k i n g 1 min to complete, i s e a s i l y followed s p e c t r o p h o t o m e t r i c a l l y , r e q u i r e s very l i t t l e enzyme (about 20Ug per a s s a y ) , and the high s o l u b i l i t y of the s u b s t r a t e makes i t p o s s i b l e t o work at s a t u r a t i n g c o n d i t i o n s . Moreover, the compound i s commercially a v a i l a b l e a t a reasonable p r i c e . However, s i n c e n i t r o p h e n y l e s t e r assays are i n h e r e n t l y u n s p e c i f i c , the use of t h i s s u b s t r a t e i s l i m i t e d to f a i r l y pure enzyme p r e p a r a t i o n s ( K i r s c h and Ingelstrom, 1966). -137-K i r s c h and Ingelstrom (1966) determined values of Km and Vmax f o r the h y d r o l y s i s of p - n i t r o p h e n y l , m-nitrophenyl, o - n i t r o p h e n y l and e t h y l e s t e r s of carbobenzoxyglycine. I n i t i a l v e l o c i t i e s were estimated using f i x e d time assays. According to the k i n e t i c data r e p o r t e d , those authors proposed t h a t enzyme and s u b s t r a t e formed a noncovalent complex i n the f i r s t stage of the r e a c t i o n , which was followed by an a c y l a t i o n step with a r a t e constant dependent upon the r e a c t i v i t y of the s u b s t r a t e . The f i n a l r e a c t i o n was the d e a c y l a t i o n of the enzyme. S i m i l a r r e s u l t s have a l s o been re p o r t e d by other r e s e a r c h e r s (Hubbard and K i r s c h , 1968; Hollaway and Hardman, 1973). Values obtained by d i f f e r e n t r e s e a r c h e r s f o r the k i n e t i c parameters Km, Vmax and Kcat (= Vmax/enzyme c o n c e n t r a t i o n ) f o r the p a p a i n - c a t a l y z e d h y d r o l y s i s of p-n i t r o p h e n y l e s t e r of carbobenzoxyglycine c a t a l y z e d are shown i n Table 13. 2. Pre-steady s t a t e k i n e t i c s f o r the papain c a t a l y z e d  h y d r o l y s i s of carbobenzoxyglycine In a recent a r t i c l e , A scenzi et a l . (1987) r e p o r t e d the steady and pre-steady s t a t e of the papain c a t a l y z e d h y d r o l y s i s of a p - n i t r o p h e n y l e s t e r of carbobenzoxyglycine over the pH range of 3 to 9.5 at 21°C. T h e i r r e s u l t suggested a minimum t h r e e - s t e p r e a c t i o n mechanism, i n v o l v i n g an a c y l enzyme i n t e r m e d i a t e . The pH p r o f i l e of the k i n e t i c parameters r e f l e c t e d the i o n i z a t i o n of two groups with pK values of 4.5+0.1 and 8.8±0.15 i n the f r e e enzyme. -138-Table 13. Michaelis-Menten parameters f o r the papain c a t a l y z e d h y d r o l y s i s of carbobenzoxyglycine p - n i t r o p h e n y l ester*-. Reference Km (UM) K c « B ,(s- 1) K i r s c h and Ingelstrom (1966) 13.0 2.73 A l e c i o et a l . (1974) 8.3 8.00 L i n et a l . (1975) 16.0 14.00 Ascenzi et a l . (1987) 16.0 12.0 13.0 3.16 *-The r e p o r t e d range of c o n d i t i o n s used d u r i n g the k i n e t i c s experiments were: temperature: 21-25°C b u f f e r : 0.02-0.1 M phosphate pH: 6-7 BKC»T = Vmax/[E] -139-I. ENZYME INHIBITION 1. D e f i n i t i o n and c l a s s i f i c a t i o n Any substance which reduces the v e l o c i t y of an enzyme-catalyzed r e a c t i o n , by whatever mechanism, i s an i n h i b i t o r (Whitaker, 1972). I n h i b i t o r s i n f l u e n c e the r a t e of the enzyme-catalyzed r e a c t i o n e i t h e r by b l o c k i n g the a c t i v e s i t e , or by b r i n g i n g about a change i n the s t r u c t u r e a f f e c t i n g the e f f i c i e n c y of the a c t i v e s i t e (Engel, 1984). Based on k i n e t i c c o n s i d e r a t i o n s i n h i b i t o r s can be c l a s s i f i e d i n t o two groups: i r r e v e r s i b l e and r e v e r s i b l e i n h i b i t o r s . The i n t e r e s t i n enzyme i n h i b i t o r s f o r c y s t e i n e proteases has grown s i g n i f i c a n t l y i n recent years, due to the f a c t t h a t some enzymes i n t h i s f a m i l y are d e g r a d a t i v e agents i n c e r t a i n d i s e a s e s t a t e s , such as muscular dystrophy, some forms of heart d i s e a s e and cancer. I n h i b i t o r s to these enzymes are t h e r e f o r e of pharmaceutical i n t e r e s t (NRC Canada, 1988; 1987). (a) I r r e v e r s i b l e i n h i b i t o r s T h i s group i n c l u d e s a l l compounds t h a t r e a c t with an enzyme to form s t a b l e c o v a l e n t bonds (Whitaker, 1972). Reagents such as i o d o a c e t i c a c i d and f l u o r o d i n i t r o b e n z e n e , whose a c t i o n i s to combine c h e m i c a l l y with s p e c i f i c amino a c i d r e s i d u e s of the enzyme, are c l a s s i c a l examples of i r r e v e r s i b l e i n h i b i t o r s (Dixon and Webb, 1964). Even though the c o v a l e n t bond formed between enzyme and i n h i b i t o r may i n some cases be r e v e r s e d by chemical -140-treatment, from a s t r i c t l y k i n e t i c p o i n t of view the i n h i b i t i o n i s c o n s i d e r e d i r r e v e r s i b l e . In some cases the i n h i b i t o r s bind so t i g h t l y to the enzyme t h a t i r r e v e r s i b l e i n h i b i t i o n k i n e t i c s can be used i n order t o c h a r a c t e r i z e the mode of i n h i b i t i o n (Ainsworth, 1977). I. K i n e t i c s of i r r e v e r s i b l e i n h i b i t i o n As mentioned by Tsou et a l . (1985), i n most textbooks of enzyme k i n e t i c s , a t t e n t i o n i s always focused on the e f f e c t of r e v e r s i b l e i n h i b i t o r s on the s t e a d y - s t a t e k i n e t i c s of enzyme c a t a l y s i s . The k i n e t i c s of the b i n d i n g of i r r e v e r s i b l e i n h i b i t o r s with the enzyme molecule u s u a l l y r e c e i v e l i t t l e more than p a s s i n g mention. I r r e v e r s i b l e i n h i b i t i o n can be seen as a second order i r r e v e r s i b l e r e a c t i o n , where the r a t e of a s s o c i a t i o n or b i n d i n g i s the second order r a t e constant (Whitaker, 1972). For a simple one-to-one b i n d i n g i r r e v e r s i b l e r e a c t i o n : E + I S ».EI (21) where E i s an enzyme, I i s the i n h i b i t o r , and EI i s a b i n a r y complex, the r a t e of the i n h i b i t i o n may be w r i t t e n as: -dE/dt = - d l / d t = dEI/dt = k [ E ] [ I ] (22) By i n t e g r a t i n g Eg.(22) under the c o n d i t i o n s t h a t a t t=0, [I ] = [ I o l or [E]=[E 0] one obt a i n s (Tonomura e t a l . , 1985; Chung and Fo l k , 1970): k t = l / ( [ I o)-[E 0] ) ln{( [ E 0 ] / [ I o ] )*( ( [ I o ] - [ E o ] ) + ( [ E t = ] / [ E o ] ) ) } (23) -141-In the case of t E o l = t I o l Eq.(23) i s s i m p l i f i e d t o : l / [ E t ] - l / [ E o ] = kt or l / [ I t ] - l / [ I o l = kt (24) U s u a l l y the amount of f r e e enzyme a t d i f f e r e n t time i n t e r v a l s [ E t ] , i s determined by enzyme a c t i v i t y (Tonomura et a l . , 1985). Since, i n enzyme k i n e t i c s t u d i e s , a c t i v i t y ( i . e . v e l o c i t y ) i s normally p r o p o r t i o n a l to the c o n c e n t r a t i o n of f r e e enzyme. Eq.(24) can a l s o by expressed i n terms of a c t i v i t i e s (Tonomura et a l . , 1985; B a r r e t t et a l . , 1982): l / a t - l / a 0 = kt (25) where a t i s a c t i v i t y at t time, and a 0 i s a c t i v i t y at zero time. If the c o n c e n t r a t i o n of i n h i b i t o r i s much g r e a t e r than the enzyme c o n c e n t r a t i o n ( [ I 0 ] >> [ E 0 ] ) , the r e a c t i o n can be assumed to be f i r s t order and the p s e u d o - f i r s t order r a t e constant, k o B a , can be c a l c u l a t e d a c c o r d i n g to the f o l l o w i n g equation: — K . O B 3 t= l n ( [ E t ] / [ E 0 ] ) (26) or i n terms of a c t i v i t i e s : In ( a t / a 0 ) (27) The apparent second order r a t e constant f o r i n a c t i v a t i o n , k, i s c a l c u l a t e d as ( B a r r e t t et a l . , 1982): K — NOBS / [ I o ] (28) where [ I o ] i s the i n i t i a l i n h i b i t o r c o n c e n t r a t i o n . When r e a c t i o n of an i r r e v e r s i b l e i n h i b i t o r with an enzyme i n v o l v e s a group near the a c t i v e s i t e of the enzyme, the presence of s u b s t r a t e (or a com p e t i t i v e i n h i b i t o r ) may change the r a t e of r e a c t i o n of i n h i b i t o r with the enzyme. In most cases, the ra t e of r e a c t i o n w i l l decrease as s u b s t r a t e c o n c e n t r a t i o n i s increased to -142-s a t u r a t i o n (Whitaker, 1972). T h i s concept of i n h i b i t i o n c o m p e t i t i o n can e q u a l l y be a p p l i e d to both r e v e r s i b l e and i r r e v e r s i b l e i n h i b i t o r s (Tsou et a l . , 1985). R e v e r s i b l e c o m p e t i t i v e , noncompetitive and uncompetitive i n h i b i t o r s are d i s t i n g u i s h e d by t h e i r e f f e c t s on Km and Vmax (see s e c t i o n R e v e r s i b l e i n h i b i t i o n ) . S i m i l a r c r i t e r i a can a l s o be d e r i v e d to d i s t i n g u i s h three types of i r r e v e r s i b l e i n h i b i t o r s from the e f f e c t of s u b s t r a t e c o n c e n t r a t i o n on the apparent r a t e constant of b i n d i n g of i r r e v e r s i b l e i n h i b i t o r s to the enzyme (Tsou et a l . , 1985) . C o n d i t i o n s to d i s t i n g u i s h three types of i r r e v e r s i b l e i n h i b i t o r s are of the same type used f o r r e v e r s i b l e i n h i b i t o r s . Whereas e q u i l i b r i u m constants are used to d i s t i n g u i s h the three types of r e v e r s i b l e i n h i b i t i o n , r a t e constants are used i n i r r e v e r s i b l e i n h i b i t i o n (Tsou et a l . , 1985). According to Tsou et a l . (1985) p l o t s of s u b s t r a t e c o n c e n t r a t i o n a g a i n s t the i n v e r s e of the second order r a t e constant can d i s t i n g u i s h these three types of i n h i b i t i o n . For competitive i n h i b i t i o n , the p l o t of 1/k a g a i n s t s u b s t r a t e c o n c e n t r a t i o n w i l l be a s t r a i g h t l i n e . For noncompetitive i n h i b i t i o n , the apparent r a t e constant i s the true r a t e constant and i s independent of s u b s t r a t e c o n c e n t r a t i o n . For uncompetitive i n h i b i t i o n , a p l o t of 1/k as a f u n c t i o n of 1/tS] w i l l be a s t r a i g h t l i n e . -143-I I . I r r e v e r s i b l e i n h i b i t o r s o£ s u l f h y d r y l enzymes A l l reagents f o r chemical m o d i f i c a t i o n of s u l f h y d r y l groups (e.g. DTNB, 2PDS, t e t r a t h i o n a t e , a l k y l a t i n g agents) a r e , from a k i n e t i c p o i n t of view, i r r e v e r s i b l e i n h i b i t o r s of s u l f h y d r y l enzymes. They have a l r e a d y been d i s c u s s e d (see the s e c t i o n " M o d i f i c a t i o n of s u l f h y d r y l groups i n p r o t e i n s " ) . An i n t e r e s t i n g i r r e v e r s i b l e i n h i b i t o r of s u l f h y d r y l enzymes i s the compound E-64, L - ( t r a n s ) - e p o x y s u c c i n y l - l e u c y l a m i d o ( 4 -guanidino) butane. T h i s i n h i b i t o r was f i r s t i s o l a t e d from the e x t r a c t of a s o l i d c u l t u r e of A s p e r g i l l u s i a p o n i c u s TPR-64 (Hanada e t a l . , 1978). Since t h i s compound combines i n an eguimolecular and i r r e v e r s i b l e form with the e s s e n t i a l -SH of papain and other s u l f h y d r y l proteases ( B a r r e t t et a l . , 1982; Hanada et a l . , 1978), i t has been used f o r a c t i v e - s i t e t i t r a t i o n , to determine the o p e r a t i o n a l m o l a r i t y of enzyme s o l u t i o n s , thus c a l i b r a t i n g r a t e assays (Ascenzi et a l . , 1987; Zucker et a l . , 1985; B a r r e t t et a l . , 1982). (b) R e v e r s i b l e i n h i b i t i o n Since many bio c h e m i c a l textbooks d i s c u s s i n d e t a i l the k i n e t i c s of r e v e r s i b l e i n h i b i t i o n , o n l y a b r i e f d i s c u s s i o n w i l l be presented here. R e v e r s i b l e i n h i b i t o r s form noncovalent complexes with the enzyme. They can be removed from an enzyme by d i a l y s i s , chromatography, e t c . , with complete r e s t o r a t i o n of the u n i n h i b i t e d r e a c t i o n r a t e (Engel, 1984). -144-I. K i n e t i c s of r e v e r s i b l e i n h i b i t i o n Three b a s i c types of r e v e r s i b l e i n h i b i t o r s are r e c o g n i z e d . They are c a l l e d c o m p e t i t i v e , noncompetitive and uncompetitive i n h i b i t o r s and they are d i s t i n g u i s h e d from one another by t h e i r e f f e c t s on the v e l o c i t y of the enzyme c a t a l y z e d r e a c t i o n when measured as a f u n c t i o n of a p a r t i c u l a r s u b s t r a t e c o n c e n t r a t i o n (Ainsworth, 1977). For the schematic r e a c t i o n : E + S* ES I-EH. + P (29) K - i I t has been found t h a t : a) Any i n h i b i t o r t h a t d i s p l a c e s the e q u i l i b r i u m E + S« *ES m o d i f i e s the value of Km. b) Any i n h i b i t o r t h a t changes the maximum c o n c e n t r a t i o n of ES m o d i f i e s Vmax. I I . Competitive i n h i b i t i o n T h i s i s the s i m p l e s t case of r e v e r s i b l e i n h i b i t i o n . The assumption here i s t h a t b i n d i n g of the i n h i b i t o r s and b i n d i n g of the s u b s t r a t e are mutually e x c l u s i v e (Engel, 1984). The b a s i s f o r t h i s i s t h a t the i n h i b i t o r has s t r u c t u r a l f e a t u r e s s u f f i c i e n t l y s i m i l a r to those of the s u b s t r a t e to enable i t to occupy a l l or p a r t of the s u b s t r a t e b i n d i n g s i t e . Since s u b s t r a t e a t i n f i n i t e c o n c e n t r a t i o n must be able to compete s u c c e s s f u l l y with i n h i b i t o r s at f i n i t e c o n c e n t r a t i o n , the maximum r a t e (Vmax) i s u n a l t e r e d by a f i x e d c o n c e n t r a t i o n of i n h i b i t o r . -145-I I I . Uncompetitive i n h i b i t i o n T h i s i s the opposite of c o m p e t i t i v e i n h i b i t i o n , s i n c e the assumption i s t h a t the i n h i b i t o r can bind o n l y to the enzyme-substrate complex (Engel, 1984). The i n h i b i t o r promotes b i n d i n g between the enzyme and s u b s t r a t e , thus d e c r e a s i n g Km. Once the enzyme-substrate complex i s formed, i t may be e i t h e r p r o d u c t i v e (form product) or non-productive (binds i n h i b i t o r ) . (Engel, 1984). IV. Noncompetitive i n h i b i t i o n In t h i s case, the i n h i b i t o r can bind with equal ease to e i t h e r the f r e e enzyme or to the enzyme-substrate complex, but the e n z y m e - s u b s t r a t e - i n h i b i t o r complex i s i n a c t i v e . Since s u b s t r a t e and i n h i b i t o r do not compete with each other f o r the same b i n d i n g s i t e on the enzyme, i n h i b i t i o n cannot be e l i m i n a t e d by adding more s u b s t r a t e , as i s the case with a c o m p e t i t i v e i n h i b i t o r (Whitaker, 1972) V. R e v e r s i b l e i n h i b i t o r s of s u l f h y d r y l enzymes Many r e v e r s i b l e i n h i b i t o r s of c y s t e i n e proteases have been r e p o r t e d . Urea and guanidine h y d r o c h l o r i d e , at low c o n c e n t r a t i o n s , i n a c t i v a t e papain i n a noncompetitive way, while cyanate ions r e a c t with papain i n a mixed type form (Nakamura and S o e j i n a , 1970). Urea at high c o n c e n t r a t i o n s (2 M), methanol, a c e t o n i t r i l e and d i m e t h y l s u l p h o x i d e e x h i b i t c o m p e t i t i v e i n h i b i t i o n (Sluyterman, 1967b). -146-,. Egg-white c y s t a t i n i s a t i g h t l y - b i n d i n g i n h i b i t o r of f i c i n , papain, c a t h e p s i n H and L and a l s o d i p e p t i d y l peptidase ( N i c k l i n and B a r r e t t , 1984). I t has been r e p o r t e d that t h i s c y s t a t i n shows a c o m p e t i t i v e r e v e r s i b l e i n h i b i t i o n of c a t h e p s i n B. With papain i t was shown to form equimolar complexes ( N i c k l i n and B a r r e t t , 1984) . Other c y s t a t i n s have been found i n mammalian t i s s u e s and body f l u i d s . T h e i r i n h i b i t o r y p r o p e r t i e s are v ery s i m i l a r to those of egg white c y s t a t i n , and amino a c i d sequences show e v o l u t i o n a r y homology ( N i c k l i n and B a r r e t t e t a l . , 1984). These i n h i b i t o r s can be grouped i n t o three f a m i l i e s , on the b a s i s of s t r u c t u r a l s i m i l a r i t i e s . Family 1 c o n s i s t s of low-molecular weight ( a l l , 0 0 0 ) i n h i b i t o r s , without a d i s u l f i d e b r i d g e , and are found mainly i n t r a c e l l u l a r l y . Family 2 c o n s i s t s of low molecular weight (»13,000) i n h i b i t o r s with two d i s u l f i d e b r i d g e s , mainly found i n body f l u i d s . R e c e n t l y three new c y s t a t i n type protease i n h i b i t o r s which belong to f a m i l y 2 have been i s o l a t e d from human s a l i v a (Isemura et a l . , 1987). Family 3 c o n s i s t s of high molecular weight i n h i b i t o r s (Isemura et a l . , 1987). Although E-64 i s an i r r e v e r s i b l e i n h i b i t o r (see I r r e v e r s i b l e i n h i b i t o r s of s u l f h y d r y l proteases) of s u l f h y d r y l p roteases, Hanada et a l . (1978) a p p l i e d r e v e r s i b l e i n h i b i t i o n k i n e t i c s to study i t s i n h i b i t i o n e f f e c t on papain, and r e p o r t e d t h a t E-64 was a noncompetitive i n h i b i t o r . T h i s r e s u l t i s s u r p r i s i n g s i n c e E-64 has been found to r e a c t with the a c t i v e s i t e of the s u l f h y d r y l -147-p r o t e a s e s , thus, i t s i n h i b i t o r y a c t i o n should be c o m p e t i t i v e . More d e t a i l e d s t u d i e s c a r r i e d out by B a r r e t t et a l . (1982) confirmed the a c t i v e - s i t e - d i r e c t e d nature of the r e a c t i o n of papain with E-64. Leupeptin, a c e t y l - L - l e u c y l - L - l e u c y l - L - a r g i n y l , i s another t i g h t l y - b i n d i n g r e v e r s i b l e i n h i b i t o r of c y s t e i n e p r o t e a s e s . T h i s i n h i b i t o r has e x h i b i t e d a p r o t e c t i v e e f f e c t a g a i n s t mouse muscular dystrophy, and i t s p o s s i b l e u s e f u l n e s s i n the treatment of muscular dystrophy i n man has been s t u d i e d (Umezawa and Aoyagi, 1983). Two other r e v e r s i b l e i n h i b i t o r s , a n t i p a p a i n and chymostatin, together with l e u p e p t i n , have an a-amino aldehyde group i n the C-terminal p a r t of t h e i r peptide molecule. The t e r m i n a l aldehyde group i s i n v o l v e d i n the s p e c i f i c b i n d i n g to enzymes, i n c l u d i n g the hydroxyl or t h i o l group of s e r i n e or c y s t e i n e p r o t e a s e s , r e s p e c t i v e l y (Umezawa and Aoyagi, 1983). -148-MATERIALS AND METHODS A. MATERIALS Papain (twice c r y s t a l l i z e d ) , c y s t e i n e - H C l , ethylene diamine-t e t r a a c e t i c disodium s a l t (EDTA), 5 , 5 1 - d i t h i o b i s 2 - n i t r o b e n z o i c a c i d (DTNB) and carbobenzoxyglycine p - n i t r o p h e n y l e s t e r were from Sigma Chemical Company ( S a i n t L o u i s , MO). Sephadex G-25 s u p e r f i n e and DEAE-Sephadex A-25 were from Pharmacia Fine Chemicals (Uppsala 1, Sweden). Sodium t e t r a t h i o n a t e was obtained from ICN Pharmaceuticals, Inc. ( P l a i n v i e w , NY). A c e t o n i t r i l e (HPLC grade, wavelength c u t o f f 190 nm), was from Caledon L a b o r a t o r i e s L t d . (Georgetown, ON). A l l reagents were of a n a l y t i c a l grade or b e t t e r . Glass d i s t i l l e d water was used i n the p r e p a r a t i o n of a l l s o l u t i o n s and b u f f e r s . Weighing i n the m i l l i g r a m range was done on an UM3 u l t r a b a l a n c e ( M e t t l e r , G r e i f e n s e e , S w i t z e r l a n d ) . B. DETERMINATION OF PROTEIN CONCENTRATION P r o t e i n c o n c e n t r a t i o n of papain s o l u t i o n s was determined from absorbance readings at 280 nm with a E ^ d cm) of 25.0 ( B r o c k l e h u r s t et a l . , 1981). A molecular weight of 23,800 d a l t o n s ( B r o c k l e h u r s t et a l . , 1981) was used f o r a l l c a l c u l a t i o n s . In some experiments, p r o t e i n was measured at l e a s t i n -149-d u p l i c a t e e i t h e r by u s i n g the dye-binding assay as d e s c r i b e d i n the Bio-Rad p r o t e i n standard assay b u l l e t i n (Bio-Rad I n s t r u c t i o n Manual 82-0275-1282, Bio-Rad L a b o r a t o r i e s , Richmond, CA) developed by Bradford (1976), or by d i g e s t i n g the samples us i n g the r a p i d M i c r o - K j e l d a h l method of Concon and S o l t e s s (1973). Digested samples were analyzed f o r t o t a l n i t r o g e n content u s i n g an Auto Analyzer II (Technicon Instruments Co., Tarrytown, NY). The crude p r o t e i n content was then c a l c u l a t e d by m u l t i p l y i n g the t o t a l n i t r o g e n content by a f a c t o r of 6.25. C. DETERMINATION OF PROTEOLYTIC ACTIVITY 1. O p t i m i z a t i o n of the c o n d i t i o n s to measure p r o t e o l y t i c  a c t i v i t y of papain I n i t i a l l y , the a b i l i t y of papain to hydrolyze c a s e i n was determined u s i n g the method of Hanada et a l . (1978). However, i t was found t h a t t h i s method had poor r e p e a t a b i l i t y . As mentioned by West (1988), enzyme assays are g e n e r a l l y not as accurate nor r e l i a b l e as good chemical a n a l y s i s . In order to in c r e a s e I t s p r e c i s i o n , an o p t i m i z a t i o n of three parameters of t h i s method: enzyme c o n c e n t r a t i o n , i n c u b a t i o n time and i n c u b a t i o n temperature, was c a r r i e d out. The o b j e c t i v e of t h i s o p t i m i z a t i o n was to minimize the standard d e v i a t i o n of the p r o t e o l y i c a c t i v i t y readings w i t h i n r e p l i c a t e s . The o p t i m i z a t i o n method used was a new o p t i m i z a t i o n approach which i n v o l v e d a c e n t r a l composite r o t a t a b l e design (CCRD) f o r -150-three v a r i a b l e s , followed by m u l t i p l e r e g r e s s i o n and computational simplex o p t i m i z a t i o n . According to the CCRD the complete experimental p l a n c o n s i s t e d of a t o t a l of 16 experiments. Table 14 shows the l e v e l s f o r each of the three f a c t o r s used. Four r e p l i c a t i o n s were done f o r each of the 16 experimental c o n d i t i o n s . P r o t e o l y t i c a c t i v i t y was measured as d e s c r i b e d below, with the m o d i f i c a t i o n t h a t papain c o n c e n t r a t i o n , i n c u b a t i o n time and i n c u b a t i o n temperature were v a r i e d a c c o r d i n g to the CCRD. Using the standard d e v i a t i o n (four r e p l i c a t e s ) of the absorbance r e a d i n g a t 280 nm of the f i l t r a t e s of each experimental c o n d i t i o n as the dependent v a r i a b l e , a q u a d r a t i c model was obtained through m u l t i p l e r e g r e s s i o n . The equation obtained was entered as pa r t of the computational simplex o p t i m i z a t i o n program. M i n i m i z a t i o n of t h i s f u n c t i o n was performed i n order to f i n d the c o n d i t i o n s a s s o c i a t e d with the s m a l l e s t standard d e v i a t i o n . 2. P r o t e o l y t i c a c t i v i t y d e t e r m i n a t i o n The method of Hanada et a l . (1978) was used to measure the p r o t e o l y t i c a c t i v i t y of n a t i v e and t e t r a t h i o n a t e modified papain, with the m o d i f i c a t i o n s i n papain c o n c e n t r a t i o n , i n c u b a t i o n time and i n c u b a t i o n temperature, which gave the s m a l l e s t standard d e v i a t i o n a c c o r d i n g to the r e s u l t s obtained with the o p t i m i z a t i o n method d e s c r i b e d above. -151-Table 14. Upper and lower l i m i t s f o r the three f a c t o r s used f o r o p t i m i z a t i o n of the p r o t e o l y t i c a c t i v i t y d e t e r m i n a t i o n of papain. L i m i t s F a c t o r Lower Upper Papain c o n c e n t r a t i o n (mg/mL) 0.03 0.10 Incubation time (min) 5.0 10.0 Incubation temperature(°C) 35 45 -152-The s u b s t r a t e s o l u t i o n was 1% (w/v) Hammersten c a s e i n (BDH Chemicals, Vancouver, B.C.) i n 0.01 M T r i s - H C l b u f f e r pH 8, which was prepared a c c o r d i n g to the Food Chemical Codex (FCC I I I , 1981). The method was as f o l l o w s : i n a stoppered t e s t tube a h a l f mL of a s o l u t i o n of papain (100 Hg/mL i n 0.05 M phosphate b u f f e r , pH 6.8) was mixed with 0.25 mL of 40 mM c y s t e i n e and 20 mM EDTA i n 0.05 M phosphate b u f f e r pH 6.8, and 0.25 mL of 0.05 M phosphate b u f f e r pH 6.8. The mixture was incubated at 35°C f o r 15 min. A f t e r t h i s p e r i o d of time, 5 mL of the 1% c a s e i n s o l u t i o n ( p r e - e q u i l i b r a t e d at 35°C) was added and the contents of the t e s t tube were mixed immediately using a Vortex mixer. The mixture was s u b j e c t e d to a f u r t h e r i n c u b a t i o n f o r 5 min at 35<>C. Then 5 mL of a 0.44 M t r i c h l o r o a c e t i c a c i d (TCA) s o l u t i o n was added to stop the enzyme r e a c t i o n and p r e c i p i t a t e the unhydrolysed s u b s t r a t e . The contents of the t e s t tubes were completely mixed. In order to separate the p r e c i p i t a t e d p r o t e i n , c e n t r i f u g a t i o n (10,000 xg, 15 min, 4<>C) followed by f i l t r a t i o n under vacuum through Whatman No.42 f i l t e r paper was c a r r i e d out. A l l f i l t r a t e s were completely c l e a r . The absorbance of the f i l t r a t e was measured at 280 nm with a Cary 210 spectrophotometer (Varian A s s o c i a t e s Inc., Palo A l t o , CA). P r o t e o l y t i c a c t i v i t i e s were expressed as I n t e r n a t i o n a l U n i t s i . e . flmoles of t y r o s i n e l i b e r a t e d min_lmg"x of sample, under assay c o n d i t i o n s . A standard curve prepared with L - t y r o s i n e plus c a s e i n s o l u t i o n under the c o n d i t i o n s f o r assay was used to convert absorbance to Hmoles of t y r o s i n e . -153-D. DETERMINATION OF THE INFLUENTIAL FACTORS FOR MAXIMUM INHIBITION AND REACTIVATION OF THE PROTEOLYTIC ACTIVITY OF PAPAIN BY TETRATHIONATE The f r a c t i o n a l f a c t o r i a l design L i s ( 2 1 B ) of Taguchi (1957) was used to determine the f a c t o r s which may s i g n i f i c a n t l y a f f e c t the i n a c t i v a t i o n of the p r o t e o l y t i c a c t i v i t y of papain by t e t r a t h i o n a t e and the subsequent r e a c t i v a t i o n of the t e t r a t h i o n a t e i n a c t i v a t e d papain by c y s t e i n e . In the i n a c t i v a t i o n experiments the f a c t o r s evaluated were: molar r a t i o of t e t r a t h i o n a t e to papain, pH, temperature and time of the i n a c t i v a t i o n r e a c t i o n . For the r e a c t i v a t i o n r e a c t i o n the same f a c t o r s were co n s i d e r e d , plus the a d d i t i o n a l f a c t o r of c y s t e i n e c o n c e n t r a t i o n d u r i n g the a c t i v a t i o n r e a c t i o n . The f a c t o r s together with t h e i r assigned l e v e l s are shown i n Table 15. The scheme used i s presented i n F i g . 23. For assay of enzyme i n h i b i t o r y a c t i v i t y , the method rep o r t e d above f o r the d e t e r m i n a t i o n of p r o t e o l y t i c a c t i v i t y was used, with the m o d i f i c a t i o n t h a t papain was f i r s t a c t i v a t e d with the cysteine-EDTA b u f f e r f o r 15 min a t 3 5 0 C , and then the s o l u t i o n was f r e e d from c y s t e i n e by passage through a Sephadex G-25 s u p e r f i n e column, p r e v i o u s l y e q u i l i b r a t e d with n i t r o g e n s a t u r a t e d 0.05 M phosphate b u f f e r pH 6.8, c o n t a i n i n g 20 mM EDTA. In order to prevent r e o x i d a t i o n of papain, the e l u t i o n was a l s o c a r r i e d out under n i t r o g e n s a t u r a t e d c o n d i t i o n s . T h i s a c t i v a t e d papain s o l u t i o n was d i l u t e d with 0.05 M phosphate b u f f e r pH 6.8 or with 0.05 M T r i s - H C l pH 10.0, to a proper c o n c e n t r a t i o n (0.1 -154-Table 15. F a c t o r s and the assigned l e v e l s i n v e s t i g a t e d f o r t h e i r p o s s i b l e i n f l u e n c e on the i n h i b i t i o n of the p r o t e o l y t i c a c t i v i t y of papain, and on the subsequent r e a c t i v a t i o n of a c t i v i t y by c y s t e i n e . L e v e l s F a c t o r Lower Upper [ T e t r a t h i o n a t e ] / [ p a p a i n ] 10 100 pH 6.8 10 Reaction time (min) 5 10 Cys t e i n e (mM)*- 20 40 Rea c t i o n temperature (°C) 22 40 "•Factor c o n s i d e r e d o n l y i n the r e a c t i v a t i o n experiments -155-[TT]/[Papain] Temperature Time [Cysteine] Figure 23. Scheme used in the Taguchi Lit fractional f a c t o r i a l experiment. The factor of cysteine concentration was only evaluated in the reactivation experiments. [TTl/[Papain] = molar ratio tetrathionate to papain in the i n h i b i t i o n reaction. -156-mg/mL) fo r the assay. To 0.25 mL of the d i l u t e d a c t i v a t e d papain s o l u t i o n was added e i t h e r 0.25 mL of n i t r o g e n s a t u r a t e d phosphate b u f f e r or a f r e s h s o l u t i o n of t e t r a t h i o n a t e i n the same b u f f e r . The c o n c e n t r a t i o n of t e t r a t h i o n a t e i n the s o l u t i o n was such that i t gave, upon d i l u t i o n , a molar r a t i o of t e t r a t h i o n a t e to papain of e i t h e r 10 or 100. The mixture was incubated under the c o n d i t i o n s of time and temperature a c c o r d i n g to the f r a c t i o n a l f a c t o r i a l d e s i g n . A f t e r the a p p r o p r i a t e i n c u b a t i o n time a d d i t i o n of c a s e i n and TCA, c e n t r i f u g a t i o n , f i l t r a t i o n and absorbance readi n g were performed as p r e v i o u s l y d e s c r i b e d . The percentage i n h i b i t i o n was c a l c u l a t e d as f o l l o w s (Hanada et a l . , 1978): % i n h i b i t i o n = 100(A-B)/A (34) where A stands f o r the absorbance without t e t r a t h i o n a t e and B f o r the absorbance with t e t r a t h i o n a t e . For the r e a c t i v a t i o n experiments the method was as f o l l o w s : a f t e r the a c t i v a t e d papain s o l u t i o n was incubated with or without t e t r a t h i o n a t e , 0.25 mL of 0.05 M phosphate b u f f e r pH 6.8 with or without c y s t e i n e was added at a c o n c e n t r a t i o n a c c o r d i n g to the f r a c t i o n a l f a c t o r i a l experiment (20 mM or 40 mM). The mixture was f u r t h e r incubated at 40°C f o r 10 min. A f t e r t h i s , 5 mL of the 1% c a s e i n s o l u t i o n was added and the other steps were c a r r i e d out as d e s c r i b e d b e f o r e . -157-The percentage r e a c t i v a t i o n was c a l c u l a t e d as f o l l o w s : % r e a c t i v a t i o n = 100(C/A) (35) where A i s the absorbance of the sample without t e t r a t h i o n a t e plus c y s t e i n e , and C i s the absorbance of the sample with t e t r a t h i o n a t e and c y s t e i n e . The values of % i n h i b i t i o n and % r e a c t i v a t i o n ( i n d u p l i c a t e s ) were analyzed u s i n g a Taguchi's f r a c t i o n a l f a c t o r i a l a n a l y s i s of v a r i a n c e computer program w r i t t e n i n IBM-BASIC (Arteaga, 1986). E. PREPARATION OF TETRATHIONATE-MODIFIED PAPAIN The t e t r a t h i o n a t e - m o d i f i e d papain (TT-papain) was prepared as f o l l o w s : papain (25 mg) was d i s s o l v e d i n 10 mL of 0.05 M phosphate b u f f e r pH 6.8, c o n t a i n i n g 20 mM EDTA and e i t h e r 40 mM c y s t e i n e or 20 mM |3-mercaptoethanol. The mixture was incubated f o r 10 min at 40°C. A f t e r t h i s p e r i o d the mixture was placed on a column of Sephadex G-25 s u p e r f i n e (2 cm x 40 cm) e q u i l i b r a t e d with n i t r o g e n s a t u r a t e d 0.05 M phosphate b u f f e r pH 6.8 c o n t a i n i n g 10 mM EDTA. P r o t e i n was e l u t e d with the same n i t r o g e n s a t u r a t e d b u f f e r at a flow r a t e of 40 mL/hr and 4 mL f r a c t i o n s were c o l l e c t e d . The absorbance at 280 nm of the e l u e n t was c o n t i n u a l l y monitored with a LKB 2138 UVICORD UV-detector (LKB-Produkter AB, Stockholm-Bromma 1, Sweden) connected to a SP-H6V p l o t t e r (Riken Denshi Co., Denmark). P r o t e i n emerged between 32-40 mL of the e f f l u e n t , and these f r a c t i o n s were pooled. The p r o t e i n c o n c e n t r a t i o n of the pooled f r a c t i o n s was immediately -158-determined by measuring absorbance at 280 nm. A predetermined amount of sodium t e t r a t h i o n a t e was added to t h i s p r o t e i n s o l u t i o n based on the p r o t e i n c o n c e n t r a t i o n . The r a t i o of moles of t e t r a t h i o n a t e to moles papain f o r the chemical m o d i f i c a t i o n v a r i e d i n the range of 10 to 500. The r e a c t i o n mixture was incubated a t room temperature f o r 15 min. A f t e r t h i s p e r i o d of time, the excess t e t r a t h i o n a t e was removed by p a s s i n g the mixture through a column of DEAE-Sephadex A-25 anion exchanger, which was e q u i l i b r a t e d with 0.05 M phosphate b u f f e r pH 6.8 c o n t a i n i n g 0.2 M NaCl. Since at t h i s pH papain has a net p o s i t i v e charge, i t d i d not bind to the anion exchange r e s i n , and i t was e l u t e d immediately from the column. The p r o t e i n f r a c t i o n s were pooled, f r e e z e - d r i e d and s t o r e d with d e s s i c a n t at -20°C u n t i l f u r t h e r a n a l y s i s . F. CIRCULAR DICHROISM C i r c u l a r d i c h r o i s m s p e c t r a were measured u s i n g a JASCO J-500A s p e c t r o p o l a r i m e t e r (Japan S p e c t r o s c o p i c Co., L t d . , Tokyo, Japan) under a constant n i t r o g e n f l u s h at 20°C. The instrument was c a l i b r a t e d by the two-point c a l i b r a t i o n technique at wavelengths of 290.5 and 192.5 nm using d-10-camphorsulfonic a c i d i n d i s t i l l e d water as r e p o r t e d by Chen and Yang (1977). -159-1. O p t i m i z a t i o n of the c o n d i t i o n s f o r measuring the CD  s p e c t r a of papain P r e l i m i n a r y experiments shoved t h a t the d e t e r m i n a t i o n of CD s p e c t r a of papain had the problem of high noise l e v e l s . Although the CD s i g n a l i s i n h e r e n t l y n o i s y (Wollmer et a l . , 1983) and i t i s w e l l known th a t t h i s problem can a f f e c t the p r e d i c t i o n of secondary s t r u c t u r e of p r o t e i n s (Hennessey and Johnson, 1982), the s e l e c t i o n of the "best" experimental c o n d i t i o n s to measure CD i s u s u a l l y done by t r i a l and e r r o r based on previous e x p e r i e n c e . I t was thought t h a t by u s i n g an o b j e c t i v e o p t i m i z a t i o n method, i t c o u l d be p o s s i b l e to i n c r e a s e the q u a l i t y of the CD spectrum of papain. Based on p r e l i m i n a r y experiments and p r e v i o u s l y p u b l i s h e d i n f o r m a t i o n , the f o l l o w i n g four f a c t o r s , which a f f e c t the de t e r m i n a t i o n of the CD s p e c t r a of p r o t e i n s , were s e l e c t e d f o r o p t i m i z a t i o n : (1) papain c o n c e n t r a t i o n , (2) bandwidth, (3) time constant, and ( 4 ) the value of the product of scan r a t e times the time c o n s t a n t . By e n t e r i n g the l i m i t s to the simplex c e n t r o i d o p t i m i z a t i o n program (SCO) (Nakai and Arteaga, 1988) d i f f e r e n t s e t s of experimental c o n d i t i o n s were obtained. Papain was d i s s o l v e d i n 0.05 M phosphate b u f f e r pH 6.8 and f i l t e r e d through a 0.44 um Millex-HA f i l t e r ( M i l l i p o r e C o r p o r a t i o n , Bedford, MA) and d i l u t e d with the same b u f f e r to the proper c o n c e n t r a t i o n ( i . e . absorbance a t 280 nm) a c c o r d i n g to the SCO. F i v e scans over the range 280-190 nm were recorded f o r each -160-experimental c o n d i t i o n and the standard d e v i a t i o n s o£ the e l l i p t i c i t y v alues (00) a t three wavelengths ( 2 2 2 , 2 0 9 , and 2 0 0 nm) were c a l c u l a t e d . Since the p h o t o m u l t i p l i e r v o l t a g e ( P M ) i s a measure of the noise l e v e l (JASCO, 1 9 7 9 ) , the P M a t those wavelengths was recorded. The o b j e c t i v e f u n c t i o n t o be minimized was a combination (product) of three n o n c o n f l l c t i n g parameters: ( 1 ) the t o t a l scanning time, ( 2 ) the mean of the standard d e v i a t i o n of e l l i p t i c i t y v alues a t the three wavelengths, and ( 3 ) the mean of the p h o t o m u l t i p l i e r v o l t a g e s at those wavelengths. In order to estimate the accurac y of each CD scan, the values of the mean r e s i d u e e l l i p t i c i t y ( [ 6 ] H R « ) at 2 2 2 , 2 0 9 and 2 0 0 nm were c a l c u l a t e d u s i n g the formula r e p o r t e d below and the values obtained were compared t o p u b l i s h e d d a t a . The c o e f f i c i e n t s of v a r i a t i o n of ( I O I M R W ) a t 2 2 2 , 2 0 9 and 2 0 0 nm were a l s o c a l c u l a t e d f o r each experimental c o n d i t i o n . 2. Ffls-yy C P Spectra ( 1 9 Q - 2 4 Q nm) CD s p e c t r a f o r n a t i v e and t e t r a t h i o n a t e - m o d i f i e d papain were scanned from 2 4 0 to 1 9 0 nm. A c e l l of 0 . 1 cm path l e n g t h was used. Sample p r e p a r a t i o n was as d e s c r i b e d above. The c o n d i t i o n s used were those obtained through the o p t i m i z a t i o n technique d e s c r i b e d above, which were: 0 . 0 2 3 mg/mL papain c o n c e n t r a t i o n , 1 . 6 nm bandwidth, 1 . 6 time constant, and 0 . 8 8 f o r the product of time constant times the scan r a t e . - 1 6 1 -The r e s u l t s of the CD a n a l y s i s were expressed i n terms of the mean r e s i d u e e l l i p t i c i t y (181M»W) i n the standard u n i t s of deq cm*/dmol. Without c o r r e c t i n g the Lorenz r e f r a c t i v e index f a c t o r , a mean r e s i d u e weight of 99 (Chang et a l . , 1978) was used f o r a l l c a l c u l a t i o n s . Each p r o t e i n s o l u t i o n was measured a minimum of three times. The b a s e l i n e spectrum f o r each p r o t e i n sample was obtained by running the b u f f e r under c o n d i t i o n s i d e n t i c a l to those used f o r the sample. The mean r e s i d u e e l l i p t i c i t y ( 9 ] M R W was c a l c u l a t e d using the f o l l o w i n g formula: [ 9 ] M » W = 9 Q»MRW (36) 10»L»c where 8° i s the observed e l l i p t i c i t y i n degrees, MRW i s the mean re s i d u e weight (taken as 99), L i s the pathlength i n cm, and c i s the p r o t e i n c o n c e n t r a t i o n i n mg/mL. 3. Near-UV Spectra (250 -350 nm) In t h i s s p e c t r a l r e g i o n the noise l e v e l of the CD s i g n a l was s m a l l , thus, there was no need to optimize the c o n d i t i o n s f o r near-UV CD measurements. A 1 cm pathlength c e l l was used with a p r o t e i n c o n c e n t r a t i o n of 1.0-2.0 mg/mL. In t h i s case, the CD r e s u l t s were expressed i n terms of the d i f f e r e n c e i n molar a b s o r p t i v i t y between l e f t and r i g h t c i r c u l a r l y p o l a r i z e d l i g h t , 5e. The fle was c a l c u l a t e d a c c o r d i n g to the f o l l o w i n g e q u a t i o n : -162-<5e = [ 9 ] M B W N 3300 (37) where N= t o t a l r e s i d u e s i n the p r o t e i n . In the case of papain, N=212 ( B r o c k l e h u r s t et a l . , 1981). G. SECONDARY STRUCTURE PREDICTION Two methods were used to p r e d i c t the secondary s t r u c t u r e f r a c t i o n s of papain based on i t s CD s p e c t r a . The c o n s t r a i n e d r e g u l a r i z a t i o n procedure of Provencher and Glockner (1981) was used by e n t e r i n g the [ 8 ] M R W at 1 nm i n t e r v a l s from 240 to 190 nm. The second method used was the procedure r e p o r t e d by S i e g e l et a l . (1980). In s p i t e of i t s s i m p l i c i t y t h i s method has been shown to be h i g h l y a c curate and v e r s a t i l e (Yada, 1984). In t h i s method, CD data i n the form of [ 8 ] M R W a t wavelengths between 210 and 240 nm provide an accurate p r e d i c t i o n of the f r a c t i o n of h e l i c a l s t r u c t u r e of a p r o t e i n . The IBM-BASIC computer program used i s r e p o r t e d i n Appendix I. H. FLUORESCENCE AND DIFFERENTIAL ABSORPTION SPECTROSCOPY The e f f e c t of pH on the f l u o r e s c e n c e emission s p e c t r a of n a t i v e and t e t r a t h i o n a t e m o d ified papain was i n v e s t i g a t e d over the pH range of 4 to 10. Fluorescence measurements were performed with a Shimadzu spectrofluorophotometer Model RF-540 (Shimadzu Co., Kyoto, Japan). -163-The emission s p e c t r a , a t room temperature (approximately 22<>C), over the range 460-300 nm at an e x c i t a t i o n wavelength of 282 nm, were measured i n the f o l l o w i n g 0.02 M b u f f e r s : sodium a c e t a t e , pH 4.0 to 5.5; sodium phosphate, pH 5.5 to 8.0; T r i s - c h l o r i d e , pH 8.0 to 9.0; g l y c i n e NaOH, pH 9.0 to 10.0. No i n f l u e n c e of b u f f e r ions on the emission s p e c t r a was observed. In a l l cases a p r o t e i n c o n c e n t r a t i o n of 0.01 mg/mL was used. The wavelength of 282 nm has been r e p o r t e d to be the maximum e x c i t a t i o n wavelength of papain. ( B a r e l and G l a z e r , 1969). A c t i v a t i o n of both n a t i v e and t e t r a t h i o n a t e - m o d i f i e d papain was c a r r i e d out by i n c u b a t i n g the enzyme with 20 mM c y s t e i n e and 10 mM EDTA i n 0.02 M phosphate b u f f e r pH 6.8 f o r 20 min a t 40<>C. A f t e r t h i s i n c u b a t i o n , a c t i v a t o r - f r e e enzyme s o l u t i o n was obtained by passage through a Sephadex G-25 s u p e r f i n e column under n i t r o g e n s a t u r a t e d c o n d i t i o n s . S o l u t i o n s of a c t i v a t e d papain were immediately d i l u t e d t o the a p p r o p r i a t e c o n c e n t r a t i o n with the corresponding b u f f e r , which had been p r e v i o u s l y s a t u r a t e d with n i t r o g e n . A b s o r p t i o n s p e c t r a measurements were made with a Cary 210 spectrophotometer (V a r i a n A s s o c i a t e s Inc., Palo A l t o , CA) i n the r e g i o n 350-260 nm. To get a b a s e l i n e , a sample cuvette c o n t a i n i n g n a t i v e papain i n 0.02 M phosphate b u f f e r pH 6.8, was scanned a g a i n s t a r e f e r e n c e c u v e t t e c o n t a i n i n g the same enzyme s o l u t i o n . The s o l u t i o n i n the sample c u v e t t e was r e p l a c e d with a s o l u t i o n of t e t r a t h i o n a t e - m o d i f i e d papain of the same c o n c e n t r a t i o n , and the d i f f e r e n t i a l a b s o r p t i o n spectrum was recorded. -164-I. DETERMINATION OF TOTAL -SH GROUPS OF PAPAIN The t h i o l group content of papain was measured a c c o r d i n g to the method of Habeeb (1972) using 5 , 5 1 - d i t h i o b i s 2-n i t r o b e n z o i c a c i d (DTNB). F i v e to ten mg of n a t i v e or t e t r a t h i o n a t e m o d ified papain was d i s s o l v e d i n 6.0 mL of a 10% s o l u t i o n of sodium dodecyl s u l f a t e (SDS) i n 0.02 M sodium phosphate pH 8, c o n t a i n i n g 20 mM EDTA. The sample was incubated a t room temperature f o r 10 min. In some experiments, before the a d d i t i o n of SDS, papain ( n a t i v e or TT-papain) was a c t i v a t e d with c y s t e i n e (40 mM) f o r 10 min f o l l o w e d by removal of c y s t e i n e by g e l f i l t r a t i o n through a Sephadex G-25 s u p e r f i n e column, under n i t r o g e n s a t u r a t e d c o n d i t i o n s as d e s c r i b e d b e f o r e . A f t e r a d d i t i o n of SDS and i n c u b a t i o n , 0.1 mL of a f r e s h l y prepared s o l u t i o n of DTNB (40 mg DTNB i n 10 mL of 0.1 M sodium phosphate b u f f e r pH 8) was added to 3 mL of the SDS-protein s o l u t i o n , and the mixture was f u r t h e r incubated a t room temperature f o r 10 min. The absorbance of the s o l u t i o n was read a t 410 nm a g a i n s t a p r o t e i n s o l u t i o n i n SDS to g i v e apparent absorbance. A reagent blank was s u b t r a c t e d from the apparent absorbance to g i v e the net absorbance. An e x t i n c t i o n c o e f f i c i e n t of 13,600 M"1 cm"1 (Hanada e t a l . , 1978) f o r the nitromercaptobenzoate anion was used f o r a l l c a l c u l a t i o n s . -165-J . INSOLUBILIZATION OF PAPAIN WITH TETRATHIONATE P r e l i m i n a r y experiments showed t h a t under c e r t a i n c o n d i t i o n s t e t r a t h i o n a t e caused p r e c i p i t a t i o n of papain. T e t r a t h i o n a t e was rep o r t e d to cause p r e c i p i t a t i o n of p i g muscle g l y c e r a l d e h y d e - 3 -phosphate dehydrogenase (Parker and A l l i s o n , 1969). In order to give more i n s i g h t on t h i s o b s e r v a t i o n the experiments d e s c r i b e d below were c a r r i e d out. 1. E f f e c t of c o n c e n t r a t i o n of t e t r a t h i o n a t e and 0-mercaptoethanol on the p r e c i p i t a t i o n of papain The e f f e c t of t e t r a t h i o n a t e and 0-mercaptoethanol c o n c e n t r a t i o n on the p r e c i p i t a t i o n of papain was s t u d i e d using response s u r f a c e methodology. A c e n t r a l composite r o t a t a b l e design (CCRD) of two v a r i a b l e s was used. The l e v e l s of t e t r a t h i o n a t e and 0-mercaptoethanol evaluated are presented i n Table 16. Papain was d i s s o l v e d i n 0.05 M sodium phosphate pH 6.8 to a c o n c e n t r a t i o n of 10 mg/mL. The r e q u i r e d 0-mercaptoethanol was added to give a f i n a l c o n c e n t r a t i o n a c c o r d i n g to the CCRD and the mixture was incubated a t 40°C f o r 10 min. Then the r e q u i r e d amount of t e t r a t h i o n a t e was added and the mixture incubated a t 60°C f o r 10 min, followed by immersion i n an i c e bath f o r 5 min. A f t e r c e n t r i f u g a t i o n (10000 xg, 20 min, 4°C) p r o t e i n content of an a l i q u o t of the supernatant was measured using the Bio-Rad method. Absorbance readings were converted to p r o t e i n c o n c e n t r a t i o n values using a standard curve prepared with n a t i v e papain. -166-Table 16. L e v e l s of the f a c t o r s used i n the RSM experiment of the p r e c i p i t a t i o n of papain by t e t r a t h i o n a t e . Coded l e v e l s F a c t o r -1.4 -1 0 +1 +1.4 [ T e t r a t h i o n a t e ] (mM)*- 0 15 50 85 100 [0-mercaptoethanol] (mM)*- 0 15 50 85 100 x F i n a l c o n c e n t r a t i o n -167-2. E f f e c t of PH and temperature on the p r e c i p i t a t i o n of papain by t e t r a t h i o n a t e Papain was d i s s o l v e d i n d i s t i l l e d water and a f t e r no pH adjustment (pH 5.5) or pH adjustment to 8.5 with NaOH, 0-mercaptoethanol (80 mM) and t e t r a t h i o n a t e ( 100 mM) were added. The mixture was Incubated f o r 10 min at 22, 40, or 60<>C, followed by immersion i n an i c e bath f o r 10 min. C e n t r i f u g a t I o n and p r o t e i n d e t e r m i n a t i o n of an a l i q u o t of the supernatant were c a r r i e d out as d e s c r i b e d above. 3. R e s o l u b i l i z a t i o n of the p r e c i p i t a t e d p r o t e i n The e f f e c t of d i f f e r e n t chemical reagents (urea, SDS, and 0-mercaptoethanol) on r e s o l u b i l i z a t i o n of the p r e c i p i t a t e d p r o t e i n was s t u d i e d . The f o l l o w i n g procedure was used: the p r e c i p i t a t e d p r o t e i n (50 mg), which was i n s o l u b l e i n water, was d i s p e r s e d i n 5 mL of 0.02 M phosphate b u f f e r pH 6.8. Then the corresponding chemical reagent was added to o b t a i n the d e s i r e d f i n a l c o n c e n t r a t i o n . The mixture was incubated f o r 60 min a t 45<>C, followed by immersion i n an i c e bath f o r 10 min. The mixture was then c e n t r i f u g e d (10000 xg; 4°C; 15 min). Samples th a t c o n t a i n e d urea were d i a l y s e d f o r 48 hr a g a i n s t three changes of d i s t i l l e d water (5 L) a t 4°C (Spectrapor membrane t u b i n g No.l, Spectrum Medical I n d u s t r i e s , Inc., Los Angeles, CA) before p r o t e i n d e t e r m i n a t i o n . An a l i q u o t of the supernatant as w e l l as the i n s o l u b l e f r a c t i o n remaining a f t e r c e n t r i f u g a t l o n were t r a n s f e r r e d to 30 mL M i c r o - K j e l d a h l f l a s k s and evaporated u n t i l dryness i n a -168-f o r c e d - a i r c o n v e c t i o n oven a t 80°C (6 h r ) . D i g e s t i o n , t o t a l n i t r o g e n d e t e r m i n a t i o n , and c a l c u l a t i o n of crude p r o t e i n c o n c e n t r a t i o n were c a r r i e d out as d e s c r i b e d b e f o r e . 4. A n a l y s i s of the p r e c i p i t a t e d p r o t e i n P r o t e i n content of the p r e c i p i t a t e was measured u s i n g the M i c r o - K j e l d a h l method as s t a t e d b e f o r e . P r o t e o l y t i c a c t i v i t y of the s o l i d was measured using c a s e i n as s u b s t r a t e as d e s c r i b e d p r e v i o u s l y . Sodium dodecyl s u l f a t e polyacrylamide g e l e l e c t r o p h o r e s i s (SDS-PAGE) of the n a t i v e and t e t r a t h i o n a t e - m o d i f i e d papain ( s o l u b l e and i n s o l u b l e ) , was c a r r i e d out u s i n g a PhastSystem* M (Pharmacia, Uppsala 1, Sweden). PhastGel g r a d i e n t 10-15% g e l s and PhastGel SDS b u f f e r s t r i p s (Pharmacia, Uppsala, Sweden) were used. The g e l s had a 13 mm s t a c k i n g g e l zone and a 32 mm continuous 10 to 15% g r a d i e n t g e l zone with 2% c r o s s l i n k i n g . The b u f f e r system i n the g e l s was 0.112 M a c e t a t e ( l e a d i n g ion) and 0.112 M T r i s , pH 6 . 4 . The b u f f e r system i n PhastGel SDS b u f f e r s t r i p s was 0.02 M t r i c i n e ( t r a i l i n g i o n ) , 0.02 M T r i s and 0.55% SDS, pH 7.5. The b u f f e r s t r i p s were made of 2% agarose. The p r e p a r a t i o n of the samples was as f o l l o w s : to 0.5 mL of the p r o t e i n s o l u t i o n (3-7 mg protein/mL) i n 0.01 M phosphate b u f f e r pH 7.0 were added 20 UL of p-mercaptoethanol and 0.2 mL of 10% SDS i n 0.01 M phosphate b u f f e r pH 7.0, and 0.3 mL of 0.01 M phosphate b u f f e r pH 7.0. The mixture was heated at 100°C f o r 5 min, the samples were cooled to room temperature, and 50 UL -169-of the t r a c k i n g dye (bromophenol blue) was added. A p p l i c a t i o n and s e p a r a t i o n of the samples were performed as recommended i n the PhastSystem manual (Pharmacia, 1986). A f t e r e l e c t r o p h o r e s i s , the g e l was immediately s t a i n e d and d e s t a i n e d u s i n g the recommended f a s t coomassie s t a i n i n g procedure (Pharmacia, 1986). -170-K. DETERMINATION OF Vmax AND Km FOR THE PAPAIN-CATALYZED HYDROLYSIS OF CARBOBENZOXYGLYCINE P-NITROPHENYL ESTER The method used was e s s e n t i a l l y the one r e p o r t e d by K i r s c h and Ingelstrom (1966). Papain was a c t i v a t e d i n f r e s h l y prepared 0.02 M phosphate b u f f e r pH 6.8, 1 mM EDTA and 0.35 mM c y s t e i n e f o r at l e a s t 1 hr at 40°C. I t has been r e p o r t e d t h a t , under the c o n d i t i o n s mentioned above, a c t i v a t i o n i s complete a f t e r 45 min and constant a c t i v i t y i s maintained f o r at l e a s t 4 hr ( K i r s c h and Ingelstrom, 1966). The p a p a i n - c a t a l y z e d h y d r o l y s i s of carbobenzoxyglycine p - n i t r o p h e n y l e s t e r was followed by f i r s t t r a n s f e r r i n g 0.2 mL of the s o l u t i o n of the n i t r o p h e n y l e s t e r i n a c e t o n i t r i l e i n t o a 1 cm quartz cuvette placed i n the c e l l compartment of a Cary 210 spectrophotometer (Varian A s s o c i a t e s Inc., Palo A l t o , CA). The r e a c t i o n was i n i t i a t e d by adding 2.8 mL of the a c t i v a t e d enzyme s o l u t i o n . During the course of the r e a c t i o n , the change i n absorbance at 400 nm was c o n t i n u o u s l y measured. The i n i t i a l v e l o c i t y f o r the enzymatic r e a c t i o n under each experimental c o n d i t i o n was c o r r e c t e d f o r the r e a c t i o n i n the absence of enzyme. Six i n i t i a l s u b s t r a t e c o n c e n t r a t i o n s were used and at l e a s t three complete r e a c t i o n curves were taken f o r each s u b s t r a t e c o n c e n t r a t i o n . A l l measurements were performed at room temperature (20-22°C). In order to o b t a i n the i n i t i a l v e l o c i t y v a l u e s (V 0) necessary -171-f o r the c a l c u l a t i o n of Km and Vmax, the l i n e a r i z a t i o n method r e p o r t e d by Durance et a l . (1986) was compared to the c o n v e n t i o n a l f i x e d time assay method. Using the computer program r e p o r t e d by Durance et a l . (1986) modified f o r an IBM-PC, the optimum value of B was found; the r e a c t i o n order with r e s p e c t to time was d e r i v e d (Eg. 20) and a c c o r d i n g to t h i s v a l u e , data was l i n e a r i z e d u s i n g Eq. 18 or Eq. 19 (see s e c t i o n "Determination of i n i t i a l v e l o c i t i e s " i n the LITERATURE REVIEW of t h i s c h a p t e r ) . The equation f o r the l i n e a r i z e d data was obtained by l i n e a r r e g r e s s i o n . I n i t i a l v e l o c i t y a t each i n i t i a l s u b s t r a t e c o n c e n t r a t i o n was taken as the f i r s t d e r i v a t i v e of the curve a t t=0. For the c o n v e n t i o n a l f i x e d time method, the i n i t i a l v e l o c i t y (Vo) was assumed to be constant over an i n i t i a l p r e d e f i n e d p e r i o d of time (Eq. 14). Since the r e a c t i o n time i n most cases was r e l a t i v e l y s h o r t (30-100 sec) the i n i t i a l time p e r i o d was taken a t 15 t o 30 sec depending on the s u b s t r a t e c o n c e n t r a t i o n . The i n i t i a l v e l o c i t i e s c a l c u l a t e d by the l i n e a r i z a t i o n procedure and by the c o n v e n t i o n a l f i x e d time method were f i t t e d to the Michaelis-Menten equation by the computer program of O e s t r e i c h e r and P i n t o (1983), modified f o r running on an IBM-PC computer. T h i s program a l s o c a l c u l a t e d the standard e r r o r and the r e s i d u a l standard e r r o r of Km and Vmax. -172-L. INHIBITION EXPERIMENTS In order to c h a r a c t e r i z e the i n h i b i t o r y e f f e c t of sodium t e t r a t h i o n a t e on the a c t i v i t y of papain, a s e t of i n h i b i t i o n experiments was c a r r i e d out. Since t e t r a t h i o n a t e r e a c t s with c y s t e i n e ( I n g l i s and L i u , 1970) present i n the a c t i v a t i o n b u f f e r , i t was necessary to i n c o r p o r a t e a g e l f i l t r a t i o n step i n order to separate c y s t e i n e from papain p r i o r to the i n h i b i t i o n experiments. Papain was a c t i v a t e d i n 0.02 M phosphate pH 6.8 c o n t a i n i n g 10 mM of EDTA and 40 mM of c y s t e i n e f o r 15 min at 40<>c. T h i s s o l u t i o n was f r e e d from c y s t e i n e by passage through a Sephadex G-25 s u p e r f i n e column p r e v i o u s l y e q u i l i b r a t e d with n i t r o g e n s a t u r a t e d 0.02 M phosphate b u f f e r pH 6.8 and 20 mM of EDTA. The e l u t i o n was c a r r i e d out under n i t r o g e n s a t u r a t e d c o n d i t i o n s i n order to prevent r e o x i d a t i o n of papain. 1. R e v e r s i b l e i n h i b i t i o n experiments T e t r a t h i o n a t e was assumed to a c t as a r e v e r s i b l e i n h i b i t o r , and the k i n e t i c parameters f o r r e v e r s i b l e i n h i b i t i o n were c a l c u l a t e d . The setup of t h i s experiment was as f o l l o w s : three d i f f e r e n t c o n c e n t r a t i o n s of the i n h i b i t o r (sodium t e t r a t h i o n a t e ) were evaluated at f i v e s u b s t r a t e c o n c e n t r a t i o n s . An a l i q u o t of 0.2 mL of the s o l u t i o n of the p - n i t r o p h e n y l e s t e r i n a c e t o n i t r i l e was t r a n s f e r r e d i n t o a 1 cm quartz cuvette placed i n the Cary 210 spectrophotometer, followed by 0.2 mL of sodium t e t r a t h i o n a t e i n 0.02 M phosphate b u f f e r pH 6.8. The r e a c t i o n was -173-i n i t i a t e d by adding 2.8 i L of the g e l f i l t e r e d a c t i v a t e d papain i n n i t r o g e n s a t u r a t e d 0.02 M phosphate b u f f e r pH 6.8 c o n t a i n i n g 20 mM of EDTA. The r e a c t i o n product was analyzed by measuring the change i n absorbance at 400 nm. A p p r o p r i a t e c o n t r o l s were run f o r a l l experiments. I n i t i a l v e l o c i t i e s were c a l c u l a t e d as mentioned before and the type of r e v e r s i b l e i n h i b i t i o n was determined using the computer program ENZYME (Lutz et a l . , 1986). T h i s program i s a weighted n o n l i n e a r l e a s t squares curve f i t t i n g computer program, implemented i n compiler BASIC f o r the IBM-PC, used to estimate the parameters of enzyme k i n e t i c s obeying Michaelis-Menten k i n e t i c s and seven i n h i b i t i o n models. 2. I r r e v e r s i b l e i n h i b i t i o n experiments (a) In the absence of s u b s t r a t e The i n a c t i v a t i o n study i n the absence of s u b s t r a t e was as f o l l o w s : 1 mL of a sodium t e t r a t h i o n a t e s o l u t i o n was added to 50 mL of a g e l f i l t e r e d a c t i v a t e d papain s o l u t i o n to s t a r t the i n a c t i v a t i o n r e a c t i o n . The mixture was kept at room temperature and the r e s i d u a l enzyme a c t i v i t y ( i . e . i n i t i a l v e l o c i t y ) was assayed every 15 sec as mentioned above, using 1.5 mM p - n i t r o p h e n y l e s t e r i n a c e t o n i t r i l e as s u b s t r a t e . A c o n t r o l mixture c o n t a i n i n g a l l of the components of the r e a c t i o n mixture except f o r the i n h i b i t o r was i n c l u d e d . The r e s u l t of the i n a c t i v a t i o n i s presented as a second order r a t e c onstant. -174-(b) In the presence of s u b s t r a t e In order to c h a r a c t e r i z e the type of i r r e v e r s i b l e i n h i b i t i o n by t e t r a t h i o n a t e , the e f f e c t of d i f f e r e n t s u b s t r a t e c o n c e n t r a t i o n s on the second order r a t e constant of i n h i b i t i o n was e v a l u a t e d . The experimental setup was as f o l l o w s . A 0.2 mL a l i q u o t of the s o l u t i o n of the p - n i t r o p h e n y l e s t e r i n a c e t o n i t r i l e was t r a n s f e r r e d i n t o a 1 cm quartz cuvette placed i n the Cary 210 spectrophotometer, followed by 0.2 mL of sodium t e t r a t h i o n a t e i n 0.02 M phosphate b u f f e r pH 6.8. The r e a c t i o n was i n i t i a t e d by adding 2.8 mL of the s o l u t i o n of the g e l f i l t e r e d , a c t i v a t e d papain i n n i t r o g e n s a t u r a t e d 0.02 M phosphate b u f f e r pH 6.8 c o n t a i n i n g 20 mM of EDTA. The r e a c t i o n product was analyzed as mentioned b e f o r e . Appropriate c o n t r o l s were run f o r a l l exper iments. Since the t e t r a t h i o n a t e c o n c e n t r a t i o n used was much higher than t h a t of papain, the r e a c t i o n was assumed to be f i r s t order. To o b t a i n the second order r a t e constants a t each s u b s t r a t e c o n c e n t r a t i o n , l i n e a r i z a t i o n of the data ( s u b s t r a t e c o n c e n t r a t i o n vs. time) was c a r r i e d out and d e r i v a t i z a t i o n of the l i n e a r i z e d equation obtained by l i n e a r r e g r e s s i o n was performed f o r the i n h i b i t e d r e a c t i o n s . From the d e r i v a t i z e d equations, the v e l o c i t y of the r e a c t i o n a t d i f f e r e n t time i n t e r v a l s was c a l c u l a t e d . -175-The r e a c t i o n r a t e s o£ the c o n t r o l r e a c t i o n s were p r a c t i c a l l y constant over the time p e r i o d analyzed (zero order with r e s p e c t to time) and were c a l c u l a t e d using the Michaelis-Menten equation (Eq. 13). The d i f f e r e n c e between the v e l o c i t i e s of the c o n t r o l r e a c t i o n and the i n h i b i t e d r e a c t i o n , at the same i n i t i a l s u b s t r a t e c o n c e n t r a t i o n , a t d i f f e r e n t time i n t e r v a l s was c a l c u l a t e d . Taking the v e l o c i t y as a measure of the a c t i v i t y of papain, s e m i l o g a r i t h m i c curves of a c t i v i t y at each s u b s t r a t e c o n c e n t r a t i o n as a f u n c t i o n of time (Eq. 13) were found to be l i n e a r and the observed (pseudo f i r s t - o r d e r ) r a t e of i n a c t i v a t i o n , k 0 b « , was taken as the value of the slope of these l i n e s . The apparent second order r a t e constant f o r i n a c t i v a t i o n , k, was taken as ( B a r r e t t et a l . , 1982): k = kob- / [I ] (38) where [ I ] = i n i t i a l c o n c e n t r a t i o n of i n h i b i t o r . M. STATISTICAL ANALYSIS C a l c u l a t i o n s were c a r r i e d out on an IBM-PC. Data hand l i n g and mathematical op e r a t i o n s were performed with LOTUS 1-2-3" (Lotus Development Co., Cambridge, MA). The STATGRAPH program (STSC, Inc., 1985) was used f o r a l l s t a t i s t i c a l e v a l u a t i o n s . -176-RESULTS AND DISCUSSION A. OPTIMIZATION OF THE CONDITIONS TO MEASURE PROTEOLYTIC ACTIVITY OF PAPAIN A c e n t r a l composite r o t a t a b l e d e sign (CCRD) f o r three v a r i a b l e s was used i n d e s i g n i n g t h i s experiment. The independent v a r i a b l e s papain c o n c e n t r a t i o n , temperature and time of i n c u b a t i o n were coded as Xi, X* and X3, r e s p e c t i v e l y . A l t o g e t h e r 16 combinations ( I n c l u d i n g a r e p l i c a t e of the c e n t r a l p o i n t ) formed the CCRD. The l e v e l s of the three independent v a r i a b l e s together with the values of the response are shown i n Table 17. The response was taken as the standard d e v i a t i o n (SD) of four r e p l i c a t e s of the absorbance at 280 nm of the T C A - f i l t r a t e at each experimental c o n d i t i o n . The data i n Table 17 was f i t t e d to a second order model commonly used i n response s u r f a c e methodology (RSM) (Nakai and Arteaga, 1988): Y= 0o + E 0 i X i + E 0 n X i * + E E PijXiX, (39) where 0 i are r e g r e s s i o n c o e f f i c i e n t s and Xi are the independent v a r i a b l e s r e l a t e d to Y. The adequacy and f i t n e s s of the model were t e s t e d by a n a l y s i s of v a r i a n c e (Table 18). The r e s u l t s showed t h a t t h i s model d i d not e x p l a i n the data v a r i a t i o n adequately. A l o g a r i t h m i c t r a n s f o r m a t i o n of the response, Ln(SD), as recommended by Draper (1985), a l s o d i d not produce a s t a t i s t i c a l l y a c c e p t a b l e model. -177-Table 17. C e n t r a l compositive r o t a t a b l e design matrix used f o r the o p t i m i z a t i o n of c o n d i t i o n s to measure p r o t e o l y t i c a c t i v i t y of papain, and r e s u l t s f o r each experiment. F a c t o r s [Papain] Temp Time Experiment (mg/mL) (°C) (min) Mean*- S.D1 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 0.050 0.050 0.050 0.050 0.090 0.090 0.090 0.090 0.100 0.030 0.070 0.070 0.070 0.070 0.070 0.070 37.0 37.0 43.0 43.0 43 . 0 37.0 37.0 43.0 40.0 40.0 45.0 35.0 40.0 40.0 40.0 40.0 6.5 9.0 6.5 9.0 6.5 9.0 6.5 9.0 7 . 5 7.5 7.5 7.5 10.0 5.0 7 . 5 7.5 0.090 0.050 0.112 0.117 0.072 0.133 0.166 0.232 0.100 0.063 0.137 0.080 0.037 0.073 0.012 0 . 013 0.008 0.007 0.009 0.021 0.002 0.030 0.022 0.009 0.016 0.012 0.021 0.011 0.017 0.012 0.003 0.005 *-Mean of the absorbance r e a d i n g a t 280 nm (n=4) B S t a n d a r d d e v i a t i o n (n=4) -178-Table 18. A n a l y s i s of v a r i a n c e of the second order model*'* Source of v a r i a t i o n DF Mean Square F-value Model 9 0.0000517 0.70 n.s E r r o r 6 0.0000752 T o t a l 15 "Model Eq. (39) B R * ( a d j u s t e d f o r DF) = 0.00 n.s not s i g n i f i c a n t a t p= 0.05 -179-I n c l u s i o n of a three term i n t e r a c t i o n (X1X2XJ) i n t o the second order model gave a h i g h l y s i g n i f i c a n t model (Table 19). The m u l t i p l e c o e f f i c i e n t of d e t e r m i n a t i o n , R», f o r t h i s m o d ified second order model was 0.95 (p<0.01), which i n d i c a t e s t h a t t h i s model accounted f o r 95% of the t o t a l v a r i a t i o n based on the r e g r e s s i o n . A p l o t of the p r e d i c t e d a g a i n s t the observed values f o r the dependent v a r i a b l e ( i . e . SD) i s shown i n F i g . 24. Included i n t h i s p l o t i s a l i n e with slope equal to 1. T h i s type of p l o t i s u s e f u l to d e t e c t cases when the v a r i a n c e i s not constant or a t r a n s f o r m a t i o n of the dependent v a r i a b l e i s needed. T h i s p l o t shows t h a t the p o i n t s are d i s t r i b u t e d f a i r l y u n i f o r m l y about the d i a g o n a l l i n e , s u g g e s t i n g t h a t the model i s reasonable. A p l o t of r e s i d u a l s versus p r e d i c t e d values (Fig.25) shows t h a t the r e s i d u a l s are randomly s c a t t e r e d , s u g g e s t i n g again the adequacy of the f i t t e d model. 1. L o c a l i z a t i o n of optimum c o n d i t i o n The common method of f i n d i n g the optimum i n RSM i s by d e r i v a t i z i n g the f i t t e d model i n r e l a t i o n to each independent v a r i a b l e , then e q u a l i z i n g the d e r i v a t i v e s to zero i n order to f i n d a s t a t i o n a r y p o i n t , and l a s t l y , s o l v i n g the system by simultaneous equations (Nakai and Arteaga, 1988). C a n o n i c a l a n a l y s i s can a l s o be performed i n order to c h a r a c t e r i z e the shape of the f i t t e d response (Nakai and Arteaga, 1988). -180-Table 19. A n a l y s i s of v a r i a n c e f o r the modified second order models-Source of v a r i a t i o n DF Mean Square F-value Model 10 0.000083 29.30*-E r r o r 5 0.0000028 T o t a l 15 *-R* (adjusted f o r ** s i g n i f i c a n t a t DF) = 0.953 p<0.01 V a r i a b l e * C o e f f i c i e n t T-value Prob(> j T j ) Xi Xj X, Xx* X,* X,* XxX* XxX3 X 2X 3 XxX2X3 -27.8002 -0.0783 -0.2414 8.7341 0.0005 0.0018 0.6642 3.6177 0.0054 -0.0900 -11.0895 -10.1408 -10.2126 5.6554 6.3621 5.8326 10.6593 11.3634 9.2726 -11.339 0.0004 0.0005 0.0005 0.0048 0.0031 0.0043 0.0004 0.0003 0.0008 0.0003 CONSTANT 2.4751 10.7857 0.0004 "•Code: Xx = X» = X 3 = [Papain] (mg/ml) temperature (<>C) time (min) -181-0.030 - i 0.025 0.020 -> q5 0 .015 -CO o 0.010 0.005 -0 .000 I i i i i i i i i i | i i i i i i i i i i i i i i i i i i i i i I I I T i i i i i 0.000 0.010 0.020 0.030 0.040 Predicted F i g u r e 24. P l o t of p r e d i c t e d values a c c o r d i n g to the modified second order model of the SD of the d e t e r m i n a t i o n of the p r o t e o l y t i c a c t i v i t y of papain a g a i n s t the corresponding experimental ones. A l i n e with s l o p e 1 i s included i n the p l o t . - 1 8 2 -20.00 -i 15.00 10.00 H I O 5.00 H x 0.00 o -5.00 H Or: -10.00 -15.00 -20.00 0.000 o o oo I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I » I » I » I » I I I 0.010 0.020 0.030 Predicted 0.040 Figure 25. Plot of residuals for the modified second order model. - 1 8 3 -However with the i n c l u s i o n o£ the three term i n t e r a c t i o n (XiXzXs), the above mathematical approach i s not p o s s i b l e s i n c e the number of v a r i a b l e s i s g r e a t e r than the number of equations. Although other mathematical procedures could be used, they are somewhat complex. Another method of l o c a l i z i n g the optimum c o n d i t i o n i s by means of the g r a p h i c a l method suggested by F l o r o s and Chinnan (1988); however, the n e c e s s i t y of p l o t t i n g d i f f e r e n t response s u r f a c e s makes t h i s method time consuming. A more e f f i c i e n t approach to f i n d the optimum c o n d i t i o n i s by using computational simplex o p t i m i z a t i o n (CSO) as d e s c r i b e d by Nakai and Arteaga (1988). By e n t e r i n g the equation, obtained using m u l t i p l e r e g r e s s i o n , as a f u n c t i o n subroutine i n the Morgan and Deming simplex program (1974), i t i s p o s s i b l e to minimize the equation, thus f i n d i n g the c o n d i t i o n s that produce the h i g h e s t r e p e a t a b i l i t y ( i . e . lowest standard d e v i a t i o n ) . The computer program used i s given i n Appendix I I . M i n i m i z a t i o n of the f i t t e d model by CSO gave the f o l l o w i n g optimum c o n d i t i o n s ( F i g . 26): papain c o n c e n t r a t i o n , 0.1 mg/mL; i n c u b a t i o n time, 5 min and temperature, 35<>C. To c o n f i r m the optimum values obtained by the CSO, response s u r f a c e s were generated ( F i g . 27-29). In a l l response s u r f a c e s , one f a c t o r was maintained constant at i t s optimum l e v e l as d e f i n e d by the CSO. T h i s "graphic o p t i m i z a t i o n " confirmed the optimum values obtained by CSO. I t i s worth n o t i n g t h a t even when the responses are r e l a t i v e l y complex, CSO i s able to o b t a i n -184-Terminating d i f f e r e n c e v a l u e - 0.0000 Lover and upper l i m i t s LL: UL: 0.070 3S.000 0.100 4S.000 5.000 10.000 I n i t i a l simplex XI X2 X3 Vertex 1 0.070 35.000 S.000 Vertex 2 0.098 37.357 6.179 Vertex 3 0.077 44.428 6.179 Vertex 4 0.077 37.357 9.714 7.750 Vertex 5 ( R e f l e c t i o n ) 0.066 28.715 Vertex 6 (Contraction - W ) 0.079 40.500 6.571 Vertex 7 ( R e f l e c t i o n ) 0.088 37.881 5.000 Vertex 8 (Expansion) 0.094 38.143 5.000 Vertex 9 ( R e f l e c t i o n ) 0.100 42.158 6.833 Vertex 10 (Contractlon - W ) 0.079 36.790 5.458 Vertex 11 ( R e f l e c t i o n ) 0.098 35.000 5.000 Vertex 12 (Expansion) 0.100 35.000 S.000 Vertex 13 ( R e f l e c t i o n ) 0.080 35.757 5.000 Vertex 14 ( R e f l e c t i o n ) 0.08S 35.000 5.306 Vertex 15 ( R e f l e c t i o n ) 0.097 35.000 5.000 Vertex 16 ( R e f l e c t i o n ) 0.100 35.000 5.204 Vertex 17 ( R e f l e c t i o n ) 0.100 35.000 5.000 Vertex 18 ( R e f l e c t i o n ) 0.098 3S.OO0 5.000 Vertex 19 (Contractlon-R) 0.099 35.000 5.000 Vertex 20 ( R e f l e c t i o n ) 0.100 35.000 5.000 Vertex 21 ( R e f l e c t i o n ) 0.100 35.000 5.000 Vertex "22 (Contraction-R) 0.100 35.000 5.000 Vertex 23 (Massive c o n t r a c t l o n - R ) 0.100 35.000 5.000 P i n a l average values 0.100 35.000 RESPONSE 0.007 0.004 0.028 0.026 Response Response Response Response Response Response Response Response Response Response Response Response Response Response Response Response Response Response Response 0.006 0.002 0.004 0.022 0.000 -0.014 -0.015 -0.002 -0.005 -0.014 -0.013 -0.015 -0.014 -0.014 -0.015 -0.015 -0.015 -0.01S Figure 26. Computational optimization used to obtain the best experimental conditions for pro t e o l y t i c a c t i v i t y determination of papain. The response is the standard deviation calculated using the modified second order model. - 1 8 5 -F i g u r e 27. Response s u r f a c e of standard d e v i a t i o n a g a i n s t i n c u b a t i o n time and enzyme c o n c e n t r a t i o n , a t a constant i n c u b a t i o n temperature of 35<>C. Standard d e v i a t i o n values are i n the range of 0.000 to 0.030 -186-F i g u r e 28. Response s u r f a c e of standard d e v i a t i o n a g a i n s t i n c u b a t i o n time and temperature, at a constant papain c o n c e n t r a t i o n of 0.1 mg/ml. Standard d e v i a t i o n values are i n the range of 0.000 to 0.030 -187-F i g u r e 29. Response s u r f a c e of standard d e v i a t i o n a g a i n s t i n c u b a t i o n temperature and enzyme c o n c e n t r a t i o n , a t a constant i n c u b a t i o n time of 5 min. Standard d e v i a t i o n values are i n the range of 0.000 to 0.030 -188-an optimum response. The optimum c o n d i t i o n s found by the CSO were v e r i f i e d by c a r r y i n g out a s e t of p r o t e o l y t i c a c t i v i t y d e t e r m i n a t i o n using the optimum computed experimental c o n d i t i o n s of papain c o n c e n t r a t i o n , temperature and i n c u b a t i o n time. The r e s u l t s of t h i s v e r i f i c a t i o n confirmed the v a l i d i t y of the o p t i m i z a t i o n technique. By using the optimum experimental c o n d i t i o n s , the standard d e v i a t i o n of four r e p l i c a t e s of absorbance at 280 nm i n the p r o t e o l y t i c a c t i v i t y d e t e r m i n a t i o n was 0.003. In summary i t can be concluded t h a t t h i s new o p t i m i z a t i o n approach of combining CCRD with CSO i s an e f f i c i e n t method of o p t i m i z a t i o n . B. DETERMINATION OF THE INFLUENTIAL FACTORS FOR MAXIMUM INHIBITION AND REACTIVATION OF THE PROTEOLYTIC ACTIVITY OF PAPAIN BY TETRATHIONATE The % i n h i b i t i o n and % r e a c t i v a t i o n data f o r the 1G experiments f o l l o w i n g a f r a c t i o n a l f a c t o r i a l experimental design L i s ( 2 1 S ) of Taguchi (1957) were analyzed by a n a l y s i s of v a r i a n c e . S i g n i f i c a n c e of the f a c t o r s was evaluated and p o s s i b l e i n t e r a c t i o n s were determined f o r the i n h i b i t i o n of the p r o t e o l y t i c a c t i v i t y of papain by t e t r a t h i o n a t e , and f o r the subsequent r e a c t i v a t i o n of the p r o t e o l y t i c a c t i v i t y of papain by c y s t e i n e . The r e s u l t s of the a n a l y s i s of v a r i a n c e on the i n a c t i v a t i o n experiments are presented i n Table 20. A l l f a c t o r s and -189-Table 20. A n a l y s i s of v a r i a n c e (Taguchi's L i s 2XB) f o r the i n h i b i t i o n of the p r o t e o l y t i c a c t i v i t y of papain by t e t r a t h i o n a t e (TT). Source of v a r i a t i o n DF Mean Square F -value Molar r a t i o TTtPapain (MR) 1 175.62 0 .28 n.s. Reaction pH (pH) 1 602.00 0 .97 n.s. Reaction time (t) 1 6.08 0 .01 n.s. Reaction temp. (T) 1 116.10 0 .19 n.s. MR x pH 1 14.68 0 .02 n.s. MR x t 1 617.38 0 .99 n.s. MR x T 1 989 .93 1 . 59 n.s. pH x t 1 220.88 0 .36 n.s. pH x T 1 42.93 0 . 07 n.s. t X T 1 116.10 0 .19 n.s. Er r o r " 2 5 621.06 T o t a l 15 n.s. not s i g n i f i c a n t at p= 0.05 eThe sums of squares values f o r the f a c t o r s and i n t e r a c t i o n s t h a t do not appear i n the ANOVA t a b l e were very low and, t h e r e f o r e , i n c o r p o r a t e d i n t o the e r r o r sums of squares. -190-i n t e r a c t i o n s were found to be n o n s i g n i f i c a n t (p>0.05). T h i s r e s u l t i n d i c a t e s t h a t under the c o n d i t i o n s t e s t e d , the i n h i b i t i o n of the p r o t e o l y t i c a c t i v i t y of papain by t e t r a t h i o n a t e was not a f f e c t e d by changes i n the l e v e l s of the f a c t o r s . In most cases the % i n h i b i t i o n was over 90%, meaning t h a t 90% of the o r i g i n a l p r o t e o l y t i c a c t i v i t y of papain was i n h i b i t e d by r e a c t i o n with e i t h e r a 10 or a 100 molar excess of t e t r a t h i o n a t e . N e i t h e r changes i n pH (6.8 or 10.0), i n c u b a t i o n time (5 or 10 min), nor temperature (22 or 40<>C) s i g n i f i c a n t l y a f f e c t e d the % i n h i b i t i o n . These r e s u l t s agree with the f a c t t h a t t e t r a t h i o n a t e r e a c t s i n a v e r y s p e c i f i c manner with s u l f h y d r y l enzymes. F i g . 30 shows the e f f e c t of the two l e v e l s of molar r a t i o of t e t r a t h i o n a t e to papain on the % i n a c t i v a t i o n . Both l e v e l s caused more than 90% i n h i b i t i o n of the p r o t e o l y t i c a c t i v i t y . The r e s u l t s of the a n a l y s i s of v a r i a n c e f o r the r e a c t i v a t i o n experiments are presented i n Table 21. The pH d u r i n g the i n a c t i v a t i o n r e a c t i o n , and c y s t e i n e c o n c e n t r a t i o n d u r i n g the r e a c t i v a t i o n r e a c t i o n were computed to be h i g h l y s i g n i f i c a n t (p<0.01) sources of v a r i a t i o n i n the r e a c t i v a t i o n r e a c t i o n by c y s t e i n e . The molar r a t i o of t e t r a t h i o n a t e to papain and r e a c t i o n time of the i n a c t i v a t i o n r e a c t i o n were a l s o found to be s i g n i f i c a n t sources of v a r i a t i o n (p<0.05). F a c t o r s i n the i n a c t i v a t i o n r e a c t i o n (molar r a t i o of t e t r a t h i o n a t e to papain, pH, temperature and time) had an e f f e c t on the r e v e r s i b i l i t y , as measured by % r e a c t i v a t i o n , of the -191-110-j 100-40-30 -1 1 1 i i 1 1 1 1 1 r 1 0 50 100 [Tetrathionate]/[Papain] (mol/mol) Figure 30. Effe c t of two levels of the molar r a t i o of tetrathionate to papain on the ina c t i v a t i o n of papain. (Mean±SEM n=8) -192-Table 21. A n a l y s i s of v a r i a n c e (Taguchi's L i s 2 1 B ) f o r the r e a c t i v a t i o n by c y s t e i n e of the p r o t e o l y t i c a c t i v i t y of t e t r a t h i o n a t e - i n a c t i v a t e d papain. Source of v a r i a t i o n DF Mean Square F-value Molar r a t i o TT: papa i n (MR)*- 1 64.56 9.44* Reaction pH (pH)*- 1 327.81 47.92" Rea c t i o n time (t)*- 1 72.36 10.58* Cyst e i n e (Cys) 1 1134.78 165.89" MR x pH 1 344.09 50.30" MR x t 1 116.95 17.10** Cys x pH 1 508.50 74.34" Cys x t 1 189.86 27.75" Cys x T B 1 349.39 51.08" pH x T 1 175.56 25.66" E r r o r 0 5 6.84 T o t a l 15 " s i g n i f i c a n t at p < 0.05 " s i g n i f i c a n t at p < 0.01 ^-Factors of the i n a c t i v a t i o n r e a c t i o n . B T = i n c u b a t i o n temperature GThe sums of squares values f o r the f a c t o r s and i n t e r a c t i o n s t h a t do not appear i n the ANOVA t a b l e were very low and, t h e r e f o r e , i n c o r p o r a t e d i n t o the e r r o r sums of squares. -193-r e a c t i o n between papain and t e t r a t h i o n a t e . Since % r e a c t i v a t i o n i s a measure of the r e v e r s i b i l i t y of the i n a c t i v a t i o n process, i t can be expected t h a t i f t e t r a t h i o n a t e , upon r e a c t i n g with papain, does not cause i r r e v e r s i b l e c o n f o r m a t i o n a l changes i n the enzyme molecules, the o r i g i n a l p r o t e o l y t i c a c t i v i t y of papain w i l l be observed ( i . e 100% r e a c t i v a t i o n ) a f t e r treatment of the i n a c t i v a t e d enzyme with the r e d u c i n g agent ( i . e . c y s t e i n e ) . In g e n e r a l , values of % r e a c t i v a t i o n higher than 85 were obtained, which i n d i c a t e s t h a t i n most cases the i n a c t i v a t i o n was r e v e r s i b l e . T h i s suggests t h a t no major c o n f o r m a t i o n a l changes i n papain took plac e due to the r e a c t i o n of papain with t e t r a t h i o n a t e . The e f f e c t curve, i n F i g . 31, shows the i n t e r a c t i o n between the molar r a t i o of t e t r a t h i o n a t e to papain and r e a c t i o n time on the % r e a c t i v a t i o n . At any l e v e l of these f a c t o r s the % r e a c t i v a t i o n was higher than 85%, i n d i c a t i n g t h a t 85% of the o r i g i n a l p r o t e o l y t i c a c t i v i t y of papain was regained a f t e r treatment with c y s t e i n e . At the lower l e v e l of the molar r a t i o of t e t r a t h i o n a t e to papain (10) a s i g n i f i c a n t l y higher r e a c t i v a t i o n was obtained when the i n a c t i v a t i o n r e a c t i o n time was o n l y f i v e minutes than when i t was 10 min. In both cases the % r e a c t i v a t i o n was g r e a t e r than 90%. A s i g n i f i c a n t i n t e r a c t i o n was a l s o found between the molar r a t i o of t e t r a t h i o n a t e to papain and pH of the i n a c t i v a t i o n r e a c t i o n . A s i g n i f i c a n t l y higher r e a c t i v a t i o n was obtained when the pH of the i n a c t i v a t i o n r e a c t i o n was 6.8 and the molar r a t i o -194-10CH c o • —~ o > o D 0) 90-80-70 Rx. time= 5 min I Rx. time= 10 min I i 50 100 [Tetrathionate]/[Papain] (mol/mol) F i g u r e 31. E f f e c t curve f o r the i n t e r a c t i o n between the molar r a t i o of t e t r a t h i o n a t e to papain and r e a c t i o n time of i n a c t i v a t i o n r e a c t i o n on the % r e a c t i v a t i o n of the p r o t e o l y t i c a c t i v i t y of papain. (Meantconfidence l i m i t s c a l c u l a t e d at p<0.05) -195-of t e t r a t h i o n a t e to papain was 10 ( F i g . 32). As expected, the c o n c e n t r a t i o n of c y s t e i n e d u r i n g the r e a c t i v a t i o n r e a c t i o n had a s i g n i f i c a n t e f f e c t on the % r e a c t i v a t i o n v a l u e . Contrary t o what was expected, a lower % r e a c t i v a t i o n was obtained when a higher l e v e l of c y s t e i n e (40 mM) was used f o r r e a c t i v a t i o n . Since the redu c i n g agent ( i . e . c y s t e i n e ) was e s s e n t i a l f o r r e a c t i v a t i o n , i t was thought th a t a higher % r e a c t i v a t i o n should be obtained a t the higher c y s t e i n e c o n c e n t r a t i o n . A p o s s i b l e e x p l a n a t i o n of t h i s phenomenon i s t h a t d i s u l f i d e interchange r e a c t i o n s may have occurred a t the higher c y s t e i n e c o n c e n t r a t i o n . T h i s c o u l d cause i r r e v e r s i b l e i n a c t i v a t i o n of some of the papain molecules, producing a lower value of % r e a c t i v a t i o n . When the r e a c t i v a t i o n r e a c t i o n was c a r r i e d out with 20 mM c y s t e i n e a 100% r e a c t i v a t i o n value was obtained, which i n d i c a t e s t h a t the i n h i b i t i o n of the p r o t e o l y t i c a c t i v i t y of papain was t o t a l l y r e v e r s i b l e . When the r e a c t i v a t i o n was c a r r i e d out with 40 mM of c y s t e i n e , the f a c t o r s i n v o l v e d i n the i n h i b i t i o n r e a c t i o n (time, pH, and temperature) had a s i g n i f i c a n t e f f e c t on the % r e a c t i v a t i o n . F i g . 33-35 show th a t at a c y s t e i n e c o n c e n t r a t i o n of 40 mM, the higher the pH or temperature and the longer the r e a c t i o n time, the lower the value of % r e a c t i v a t i o n . The o v e r a l l r e s u l t s of the f r a c t i o n a l f a c t o r i a l experiments i n d i c a t e t h a t the r e a c t i o n of papain with t e t r a t h i o n a t e , under the c o n d i t i o n s s t u d i e d , caused a complete i n h i b i t i o n of the -196-110-, 80-70 H 1 1 1 1 1 1 i 1 1 1 1 0 50 100 [Tetrathionate]/[Papain] (mol/mol) F i g u r e 32. E f f e c t curve f o r the i n t e r a c t i o n between the molar r a t i o of t e t r a t h i o n a t e to papain and pH of the i n a c t i v a t i o n r e a c t i o n on the % r e a c t i v a t i o n of the p r o t e o l y t i c a c t i v i t y of papain. (Meantconfidence l i m i t s c a l c u l a t e d at p<0.05) -197-110-i 100-90 80-70-60-50 pH 10.0 I 30 i 35 —r~ 40 15 20 25 45 [Cysteine] (mM) ure 3 3 . Effect curve for the interaction between the cysteine concentration during the reactivation and pH of the inactivation on the % reactivation of the proteolytic a c t i v i t y of papain. (Mean+confidence l i m i t s calculated at p<0.05) -198-F i g u r e 34. E f f e c t curve f o r the i n t e r a c t i o n between the c y s t e i n e c o n c e n t r a t i o n d u r i n g r e a c t i v a t i o n and temperature of i n a c t i v a t i o n on the % r e a c t i v a t i o n of the p r o t e o l y t i c a c t i v i t y of papain. (Mean±Confidence l i m i t s c a l c u l a t e d a t p<0.05) -199-110-. 70-60-| -i 1 1 1 1 1 15 20 25 30 35 40 45 [Cysteine] (mM) Figure 35. Effect curve for the interaction between the cysteine concentration during reactivation and time of inactivation on the % reactivation of the proteolytic a c t i v i t y of papain. (Meantconfidence l i m i t s calculated at p<0.05) -200-not s i g n i f i c a n t l y a f f e c t e d by changes i n the molar r a t i o of papain to t e t r a t h i o n a t e , pH, temperature or time of the i n h i b i t i o n r e a c t i o n . In order to o b t a i n t o t a l r e v e r s i b i l i t y of the i n h i b i t i o n , the i n h i b i t i o n r e a c t i o n must be c a r r i e d out at pH 6.8, and a t a temperature of 22°C ( F i g . 36), and the r e a c t i v a t i o n r e a c t i o n must be c a r r i e d out with 20 mM c y s t e i n e . C. OPTIMIZATION OF CONDITIONS FOR MEASURING THE CD SPECTRA OF PAPAIN Experiments were performed a c c o r d i n g to the SCO as p r e v i o u s l y d e s c r i b e d . SCO with the four f a c t o r s was i n i t i a t e d by performing f i v e experiments c a l l e d " i n i t i a l simplex". A f t e r o b t a i n i n g the response v a l u e s , which were i n t h i s case the combination of three n o n c o n f l i c t i n g parameters, the response values were re p o r t e d back to the computer to o b t a i n new experimental c o n d i t i o n s . Table 22 shows the s e r i e s of the o p t i m i z a t i o n experiments f o r a c h i e v i n g the best c o n d i t i o n s f o r measuring the CD s p e c t r a of papain. A t o t a l of 18 experiments were c a r r i e d out, with v e r t e x number 15 g i v i n g the lowest response ( i . e . best s c a n ) . Since the s i z e s of s c a l e s of the three parameters t h a t were i n c l u d e d i n the evaluated response were d i f f e r e n t , i t was necessary to transform two of the responses; the n a t u r a l l o g a r i t h m of the t o t a l scan time was used and the mean of the p h o t o m u l t i p l i e r v o l t a g e at 222, 209, and 200 nm was d i v i d e d by 100 (Table 22). These t r a n s f o r m a t i o n s were a r b i t r a r i l y s e l e c t e d . Two c y c l e s of c e n t r o i d , r e f l e c t i o n and curve f i t t i n g plus an a d d i t i o n a l c e n t r o i d were done. Mapping was performed by p l o t t i n g -201-A f , ) not s i g n i f i c a n t l y a f f e c t e d by changes i n the molar r a t i o of papain t o t e t r a t h i o n a t e , pH, temperature or time of the i n h i b i t i o n r e a c t i o n . In order to o b t a i n t o t a l r e v e r s i b i l i t y of the i n h i b i t i o n , the i n h i b i t i o n r e a c t i o n must be c a r r i e d out at pH 6.8, and a t a temperature of 22<>C ( F i g . 36), and the r e a c t i v a t i o n r e a c t i o n must be c a r r i e d out with 20 mM c y s t e i n e . C. OPTIMIZATION OF CONDITIONS FOR MEASURING THE CD SPECTRA OF PAPAIN Experiments were performed a c c o r d i n g to the SCO as p r e v i o u s l y d e s c r i b e d . SCO with the four f a c t o r s was i n i t i a t e d by performing f i v e experiments c a l l e d " i n i t i a l simplex". A f t e r o b t a i n i n g the response v a l u e s , which were i n t h i s case the combination of three n o n c o n f l i c t i n g parameters, the response values were r e p o r t e d back to the computer to o b t a i n new experimental c o n d i t i o n s . Table 22 shows the s e r i e s of the o p t i m i z a t i o n experiments f o r a c h i e v i n g the best c o n d i t i o n s f o r measuring the CD s p e c t r a of papain. A t o t a l of 18 experiments were c a r r i e d out, with v e r t e x number 15 g i v i n g the lowest response ( i . e . best s c a n ) . Since the s i z e s of s c a l e s of the three parameters t h a t were i n c l u d e d i n the eval u a t e d response were d i f f e r e n t , i t was necessary to transform two of the responses; the n a t u r a l l o g a r i t h m of the t o t a l scan time was used and the mean of the p h o t o m u l t i p l i e r v o l t a g e a t 222, 209, and 200 nm was d i v i d e d by 100 (Table 22). These t r a n s f o r m a t i o n s were a r b i t r a r i l y s e l e c t e d . Two c y c l e s of c e n t r o i d , r e f l e c t i o n and curve f i t t i n g p l u s an a d d i t i o n a l c e n t r o i d were done. Mapping was performed by p l o t t i n g -202-Table 22. S i m p l e x - c e n t r o i d o p t i m i z a t i o n of the c o n d i t i o n s f o r CD spectrophotometry of papain. F a c t o r s Papain Bandwidth TC*- P r o d u c t 3 Technique Vertex (Aieo) (nm) (sec) (nm) RES C Super Simplex I n i t i a l Simplex 1 0. .500 1. .000 1, .000 0, .100 26. .00 2 1. ,888 1, ,219 2, ,530 0, .297 123. ,00 3 0, .828 1, .926 2, .530 0, .297 16. .00 4 0. ,828 1, ,219 2. , 530 0, ,933 16. ,80 5 0. .828 1, .219 7, .479 0, . 297 32. .00 C e n t r o i d 6 0. ,746 1, .341 3. ,385 0, .407 40. ,24 R e f l e c t i o n 7 0. . 500 1, .463 4 , .240 0 , .517 8. .40 Curve f i t t e d 8 0. ,500 1. , 478 4 . , 347 0. .530 8. , 39 C e n t r o i d 9 0, .664 1, .406 2, . 601 0, .465 37. . 22 R e f l e c t i o n 10 0 . . 500 1. ,593 1. ,000 0 , .633 6. ,80 Curve f i t t e d 11 0. .500 1, .780 1, . 000 0 , . 802 7. .19 C e n t r o i d Search I n i t i a l Simplex 12 0. 450 1. 500 1. 000 0 . 500 29 . 60 13 0. 959 1. 609 1. 656 0 .609 32. 70 14 0. 570 1. 963 1. 656 0 .609 12. 24 15 0. 570 1. 609 1. 656 0 .963 6. 00 C e n t r o i d Search 16 0. 570 1. 609 3. 777 0 .609 25. 60 17 0. 540 1. 670 2 . 022 0 .670 17. 30 18 0. 563 1. 713 2. 277 0 .713 17. 00 *• TC = time constant B Product = TC (min) x Scan r a t e (nm/min) e RE = Response = L n ( t o t a l scan time (min) x A x _B 100 where A = E S.D. (n=4) of [6] at 200 f 209 and 22 nm 3 B = E PV at 200, 209 and 222 nm 3 S.D = standard d e v i a t i o n [9] = e l l i p t i c i t y (m<>) PV = p h o t o m u l t i p l i e r v o l t a g e (V) -203-the response values obtained a g a i n s t each f a c t o r . The response s u r f a c e s appeared to d i r e c t the search f o r a lower response toward lower p r o t e i n c o n c e n t r a t i o n , longer band width and higher v a l u e s of the product of time constant times the scan r a t e . A f t e r the c e n t r o i d search, the optimum c o n d i t i o n s were found to be as f o l l o w s : papain c o n c e n t r a t i o n , 0.23 mg/mL; band width, 1.6 nm; time co n s t a n t , 2 sec, and the value of the product of time constant times the scan r a t e 0.8 nm. F i g . 37 shows the a c t u a l t r a c e s of the CD scan a t v e r t i c e s 1 and 15. I t i s evi d e n t t h a t a s i g n i f i c a n t improvement i n the q u a l i t y of the CD scan was achieved by using SCO. Johnson (1985) recommended t h a t f o r optimum CD measurements the p r o t e i n c o n c e n t r a t i o n should be such t h a t the absorbance a t 280 nm(l cm pathlength) i s l e s s than 1. In the present study the optimum c o n c e n t r a t i o n gave an absorbance a t 280 nm of 0.575. Hennessey and Johnson (1982) recommended t h a t the product of time constant times the scan r a t e should be not more than 0.33 nm. The o p e r a t i o n manual f o r the s p e c t r o p o l a r i m e t e r (Jasco, 1979), however,indicated t h a t values i n the range of 3.33 to 13.33 nm were adequate. Su and Ji r g e n s o n s (1977) used a value of 0.42 nm fo r the above parameter when measuring the CD s p e c t r a of many p r o t e i n s , i n c l u d i n g papain. The noise l e v e l of the scan decreased as the time constant i n c r e a s e d . However a t high time c o n s t a n t , i t was necessary to decrease the scan r a t e i n order t o compensate f o r the readi n g d e l a y of the instrument. -204-Figure 37. Trace of the far-UV CD spectrum of papain measured under d i f f e r e n t c o n d i t i o n s . (A) CD spectrum measured under the c o n d i t i o n s of v e r t e x 1. (B) CD spectrum measured under the optimum c o n d i t i o n s (Vertex 15). Band width i s r e l a t e d to the amount of l i g h t t h a t reaches the p h o t o m u l t i p l i e r , and i n many cases longer band widths give lower no i s e l e v e l s . I n c r e a s i n g the band width to more than 2 nm i s not recommended (Johnson, 1985) s i n c e a r t i f a c t s i n the s i g n a l are produced. I t should be pointed out t h a t the optimum c o n d i t i o n s found are o n l y f o r measuring the CD of papain. The same methodology can be a p p l i e d to f i n d the optimum c o n d i t i o n s f o r the CD d e t e r m i n a t i o n of other p r o t e i n s . Not a l l p r o t e i n s produce a CD scan with high noise l e v e l s , and t h e r e f o r e an o p t i m i z a t i o n of the experimental c o n d i t i o n s to measure the CD may not always be r e q u i r e d . The high noise l e v e l s i n the CD scan of papain are due to the high a b s o r p t i o n c o e f f i c i e n t [E3-* (1 cm) =25.0) and r e l a t i v e l y weak CD bands of t h i s enzyme. In p r o t e i n s where the a b s o r p t i o n c o e f f i c i e n t i s low or the e l l i p t i c i t y values high, the noise l e v e l s of the CD scan are lower. Table 23 shows the numerical values of [ 8 ] M R W at 222, 209 and 200 nm, together with the corresponding c o e f f i c i e n t of v a r i a t i o n f o r each ve r t e x of the SCO. Reference values f o r [ 0 ] M R W at those wavelengths f o r papain are a l s o i n c l u d e d . As expected, higher c o e f f i c i e n t s of v a r i a t i o n were obtained at the s h o r t e r wavelength (200 nm) and i n some cases c o e f f i c i e n t s of v a r i a t i o n of more than 100% o c c u r r e d . T h i s t a b l e demonstrates the importance of s e l e c t i n g the optimum c o n d i t i o n s f o r measuring the CD spectrum of papain; changes i n p r o t e i n c o n c e n t r a t i o n , band width, time constant and the product of time constant times scan r a t e had an -206-T a b l e 23. Values of mean r e s i d u e e l l i p t i c i t y * - (181M**) and corresponding c o e f f i c i e n t of v a r i a t i o n (C.V)» a t three wavelengths f o r the d i f f e r e n t v e r t i c e s of the s l m p l e x - c e n t r o i d o p t i m i z a t i o n . W a v e l e n g t h (nm) 222 209 200 V e r t e x [ 6 ] „ . C .V (% ) [ 6 ) « . C . V ( % ) [9]*.. C . V ( % ) 1 - 7 .27 8. 51 - 8 . 74 14 .16 - 5 . 41 68 . 57 2 -8 . 89 17 . 78 - 1 5 . 47 8 . 33 - 1 0 . 83 9 5 . 24 3 - 7 .28 1. 31 - 1 3 . 77 1 .69 -6 . 40 30 . 60 4 - 7 .04 0 . 68 - 1 3 . 77 1 .69 - 7 . 89 56 . 88 5 - 7 .38 0. 65 - 14 . 15 1 .64 -7 . 29 4 5 . 95 6 -8 .70 0 . 4 4 -14 . 85 1 .04 - 1 1 . 34 38 . 18 7 -9 . 50 0. 6 0 - 1 0 . 83 0 .48 - 7 . 27 2 5 . 53 8 -9 . 50 0. 30 - 1 0 . 83 0 .48 -7 . 27 2 5 . 53 9 - 8 .78 5. 07 - 1 2 . 68 4 . 07 . - 7 . 22 78 . 57 10 -9 .40 3. 03 - 1 1 . 24 3 .21 - 5 . 8 3 79 . 65 11 -9 .40 1. 82 - 1 1 . 34 9 .09 -6 . 70 6 5 . 38 12 -8 .25 4 . 00 -9 . 24 14 . 29 - 6 . 77 1 1 3 . 82 13 -8 .52 2. 42 -9 . 55 8 • 65 - 3 . 10 250 . 00 14 -8 .68 1. 50 -9 . 12 4 .76 -6 . 51 40 . 00 15 -8 .96 0. 50 - 1 0 . 40 1 .70 -7 . 70 2 0 . 30 16 -9 . 00 3. 00 - 1 1 . 30 4 .10 -8 . 00 37 . 75 17 -9 .17 0. 98 - 1 1 . 00 2 .40 -8 . 20 4 5 . 00 L8 -9 .17 0. 98 - 1 0 . 60 2 .00 -8 . 76 4 5 . 00 R e f e r e n c e v a l u e s 1 - 11 .70 - - 1 2 . 60 - - 8 . 88 *-Units: (10*") deg cm* drool - 1 • C o e f f i c i e n t of v a r i a t i o n (%) = Standard d e v i a t i o n x Mean °Source: Yang et a l . (1986) - 2 0 7 -important e f f e c t on the r e p r o d u c i b i l i t y . As Hennessey and Johnson (1982) have i n d i c a t e d , " I f CD spectroscopy i s to go beyond e m p i r i c a l c o r r e l a t i o n s so t h a t the s p e c t r a themselves can be used to determine secondary s t r u c t u r e , a higher degree of accuracy i s r e q u i r e d . " The r e s u l t s of t h i s study show the u s e f u l n e s s of SCO i n f i n d i n g the best c o n d i t i o n s to c a r r y out the d e t e r m i n a t i o n of the CD of papain. A s m a l l number of experiments were needed and a s i g n i f i c a n t improvement i n the q u a l i t y of the CD spectrum of papain was obtained. Furthermore, t h i s same approach can be used f o r other p r o t e i n s a n d a l s o f o r o p t i m i z a t i o n of other a n a l y t i c a l t e c h niques. D. CD OF NATIVE AND TETRATHIONATE MODIFIED PAPAIN T y p i c a l far-UV CD s p e c t r a f o r n a t i v e and t e t r a t h i o n a t e - m o d i f i e d papain (TT-papain) (molar r a t i o of t e t r a t h i o n a t e to papain f o r the m o d i f i c a t i o n =200) are shown i n F i g . 38. The two s p e c t r a are superimposable, which suggests t h a t no change i n the secondary s t r u c t u r e of papain occurred upon the m o d i f i c a t i o n . S i m i l a r r e s u l t s were obtained when the m o d i f i c a t i o n was c a r r i e d out with other molar r a t i o s of t e t r a t h i o n a t e t o papain. The CD s p e c t r a of both papains showed negative peaks a t about 222 and 209 nm, and a s m a l l shoulder a t about 205 nm, with [ 8 ] H R « of -9.2x10', -10.3x10' and -7x10' deg cm* dmol, r e s p e c t i v e l y . The -208-F i g u r e 38. Far-UV CD s p e c t r a of n a t i v e and TT-papain. (molar r a t i o of t e t r a t h i o n a t e to papain d u r i n g r e a c t i o n = 200). Note: both p r o t e i n s gave the same s p e c t r a . -209-p o s i t i o n of the peaks and the o v e r a l l shape of the CD s p e c t r a are v e r y s i m i l a r to the one p r e v i o u s l y r e p o r t e d by Su and J i r g e n s o n (1977); however, somewhat s m a l l e r [ 9 J M » W values were obtained i n the present study. Since t e t r a t h i o n a t e i s thought to r e a c t s p e c i f i c a l l y with the o n l y c y s t e i n e r e s i d u e of papain, Cys-25, i t i s very u n l i k e l y t h a t major c o n f o r m a t i o n a l changes would occur upon t h i s m o d i f i c a t i o n . The f a c t t h a t n a t u r a l , r e v e r s i b l y and i r r e v e r s i b l y i n a c t i v a t e d papain have the same CD s p e c t r a as f u l l y a c t i v a t e d papain (Sluyterman, 1967c) supports t h i s o b s e r v a t i o n . In the near u l t r a v i o l e t r e g i o n the three aromatic r e s i d u e s , p h e n y l a l a n i n e , t y r o s i n e and tryptophan give r i s e to CD s i g n a l s when t h e i r s i d e chains are i n asymmetric surrounding or i n v o l v e d i n i n t e r a c t i o n s i n the p r o t e i n s . Furthermore, d i s u l f i d e b r i d g e s may c o n t r i b u t e s i g n i f i c a n t l y between 250 and 300 nm (Heindl e t a l . , 1980; S t r i c k l a n d , 1974). T y p i c a l near-UV CD s p e c t r a f o r n a t i v e and t e t r a t h i o n a t e -modified papain (molar r a t i o t e t r a t h i o n a t e to papain d u r i n g the m o d i f i c a t i o n =200) are shown i n F i g . 39. The two p r o t e i n s had n e a r l y i d e n t i c a l s p e c t r a . Since the near-UV CD spectrum i s r e l a t e d to the t e r t i a r y s t r u c t u r e of p r o t e i n s ( S t r i c k l a n d , 1974), t h i s r e s u l t suggests t h a t l i t t l e , i f any, change occurred i n the t e r t i a r y s t r u c t u r e of papain due to m o d i f i c a t i o n with t e t r a t h i o n a t e . S i m i l a r r e s u l t s were obtained when m o d i f i c a t i o n was c a r r i e d out with other molar r a t i o s of t e t r a t h i o n a t e to papain. -210-Wavelength (nm) F i g u r e 3 9 . Near-UV CD s p e c t r a of n a t i v e ( .) and TT-papain (- - -) (molar r a t i o of t e t r a t h i o n a t e to papain d u r i n g r e a c t i o n = 200). -211-F o l l o w i n g the approach o£ S t r i c k l a n d (1974), the peaks at 262 and 268 nm may be assigned to p h e n y l a l a n i n e r e s i d u e s , while the one at 277 nm i s due to t y r o s i n e r e s i d u e s . Assignment of the negative peak at 290 nm i s more d i f f i c u l t s i n c e i t could be due to e i t h e r tryptophan or d i s u l f i d e r e s i d u e s . U s u a l l y the d i s u l f i d e CD begins at longer wavelengths (320 to 350 nm) and g r a d u a l l y i n t e n s i f i e s to give one or two bands l o c a t e d above 240 nm. D i s u l f i d e bands are much broader than the CD bands observed f o r the aromatic amino a c i d s i d e c h a i n s , and the longest wavelength of d i s u l f i d e CD band can be expected to peak below 290 nm ( S t r i c k l a n d , 1974). For many p r o t e i n s the peak of the d i s u l f i d e CD band at the l o n g e s t wavelength i s n e g a t i v e . The near-UV CD spectrum obtained i n t h i s experiment i s very s i m i l a r to the one r e p o r t e d by Su and J i r g e n s o n (1977). E. SECONDARY STRUCTURE OF NATIVE AND TETRATHIONATE-MODIFIED PAPAIN Table 24 shows the p r e d i c t e d values obtained through the use of two p u b l i s h e d a l g o r i t h m s , f o r the d i f f e r e n t secondary s t r u c t u r e f r a c t i o n s of n a t i v e papain and t e t r a t h i o n a t e - m o d i f i e d papain (molar r a t i o of t e t r a t h i o n a t e to papain f o r the m o d i f i c a t i o n = 200). The r e s u l t s obtained confirm the f a c t t h a t no change i n the secondary s t r u c t u r e of papain occurred upon m o d i f i c a t i o n with t e t r a t h i o n a t e . As r e p o r t e d by Yada (1984), the a l g o r i t h m of Provencher and Glockner (1981) when a p p l i e d to the CD data p r e d i c t e d more c l o s e l y than the a l g o r i t h m of S i e g e l et -212-Table 24. P r e d i c t e d secondary s t r u c t u r e f r a c t i o n s of n a t i v e and t e t r a t h i o n a t e - m o d i f i e d papain* (TT-papain) based on CD data, using two a l g o r i t h m s , and X-ray determined secondary s t r u c t u r e f r a c t i o n s . Secondary s t r u c t u r e f r a c t i o n H e l i x 0-sheet 0-turn Random P r o t e i n 1 B 2 3 1 2 3 1 3 1 3 Native .28 .40 .22 .14 .31 .10 .17 .28 .47 .39 papain TT-papain - .40 .22 - .31 .10 - .28 - .39 *Molar r a t i o of t e t r a t h i o n a t e to papain d u r i n g m o d i f i c a t i o n = 200. S i m i l a r r e s u l t s r e s u l t s were found at a l l r a t i o s t e s t e d (10-500). BMethods: 1. X-ray (Chang et a l . , 1978). 2. S i e g e l et a l . (1980) . 3. Provencher and Glockner (1981). Note: The method of S i e g e l et a l . (1980) does not estimate 0-turn or random f r a c t i o n s . -213-a l . (1980) the secondary s t r u c t u r e f r a c t i o n s obtained with X-ray c r y s t a l l o g r a p h y . F. FLUORESCENCE AND UV-ABSORPTION OF NATIVE AND TETRATHIONATE MODIFIED PAPAIN Na t i v e , a c t i v a t e d papain ( i . e . papain that was incubated with 20 mM of c y s t e i n e and 4 mM of EDTA p r i o r to a n a l y s i s ) showed an i n f l e c t i o n p o i n t at about pH 8.0 i n the pH-fluorescence p r o f i l e , and an in c r e a s e i n the f l u o r e s c e n c e i n t e n s i t y with f u r t h e r i n c r e a s e i n pH, re a c h i n g a maximum a t pH 9.5 ( F i g . 40). In c o n t r a s t , the pH-fluorescence p r o f i l e of u n a c t i v a t e d , n a t i v e papain and t e t r a t h i o n a t e - m o d i f i e d papain d i d not show any i n f l e c t i o n p o i n t at pH 8.0 ( F i g . 40-41). The pH-fluorescence p r o f i l e of n a t i v e a c t i v a t e d papain i s i n e x c e l l e n t agreement with the one p r e v i o u s l y r e p o r t e d by B a r e l and Glazer (1969). According to B a r e l and Glazer (1969), the presence of an i n f l e c t i o n p o i n t at pH 8.0 i n the pH-fluorescence p r o f i l e of a c t i v a t e d papain i s due to i o n i z a t i o n of the s u l f h y d r y l group at the a c t i v e s i t e of the enzyme. Since u n a c t i v a t e d , n a t i v e papain has the s u l f h y d r y l group p a r t l y blocked i n the form of a mixed d i s u l f i d e with h a l f - c y s t i n e or p a r t l y e x i s t i n g at the o x i d a t i o n s t a t e l e v e l of s u l f e n i c a c i d ( B r o c k l e h u r s t et a l . , 1981), no f r e e s u l f h y d r y l group i s present and hence no i n f l e c t i o n p o i n t at pH 8.0 i s observed. The o b s e r v a t i o n t h a t t e t r a t h i o n a t e modified papain (molar r a t i o of t e t r a t h i o n a t e to papain f o r the chemical m o d i f i c a t i o n r e a c t i o n = 10 to 500) d i d not show an i n f l e c t i o n p o i n t a t pH 8.0 ( F i g . 41) was a c l e a r i n d i c a t i o n that the -214-120-1 F i g u r e 40. E f f e c t of pH on the r e l a t i v e fluorescence i n t e n s i t y at 22°C of native papain. Unactivated (• ), and a c t i v e papain ( o ) . - 2 ] 5 -1 0 0 - 1 1 1 1 1 1 1 1 1 1 3 4 5 6 7 8 9 10 11 pH Figure 41. E f f e c t of pH on the r e l a t i v e fluorescence intensity at 22°C of TT-papain (molar r a t i o of tetrathionate to papain during reaction - 200). Unactivated (O), and active papain (o). -216-s u l f h y d r y l group of papain was blocked upon r e a c t i o n with t e t r a t h i o n a t e , as expected. A l k y l a t i o n of papain with e i t h e r i o d o a c e t a t e or iodoacetamide was r e p o r t e d ( B a r e l and G l a z e r , 1969) to produce a s i m i l a r e f f e c t . When the t e t r a t h i o n a t e - m o d i f i e d papain was incubated ( i . e . a c t i v a t e d ) with c y s t e i n e plus EDTA, a very s i m i l a r pH-fluorescence p r o f i l e to the one of a c t i v a t e d n a t i v e papain was obtained ( F i g . 41). F i g u r e 42 shows the e f f e c t of the molar r a t i o of t e t r a t h i o n a t e to papain f o r the m o d i f i c a t i o n , on the f l u o r e s c e n c e i n t e n s i t y at 352 nm. A quenching e f f e c t was observed when the chemical m o d i f i c a t i o n was c a r r i e d out with more than a 50 molar excess of t e t r a t h i o n a t e over papain. Since tryptophan r e s i d u e s c o n t r i b u t e a major p r o p o r t i o n of the f l u o r e s c e n c e i n t e n s i t y of papain, t h i s o b s e r v a t i o n suggests t h a t some tryptophan r e s i d u e s were modified by t e t r a t h i o n a t e . The f a c t t h a t t e t r a t h i o n a t e has been rep o r t e d to r e a c t with tryptophan r e s i d u e s i n p r o t e i n s ( I n g l i s and L i u , 1970) g i v e s support to t h i s h y p o t h e s i s . However, s i n c e amino a c i d a n a l y s i s of the modified papain was not c a r r i e d out, no d e f i n i t i v e c o n c l u s i o n can be drawn. S i m i l a r to the CD r e s u l t s , no d i f f e r e n c e s i n the absorbance s p e c t r a was obtained between n a t i v e and t e t r a t h i o n a t e - m o d i f i e d papain (molar r a t i o of t e t r a t h i o n a t e to papain f o r the chemical m o d i f i c a t i o n ^ 10 to 500). T h i s data a l s o suggests t h a t no c o n f o r m a t i o n a l change occurred i n papain upon chemical m o d i f i c a t i o n with t e t r a t h i o n a t e . A c t i v a t i o n with c y s t e i n e d i d not a f f e c t the u l t r a v i o l e t -217-J H 1 0 0 - 0 CD 20-j 1 1 1 1 1 1 1 1 1 1 1 0 100 200 300 400 500 [Tetrathionate]/[Papain] (mol/mol) Figure 42. Effect of the molar ratio of tetrathionate to papain during the chemical modification on the relative fluorescence intensity at 352 nm (pH 6.8 and 22°C). -218-spectrum of t e t r a t h i o n a t e m o dified papain. As r e p o r t e d by B a r e l and Glaser (1969) the same was a l s o true f o r n a t i v e papain. G. DETERMINATION OF SH GROUPS IN NATIVE AND TETRATHIONATE-MODIFIED PAPAIN Table 25 summarizes the r e s u l t s of SH d e t e r m i n a t i o n of n a t i v e and t e t r a t h i o n a t e - m o d i f i e d papain. Native, u n a c t i v a t e d papain ( i . e . n a t i v e papain t h a t was not a c t i v a t e d with c y s t e i n e and EDTA) showed a content of 0.1+0.02 mole of SH/mole of papain. As expected, upon a c t i v a t i o n with c y s t e i n e , the content of SH groups i n c r e a s e d s i g n i f i c a n t l y to 0.7+0.06 mole of SH/mole of papain. Since no a f f i n i t y chromatography technique was used to separate a c t i v e from i n a c t i v e papain, i t was not p o s s i b l e to prepare papain with 1 mole of SH/mole of p r o t e i n . The r e a c t i o n of a c t i v a t e d papain ( f r e e d from a c t i v a t o r s by g e l f i l t r a t i o n ) with t e t r a t h i o n a t e (molar r a t i o of t e t r a t h i o n a t e to papain = 10 to 500) caused the complete disappearance of SH. Incubation of t h i s m o d ified papain with 40 mM c y s t e i n e and 10 mM EDTA r e s t o r e d the content of SH group of the m o d i f i e d papain to a l e v e l v ery s i m i l a r to t h a t found i n n a t i v e , a c t i v a t e d papain. These r e s u l t s c o n f i r m the f a c t t h a t t e t r a t h i o n a t e r e a c t s i n a r e v e r s i b l e manner with the s u l f h y d r y l group of papain. -219-Table 2 5 . S u l f h y d r y l content of n a t i v e and t e t r a t h i o n a t e - m o d i f i e d papain (TT-papain)*-. P r o t e i n SH con t e n t * 3 ' 0 (moles SH/ mol p r o t e i n ) N o n - a c t i v a t e d 0 Native papain TT-papain 0.1 ±0.02 0.0 ±0.00 Activated™ Native papain TT-papain 0.7 ±0.06 0.6 ±0.10 *-Molar r a t i o t e t r a t h i o n a t e papain d u r i n g i n a c t i v a t i o n = 200. B A molecular weight of 23,800 was used f o r a l l c a l c u l a t i o n s . °Mean ±S.D (n= 4 ) D N o n - a c t i v a t e d i n d i c a t e s samples t h a t were not incubated with c y s t e i n e and EDTA p r i o r to the assay. " A c t i v a t e d i n d i c a t e s samples t h a t were incubated with c y s t e i n e and EDTA, and then f r e e d from c y s t e i n e , under n i t r o g e n s a t u r a t e d c o n d i t i o n s , by g e l f i l t r a t i o n , p r i o r to the assay. -220-H. INSOLUBILIZATION OF PAPAIN WITH TETRATHIONATE P r e l i m i n a r y experiments showed t h a t when a papain s o l u t i o n was heated to 60°C and 0-mercaptoethanol and t e t r a t h i o n a t e were added, p r e c i p i t a t i o n of the p r o t e i n o c c u r r e d . I t i s important to p o i n t out t h a t t h i s i n s o l u b i l i z a t i o n of papain with t e t r a t h i o n a t e was not an o b j e c t i v e of t h i s t h e s i s r e s e a r c h ; moreover, i t ' s occurrence was not expected. Since t h i s p r e c i p i t a t i o n was an e f f e c t of the chemical m o d i f i c a t i o n of papain by t e t r a t h i o n a t e , a s e r i e s of experiments were conducted i n order to g i v e more i n s i g h t i n t o t h i s phenomenon. The e f f e c t of c o n c e n t r a t i o n of t e t r a t h i o n a t e and 0-mercaptoethanol on the p r e c i p i t a t i o n of papain i s d e p i c t e d i n F i g . 43. T h i s response s u r f a c e was generated from the q u a d r a t i c model f i t t e d to the data obtained u s i n g RSM (Table 26). The f i t t e d model was shown to be h i g h l y s i g n i f i c a n t (Table 27). Up to 90% of the o r i g i n a l papain was p r e c i p i t a t e d with the combined a d d i t i o n of 100 mM 0-mercaptoethanol and 50 mM t e t r a t h i o n a t e . A d d i t i o n of 0-mercaptoethanol alone d i d not produce high l e v e l s of p r e c i p i t a t i o n ( F i g . 43). Regarding the e f f e c t of pH and temperature on the p r e c i p i t a t i o n of papain with t e t r a t h i o n a t e , i t was found t h a t p r a c t i c a l l y no p r e c i p i t a t i o n occurred a t a b a s i c pH (pH >8) at a l l the i n c u b a t i o n temperatures t e s t e d . At a n e u t r a l pH (pH 6.8), very s l i g h t p r e c i p i t a t i o n occurred a t room temperature. The h i g h e s t l e v e l of p r o t e i n p r e c i p i t a t i o n occurred when the pH -221-- 2 2 2 -Table 26. Experimental data f o r the two-factor, f i v e - l e v e l response s u r f a c e a n a l y s i s of the e f f e c t of t e t r a t h i o n a t e (TT) and 6-mercaptoethanol (13-ME) c o n c e n t r a t i o n on the p r e c i p i t a t i o n of papain. F a c t o r , mM TT 13-ME % P r o t e i n Treatment p r e c i p i t a t e d * " B 1 15 15 47. 21 2 85 15 77.28 3 15 85 67.43 4 85 85 88.63 5 50 100 90.20 6 50 100 62.00 7 100 50 81.90 8 0 50 17.39 9 50 50 81.39 10 50 50 83.81 ^ P r o t e i n p r e c i p i t a t e d was c a l c u l a t e d as f o l l o w s : (1 - mg p r o t e i n i n supernatant a f t e r treatment ) x 100 i n i t i a l mg p r o t e i n i n s o l u t i o n s P r o t e i n was determined u s i n g the Bio-Rad p r o t e i n assay -223-Table 27. A n a l y s i s of v a r i a n c e f o r the second order model*-for the p r e c i p i t a t i o n of papain by t e t r a t h i o n a t e , obtained u s i n g backward stepwise m u l t i p l e r e g r e s s i o n . Source of v a r i a t i o n DF Mean Square F-value Model E r r o r T o t a l 5 13219.308 4 60.724 9 217.695* A R * ( a d j u s t e d f o r DF) = 0.990 " s i g n i f i c a n t at p<0.001 Var i a b l e J C o e f f i c i e n t T-value P r o b O |Tj ) X* X** XiX 2 0.481 1.956 -0.012 -0.004 9.574 4.783 •4.981 -1.870 0.0004 0.0031 0.0025 0.1107 *Code: Xi X2 (0-mercaptoethanol] (mM) [ T e t r a t h i o n a t e ] (mM) -224-of the papain s o l u t i o n was not adju s t e d with NaOH (pH 5.0) (Table 28). I t was a l s o observed t h a t p r e c i p i t a t i o n occurred almost i n s t a n t a n e o u s l y a t 60°C. Upon a d d i t i o n of t e t r a t h i o n a t e to the s o l u t i o n of papain c o n t a i n i n g 0-mercaptoethanol, a drop i n pH occurred ( F i g . 44) , p o s s i b l y due to the formation of some H2S v i a r e d u c t i o n of the t e t r a t h i o n a t e with 0-mercaptoethanol. Experiments i n which the pH of a papain s o l u t i o n with 0-mercaptoethanol was decreased by a d d i t i o n of 0.1 N HC1, showed t h a t the drop of pH per se was not r e s p o n s i b l e f o r the p r e c i p i t a t i o n . A d d i t i o n of thioacetamide, a compound which y i e l d s H2S upon s o l u b i l i z a t i o n i n water, to a papain s o l u t i o n with 0-mercaptoethanol d i d not produce any p r e c i p i t a t i o n . Thus, the p o s s i b i l i t y t h a t the drop i n pH or the formation of H2S was r e s p o n s i b l e f o r the p r e c i p i t a t i o n of the enzyme was r u l e d out. P r o t e i n a n a l y s i s of the p r e c i p i t a t e d m a t e r i a l by m i c r o - K j e l d a h l i n d i c a t e d t h a t i t c o n s i s t e d of almost pure p r o t e i n (70-90% p r o t e i n d.b.). Ne i t h e r urea nor SDS at a f i n a l c o n c e n t r a t i o n of 8 M and 10% r e s p e c t i v e l y , were able to r e s o l u b i l i z e the p r e c i p i t a t e d p r o t e i n . A d d i t i o n of 0-mercaptoethanol to a f i n a l c o n c e n t r a t i o n of 100 mM s o l u b i l i z e d approximately 70% of the i n s o l u b l e p r o t e i n . A d d i t i o n of SDS (10% f i n a l c o n c e n t r a t i o n ) together with 100 mM of 0-mercaptoethanol was the c o n d i t i o n i n which the hi g h e s t l e v e l of s o l u b i l i z a t i o n was achieved (Table 29). -225-Table 28. E f f e c t of temperature and pH on the p r e c i p i t a t i o n of papain by t e t r a t h i o n a t e * . C o n d i t i o n s of i n c u b a t i o n % P r o t e i n pH Temperature (<>C) p r e c i p i t a t e d * * ' 0 ' 0 5.0 22 10.5 ± 1.9 5.0 40 30.3 ± 2.0 5.0 60 82.8 ± 2.9 6.8 22 4.7 ± 1.0 6.8 40 13.9 ±- 3.6 6.8 60 23.5 + 2.2 8.0 22 3.0 ± 0.7 8.0 40 2.2 + 0.3 8.0 60 3.3 + 0.1 10.0 60 2.0 + 1.0 *A11 samples were incubated with 100 mM of 3-mercaptoethanol and 100 mM of t e t r a t h i o n a t e . (See MATERIALS AND METHODS f o r d e t a i l s ) B P r o t e i n p r e c i p i t a t e d was c a l c u l a t e d as f o l l o w s : (1 - mg p r o t e i n i n supernatant a f t e r treatment ) x 100 i n i t i a l mg p r o t e i n i n s o l u t i o n °Protein was determined u s i n g the Bio-Rad P r o t e i n assay. "Values are Mean±S.D (n=3) -226-2.00 | i i i i i i i i i | i i i i i i i i i i i 0 100 200 i i i i i i i i i i i i i i i i i I 300 400 Reaction time (sec) F i g u r e 44. Change of pH d u r i n g the r e a c t i o n between t e t r a t h i o n a t e , 0-mercaptoethanol and papain. F i n a l [ t e t r a t h i o n a t e ] = [0-mercaptoethanol] = 100 mM, F i n a l [papain] = 10 mg/mL. - 2 2 7 -Table 29. E f f e c t of d i f f e r e n t reagents on the r e s o l u b i l i z a t i o n of the p r e c i p i t a t e d papain. Reagent F i n a l c o n c e n t r a t i o n % R e s o l u b i l i z a t i o n * " B ' a Urea 8 M 3.0 SDS 10 % 4.9 0-ME** 100 mM 75.3 Urea - SDS 8 M, 10% 5.3 Urea - 0-ME 8 M, 100 mM 72.9 0-ME - SDS 100 mM, 10% 80.3 *•% R e s o l u b i l i z a t i o n was c a l c u l a t e d as f o l l o w s : ( mg p r o t e i n i n supernatant a f t e r treatment ) x 100 i n i t i a l mg p r o t e i n i n the p r e c i p i t a t e B P r o t e i n was determined u s i n g M i c r o - K j e l d a h l . cMean of two r e p l i c a t e s . -228-The I n s o l u b l e p r o t e i n was found to have no p r o t e o l y t i c a c t i v i t y (Table 30). However, i n c u b a t i o n of the i n s o l u b l e p r o t e i n with 0-mercaptoethanol, i n a d d i t i o n to r e s o l u b i l i z a t i o n , caused a p a r t i a l r e a c t i v a t i o n of the p r o t e o l y t i c a c t i v i t y . But on a p r o t e i n b a s i s , the p r o t e o l y t i c a c t i v i t y of the i n s o l u b l e papain a f t e r i n c u b a t i o n with 0-mercaptoethanol was much l e s s (approximately 20 times) when compared to n a t i v e papain (Table 30). SDS-Gel e l e c t r o p h o r e s i s of the i n s o l u b l e papain produced a s i m i l a r p a t t e r n to t h a t of n a t i v e papain ( F i g . 45). As i n d i c a t e d i n the MATERIALS AND METHODS s e c t i o n , p r e p a r a t i o n of the samples f o r the e l e c t r o p h o r e s i s i n c l u d e d a d d i t i o n of 0-mercaptoethanol together with SDS and h e a t i n g i n b o i l i n g water. T h i s treatment caused complete r e s o l u b i l i z a t i o n of the i n s o l u b l e p r o t e i n . T h i s r e s u l t g i v e s support to the hypothesis t h a t d i s u l f i d e bond formation caused p r e c i p i t a t i o n of papain. At t h i s moment i t can not be s a i d i f i n t r a - or i n t e r - molecular bond formation was i n v o l v e d . Taking i n t o c o n s i d e r a t i o n t h a t papain has o n l y one f r e e s u l f h y d r y l group per molecule, together with the r e s i s t a n c e of i t s d i s u l f i d e bonds to r e d u c t i o n , i t i s more l i k e l y t h a t i n t e r -molecular d i s u l f i d e bonds were formed. E l e c t r o p h o r e s i s was a l s o t r i e d without re d u c i n g the i n s o l u b l e p r o t e i n ( i . e . without i n c u b a t i o n with 0-mercaptoethanol), however, s i n c e no r e s o l u b i l i z a t i o n was achieved i n the presence of SDS alone, i t was impossible to c a r r y out the e l e c r o p h o r e s i s . -229-Table 3 0 . P r o t e o l y t i c a c t i v i t y of n a t i v e and i n s o l u b l e papain. P r o t e i n % P r o t e o l y t i c a c t i v i t y ' Native non-activated° 2.0 a c t i v a t e d * 5 100.0 I n s o l u b l e n o n - a c t i v a t e d 0.0 a c t i v a t e d 6.5 a c t i v a t e d 3 12.5 *•% P r o t e o l y t i c a c t i v i t y (P.A) was c a l c u l a t e d as f o l l o w s : P.A of sample x 100 P.A of n a t i v e , a c t i v a t e d papain P.A was expressed i n )ig t y r o s i n e min"1 mg p r o t e i n " 1 BMean of two r e p l i c a t e s . °Non-activated i n d i c a t e s samples t h a t were not incubated with 0-mercaptoethanol and EDTA p r i o r to the assay. D A c t i v a t e d i n d i c a t e s samples t h a t were incubated with 0-mercaptoethanol (10 mM) and EDTA (10 mM) p r i o r to to the assay. "Sample t h a t was a c t i v a t e d with 100 mM of 0-mercaptoethanol and 10 mM EDTA p r i o r to the asay. -230-I 1. 4 * - . • -t. . '-. 1 2 F i g u r e 45. Electrophoretogram of s o l u b l e and i n s o l u b l e papaya - 2 3 1 -P a r a l l e l experiments showed that t e t r a t h i o n a t e a l s o caused the p r e c i p i t a t i o n of the p r o t e i n components of papaya l a t e x (prepared as d e s c r i b e d i n MATERIALS AND METHODS of CHAPTER 1) and of a sample of commercial papain (PANOL, Enzyme Technology Corp. New York, NY) under the same c o n d i t i o n s t h a t caused p r e c i p i t a t i o n of pure papain. SDS-gel e l e c t r o p h o r e s i s of the i n s o l u b l e m a t e r i a l r e s u l t i n g from the r e a c t i o n of t e t r a t h i o n a t e with papaya l a t e x and the commercial papain sample a l s o gave p a t t e r n s s i m i l a r to the corresponding s o l u b l e m a t e r i a l s ( F i g . 46). SDS-gel e l e c t r o p h o r e s i s under non-reducing c o n d i t i o n s of s o l u b l e c h e m i c a l l y modified papain (molar r a t i o of t e t r a t h i o n a t e to papain = 200) showed the formation of a high molecular weight f r a c t i o n not o r i g i n a l l y present i n n a t i v e papain ( F i g . 47). I t appears t h a t even under c o n d i t i o n s where p r e c i p i t a t i o n d i d not occur t e t r a t h i o n a t e caused formation of high molecular weight aggregates. These r e s u l t s suggest t h a t i n s o l u b i l i z a t i o n or p r e c i p i t a t i o n of papain with t e t r a t h i o n a t e was mainly due to the formation of aggregates v i a d i s u l f i d e interchange s i n c e o n l y (3-mercaptoethanol produced a s i g n i f i c a n t r e s o l u b i l i z a t i o n of the p r e c i p i t a t e d papain. D i s u l f i d e interchange u s u a l l y occurs at higher r a t e s when the pH of the r e a c t i o n i s b a s i c (pH>8). In t h i s case, no p r e c i p i t a t i o n occurred when the pH of the r e a c t i o n was b a s i c . Moreover, g r e a t e r p r e c i p i t a t i o n occurred at pH 5.0. A p o s s i b l e -232-2 3 4 5 6 7 8 F i g u r e 46. Electrophoretogram of n a t i v e (1) and i n s o l u b l e (2) papain. (1) s o l u b l e commercial papain (Calbiochem p a p a i n ) . (2) i n s o l u b l e commercial papain (Calbiochem p a p a i n ) . (3) i n s o l u b l e commercial papain (PANOL). (4) s o l u b l e commercial papain (PANOL). (5) s o l u b l e pure papain. (6) and (7) i n s o l u b l e papaya l a t e x . (8) s o l u b l e papaya l a t e x . -233-1 2 3 4 5 6 7 8 F i g u r e 4 7 . Electrophoretogram under non-reducing c o n d i t i o n s of s o l u b l e t e t r a t h i o n a t e - m o d i f i e d pure papain and of t e t r a t h i o n a t e - t r e a t e d commercial papain and papaya l a t e x . (1) t e t r a t h i o n a t e - m o d i f i e d pure papain (molar r a t i o of t e t r a t h i o n a t e to papain = 200). (2) t e t r a t h i o n a t e - m o d i f i e d pure papain (molar r a t i o of t e t r a t h i o n a t e to papain = 100). (3) c o n t r o l pure papain. (4) and (5) t e t r a t h i o n a t e - t r e a t e d (1%, w/w) commercial papain (PANOL) (6) t e t r a t h i o n a t e - t r e a t e d (1%, w/w) papaya l a t e x . (7) p r o t e i n s t a n d a r s . (8) c o n t r o l papaya l a t e x . -234-e x p l a n a t i o n of t h i s pH e f f e c t can be r e l a t e d to the s t a b i l i t y of papain a t d i f f e r e n t pH's. I t i s w e l l known t h a t papain i s u n s t a b l e a t a c i d i c pH (Arnon, 1970). Therefore i t i s p o s s i b l e t h a t the a c i d i c pH caused d e s t a b i l i z a t i o n of the papain molecule which f a c i l i t a t e d the formation of aggregates. Reduction of papain with 0-mercaptoethanol p r i o r to the a d d i t i o n of t e t r a t h i o n a t e was e s s e n t i a l f o r p r e c i p i t a t i o n , i n d i c a t i n g t h at e i t h e r the presence of 0-mercaptoethanol and/or the reduced papain molecule were i n v o l v e d i n the p r e c i p i t a t i o n mechanism. Although r e s o l u b i l i z a t i o n of the p r e c i p i t a t e d papain was p o s s i b l e , the lower p r o t e o l y t i c a c t i v i t y of the r e s o l u b i l i z e d papain compared to n a t i v e papain suggested t h a t i r r e v e r s i b l e d e n a t u r a t i o n had o c c u r r e d . Taking i n t o c o n s i d e r a t i o n the high s t a b i l i t y of papain to temperature and d e n a t u r i n g agents such as urea, t h a t t e t r a t h i o n a t e induced p r e c i p i t a t i o n of papain under r e l a t i v e l y m i l d c o n d i t i o n s was not expected. F u r t h e r r e s e a r c h i s needed i n order to have a b e t t e r understanding of t h i s phenomenon. -235-K. KINETIC PARAMETERS FOR THE PAPAIN CATALYZED HYDROLYSIS OF CARBOBENZOXYGLYCINE P-NITROPHENYL ESTER The r e a c t i o n s of the enzyme with the n i t r o p h e n y l e s t e r of carbobenzoxyglycine at s e v e r a l d i f f e r e n t i n i t i a l e s t e r c o n c e n t r a t i o n s are shown i n F i g . 48. Since the r e a c t i o n r a t e f o r t h i s enzymatic r e a c t i o n was r e l a t i v e l y high, i t was necessary to design a system that permitted r a p i d a d d i t i o n of the enzyme s o l u t i o n i n t o the cuvette placed i n the c e l l compartment of the spectrophotometer, i n order to o b t a i n accurate r e s u l t s . The system developed i s shown i n F i g . 49. A d d i t i o n of the enzyme s o l u t i o n i n t o the cuvette c o n t a i n i n g the s u b s t r a t e s o l u t i o n p laced i n the c e l l compartment of the spectrophotometer was performed with an automatic p i p e t t o r ( D i s p e n s e t t e , Brinkmann Brand, W. Germany), c a l i b r a t e d to dispense e x a c t l y 2.8 mL of the papain s o l u t i o n . As d e p i c t e d i n F i g . 50 t h i s system permitted an accurate d e t e r m i n a t i o n of the zero time of the r e a c t i o n . As r e p o r t e d by K i r s c h and Ingelstrom (1966) the i n i t i a l r a t e s of the r e a c t i o n s were not very s e n s i t i v e to s u b s t r a t e c o n c e n t r a t i o n , i n d i c a t i n g t h a t the Km was very low. As expected, the r a t e of appearance of n i t r o p h e n o l i n the absence of enzyme was a p p r e c i a b l e and was dependent upon the c o n c e n t r a t i o n of the s u b s t r a t e . Values of B and r e a c t i o n order f o r each i n i t i a l s u b s t r a t e c o n c e n t r a t i o n c a l c u l a t e d using the l i n e a r i z a t i o n procedure are shown i n Table 31. For the c a l c u l a t i o n of B v a l u e s , o n l y the data of s u b s t r a t e c o n c e n t r a t i o n obtained every 2 sec up to 20-30 sec were used. The r e a c t i o n s were 80-95% completed -236-0.40 -, Time (sec) Figure 48. Papain-catalyzed hydrolysis of carbobenzoxyglycine p-nitrophenyl ester at pH 6.8 and 22°C. The reaction mixture contained 0.02 M sodium phosphate buffer, 1 mM EDTA, 0.35 mM cysteine, 6.7% (v/v) a c e t o n i t r i l e and 3.3 x 10-"7 papain. I n i t i a l substrate concentration as shown. -237-Cell compartment of the Spectophotometer Tygon tubing r~ \ Cuvette with substrate Nitrogen v • ^et J Automatic pipette Papain solution F i g u r e 49. Schematic diagram of the system used for the enzyme k i n e t i c s experiments. -238-O time zero Reaction Time Figure 50. Idealized progress reaction curve (A 4 0o vs. time) for the papain-catalyzed hydrolysis of carbobenzoxyglycine p-nitrophenyl ester. -239-Table 31. Values of B and corresponding e m p i r i c a l r e a c t i o n order with r e s p e c t to time ( n T ) at d i f f e r e n t i n i t i a l s u b s t r a t e c o n c e n t r a t i o n s f o r the r e a c t i o n of papain-carbobenzoxyglycine p - n i t r o p h e n y l e s t e r . [ I n i t i a l ] Best estimate 10"5M of B n T 19 .0 -0.19 1.19 9 . 52 -0.05 1.05 6.66 -0.20 1.20 4.76 1.00 0. 00 2.76 1.00 0.00 0.92 1.00 0.00 -240-w i t h i n t h i s p e r i o d . T h i s agrees with the recommendation o£ Durance et a l . (1986) who i n d i c a t e d t h a t , f o r accurate d e t e r m i n a t i o n of the r e a c t i o n r a t e s using t h i s l i n e a r i z a t i o n approach, the s u b s t r a t e d e p l e t i o n data used f o r c a l c u l a t i o n s should i n c l u d e data when at l e a s t 80% of the i n i t i a l s u b s t r a t e has been transformed to the corresponding product. Regression equations using o n l y data f o r the i n i t i a l 20-30 sec of the r e a c t i o n and t h e i r r* values are shown i n Table 32, together with the i n i t i a l v e l o c i t i e s d e r i v e d from those equations. The d e r i v e d V 0 values and V 0 values estimated by f i x e d time assay were used to compute Km and Vmax (Table 33). Both methods gave s i m i l a r r e s u l t s . No r e d u c t i o n i n the standard e r r o r was obtained u s i n g the l i n e a r i z a t i o n procedure. The Km and Vmax values obtained i n t h i s experiment were w i t h i n the range of p r e v i o u s l y r e p o r t e d values (Table 13). Although the l i n e a r i z a t i o n procedure of Durance et a l . (1986) d i d not improve the accuracy of the de t e r m i n a t i o n of the k i n e t i c parameters Km and Vmax i n t h i s case, through the use of t h i s methodology i t was p o s s i b l e to o b t a i n the equation t h a t r e l a t e s the change of v e l o c i t y of the r e a c t i o n to time. T h i s r e l a t i o n s h i p was used to determine the r a t e constant of i n a c t i v a t i o n . T h i s approach can o n l y be used when product i n h i b i t i o n does not occur and the s u b s t r a t e c o n c e n t r a t i o n i s high enough to remain at s a t u r a t i n g c o n d i t i o n s d u r i n g the course of a l l of the r e a c t i o n , so t h a t decrease i n v e l o c i t y ( i . e . a c t i v i t y ) i s due to the r e a c t i o n of the i n h i b i t o r with the enzyme and not to the -241-Table 32. R e s u l t s of curve f i t t i n g the k i n e t i c data f o r the r e a c t i o n of papain-carbobenzoxyglycine p - n i t r o p h e n y l e s t e r a t v a r i o u s i n i t i a l s u b s t r a t e c o n c e n t r a t i o n s . I n i t i a l v e l o c i t i e s (V 0) determined by the f i x e d time assay method are i n c l u d e d f o r comparative purposes. [ I n i t i a l ] (10-»M) Regression Equation*' r * Derived F i x e d time 19 .00 y-o.it - 0.0092X + 5 .105 0.99 1.80 1.49 9.50 y-o. so— 0.0016X + 1 .590 0.99 1.80 1.38 6.60 yo.io = -7.0 x 10"«X + 0.1457 0.99 1.58 1.38 4.76 Y= -1 .2 x 10"SX + 4.5 x 10"s 0.96 1.20 1.24 2.86 Y= -1 .1 x 10-«X + 3.3 x 10-9 0.96 1.10 1.04 0.92 Y= -7 .2 x 10-TX + 2.6 x 10"s 0.93 0.72 0.61 x U n i t s : Y, 10-SM r e s i d u a l s u b s t r a t e ; X, sec B U n i t s : (10- 8M)/sec -242-Table 33. K i n e t i c parameters of the papain-carbo-benzoxyglycine p - n i t r o p h e n y l e s t e r r e a c t i o n (± standard e r r o r ) , computed with the program of O e s t r e i c h e r and P i n t o (1983) using i n i t i a l v e l o c i t i e s estimated by f i x e d time assays or d e r i v e d from e x p e r i m e n t a l l y determined curves. Parameter Fi x e d time method D i f f e r e n t i a l method Km* 1.53 ± 0.5 1.70 ± 0.6 Vmax B 1.63 ± 0.2 1.95 ± 0.2 Kcat° 8.15 ± 0.2 9.75 ± 0.3 * 10-'M B (10- 6M)/sec c sec -x -243-decrease i n s u b s t r a t e c o n c e n t r a t i o n . L. CHARACTERIZATION OF THE INHIBITORY EFFECT OF TETRATHIONATE 1. R e v e r s i b l e i n h i b i t i o n Since l i n e a r i z a t i o n d i d not improve the accuracy of the det e r m i n a t i o n of Km and Vmax, the fi x e d - t i m e method was used to estimate the i n i t i a l v e l o c i t i e s i n these experiments. Assuming t h a t t e t r a t h i o n a t e acted as a r e v e r s i b l e i n h i b i t o r of papain, i t was found t h a t the r a t e of i n h i b i t i o n or i n a c t i v a t i o n of papain by t e t r a t h i o n a t e decreased as the s u b s t r a t e c o n c e n t r a t i o n i n c r e a s e d . Using the computer program ENZYME (Lutz et a l . , 1986) to analyze the experimental data i t was found t h a t t e t r a t h i o n a t e s i g n i f i c a n t l y a f f e c t e d the Km (p<0.005) but not Vmax, i n d i c a t i n g t h a t t e t r a t h i o n a t e acted as a competitive i n h i b i t o r of papain. T h i s r e s u l t i s c o n s i s t e n t with the nature of the r e a c t i o n of t e t r a t h i o n a t e with Cys-25, which i s pa r t of the a c t i v e - s i t e of papain. Most r e f e r e n c e s i n d i c a t e t h a t i f an i r r e v e r s i b l e i n h i b i t o r r e a c t s with a group near or i n the a c t i v e s i t e , such as t e t r a t h i o n a t e with the onl y c y s t e i n e r e s i d u e of papain, i t s mode of i n h i b i t i o n should be c o m p e t i t i v e . The r e s u l t s i n t h i s study support t h i s suggested mechanism. -244-A Lineweaver-Burk p l o t of the enzyme a c t i v i t y of papain a t three d i f f e r e n t c o n c e n t r a t i o n s of t e t r a t h i o n a t e i s shown i n F i g . 51. I t c l e a r l y shows the c o m p e t i t i v e i n h i b i t i o n nature of t e t r a t h i o n a t e on the a c t i v i t y of papain. I t was necessary to use r e l a t i v e l y high c o n c e n t r a t i o n s of t e t r a t h i o n a t e i n order to measure changes i n the i n i t i a l v e l o c i t i e s a t the d i f f e r e n t s u b s t r a t e c o n c e n t r a t i o n s e v a l u a t e d . A i n h i b i t i o n or d i s s o c i a t i o n constant (Ki) of 6.6 X 10~4 was obtained by means of the computer program ENZYME. T h i s value i n d i c a t e s t h a t t e t r a t h i o n a t e d i d not bind t o papain as t i g h t l y as other i n h i b i t o r s such as E-64 (Ki=1.8 X 10"«) (Hanada et a l , , 1978) or c y s t a t i n (Ki < 5"") ( N i c k l i n and B a r r e t t , 1984). Since t e t r a t h i o n a t e i s an i n o r g a n i c compound, I t may not have as high a f f i n i t y f o r the a c t i v e s i t e of papain as more complex molecules which a c t as s u b s t r a t e analogs of papain. 2. I r r e v e r s i b l e i n h i b i t i o n (a) In the presence of s u b s t r a t e F i g u r e 52 shows the i n h i b i t o r y e f f e c t of a constant c o n c e n t r a t i o n of t e t r a t h i o n a t e a t three d i f f e r e n t c o n c e n t r a t i o n s of s u b s t r a t e . I t i s evident t h a t a high c o n c e n t r a t i o n of s u b s t r a t e p r o t e c t e d papain from i n h i b i t i o n . Since a r e l a t i v e l y high c o n c e n t r a t i o n of the i n h i b i t o r was used, the r e a c t i o n was assumed to be f i r s t order (Eq. 5) and the p s e u d o - f i r s t order r a t e constant of i n h i b i t i o n at each s u b s t r a t e c o n c e n t r a t i o n was -245-Figure 51. Lineveaver-Burk plot for the papain a c t i v i t y at different concentrations of tetrathionate. 10.6 x IO'4 M (•) 20.6 x 10"* M («A) 31.8 x IO"4 M (O) Without tetrathionate (O) -246-F i g u r e 52. I n h i b i t o r y e f f e c t of t e t r a t h i o n a t e (TT) i n the presence of three d i f f e r e n t i n i t i a l s u b s t r a t e c o n c e n t r a t i o n a t a constant TT c o n c e n t r a t i o n (1.06 x 1 0 - 3 M. The three top l i n e s are the r e a c t i o n s without TT. The i n i t i a l s u b s t r a t e s c o n c e n t r a t i o n were: 1.90 x 10-" ( O ,e ) 0.95 x 10-" (Q ,0 ) 0.48 x 10- 4 ( A ,if ) - 2 4 7 -c a l c u l a t e d . The second order r a t e constant was obtained i n the usual way by d i v i d i n g the p s e u d o - f i r s t order constant by the i n h i b i t o r c o n c e n t r a t i o n . In order to c h a r a c t e r i z e the i r r e v e r s i b l e mode of i n h i b i t i o n of papain by t e t r a t h i o n a t e , a p l o t of the i n v e r s e of the second order r a t e constant (k) a g a i n s t s u b s t r a t e c o n c e n t r a t i o n as suggested by Tsou e t a l . (1985) was e v a l u a t e d . F i g . 53 shows a l i n e a r r e l a t i o n s h i p (r*=0.98) which i n d i c a t e s t h a t t e t r a t h i o n a t e i s an i r r e v e r s i b l e c o m p e t i t i v e i n h i b i t o r of papain. (b) In the absence of s u b s t r a t e F i g . 54-56 summarize the changes i n the c a t a l y t i c a c t i v i t y of papain which occurred upon treatment of t h i s enzyme with t e t r a t h i o n a t e i n the absence of s u b s t r a t e . A p e r i o d of about 10 min was r e q u i r e d f o r 90-95% i n a c t i v a t i o n at l e v e l s of 1 mole of t e t r a t h i o n a t e per mole of papain ( F i g . 54). At l e v e l s of l e s s than 3 moles of t e t r a t h i o n a t e per mole of enzyme, the degree of i n a c t i v a t i o n obtained a f t e r 30 sec of i n c u b a t i o n appeared to be l i n e a r l y r e l a t e d to the r a t i o of reagent to enzyme ( F i g . 55). The k i n e t i c s of papain i n a c t i v a t i o n by t e t r a t h i o n a t e a t l e v e l s of 1-3 moles of t e t r a t h i o n a t e per mole of enzyme at 22<>C was second order. Using Eq. 11 ( when mole t e t r a t h i o n a t e / m o l e papain =1) or Eq. 12 (when mole t e t r a t h i o n a t e / m o l e papain >1) the value of k (second order r a t e constant f o r t e t r a t h i o n a t e i n a c t i v a t i o n ) was 16,919 M-Xsec'1 ( F i g . 56). -248-F i g u r e 53. E f f e c t of carbobenzoxyglycine p - n i t r o p h e n y l on the second order r a t e constant of i n a c t i v a t i o n of papain by t e t r a t h i o n a t e . (Mean±S.D., n=3). The r e g r e s s i o n l i n e i s shown. -249-100.00 H . -o 0) c LY > o < 80.00 -Molar ratio Tetrathionate:Papain 0 1:1 2:1 3:1 60.00 40.00 -20.00 0.00 100 200 Time (sec) i — i — i — i — i 300 400 Figure 54. Change i n the c a t a l y t i c a c t i v i t y of papain as as a r e s u l t of the r e a c t i o n with t e t r a t h i o n a t e A c t i v i t y was measured with carbobenzoxyglycine p - n i t r o p h e n y l e s t e r as the s u b s t r a t e . -250-F i g u r e 55. E f f e c t of the molar r a t i o of t e t r a t h i o n a t e to papain on the c a t a l y t i c a c t i v i t y of papain at two r e a c t i o n times. A c t i v i t y was measured with carbobenzoxyglycine p - n i t r o p h e n y l e s t e r as the s u b s t r a t e . -251-0 . 1 2 - i 0 . 1 0 i . 0 . 0 8 Molar ratio Enzyme : Tetrathionate 0 1 : 1 A 1 : 2 1 : 3 O ° - 0 6 0 . 0 4 -0 . 0 2 z 0 . 0 0 0 i i i i I i i 1 0 0 1 I I I I I I I I I I I I I 2 0 0 3 0 0 4 0 0 Time (sec) i i i i i i i i * 1 * * i 5 0 0 6 0 0 7 0 0 F i g u r e 56. Progress of i n a c t i v a t i o n of papain, by d i f f e r e n t l e v e l s of t e t r a t h i o n a t e (TT). The value of Y was c a l c u l a t e d based on E q . l l when the molar r a t i o of TT to papain was 1:1. For the other molar r a t i o s Eq.12 was used. A c t i v i t y was measured with carbobenzoxyglycine p - n i t r o p h e n y l e s t e r as the s u b s t r a t e . -252 Table 34 presents values o£ second order r a t e constants f o r d i f f e r e n t i r r e v e r s i b l e i n a c t i v a t o r s of papain and a l s o f o r the i n a c t i v a t i o n of guinea p i g l i v e r transglutaminase and glyceraldehyde-3-dehydrogenase with t e t r a t h i o n a t e . With the ex c e p t i o n of E-64 (k= 638,000 M^sec - 1) and c y s t a t i n (k= 1.0 X 10 7 M _ 1sec _ x), papain r e a c t s f a s t e r with t e t r a t h i o n a t e than with other i n a c t i v a t o r s such as iodoacetamide or hydrogen peroxide. According to the values of k r e p o r t e d f o r the i n a c t i v a t i o n of other enzymes with t e t r a t h i o n a t e , i t would appear that t e t r a t h i o n a t e r e a c t s f a s t e r with papain than with transglutaminase and dehydrogenase. In a recent a r t i c l e , Prasad and Horowitz (1987) r e p o r t e d t h a t i n a c t i v a t i o n of bovine l i v e r rhodanase (EC 2.8.1.1) with t e t r a t h i o n a t e was instantaneous and a l i n e a r f u n c t i o n of t e t r a t h i o n a t e c o n c e n t r a t i o n . Although no k was r e p o r t e d , the term "instantaneous" could i n d i c a t e t h a t the k f o r the r e a c t i o n of rhodanase with t e t r a t h i o n a t e was s i g n i f i c a n t l y higher than the r e a c t i o n of t e t r a t h i o n a t e with papain. Since the r e a c t i v i t y of c y s t e i n e r e s i d u e s i n p r o t e i n s i s a f f e c t e d by neighboring groups (Means and Feeney, 1971) i t i s w e l l known t h a t p r o t e i n s r e a c t a t d i f f e r e n t r a t e s with reagents t h a t modify these r e s i d u e s . T h i s c o u l d account f o r the d i f f e r e n t values of k f o r the i n a c t i v a t i o n of s u l f h y d r y l enzymes with t e t r a t h i o n a t e . -253-Table 34. Second order r a t e c o n s t a n t s of i n a c t i v a t i o n f o r some papain i n h i b i t o r s and f o r t e t r a t h i o n a t e with two other enzymes. k Enzyme I n h i b i t o r (M*1 s"x) Reference Papain N-ethylmaleimide 166.00 (1) Papain N-heptylmaleimide 3.05 x 10' (1) Papain N-decylmaleimide 21.45 x 10 1 (1) Papain C h l o r o a c e t y l amino a c i d 0.38-5.24 (2) Papain Cyanate 156.70 (3) Papain C y s t a t i n 1.00 x 10' (4) Papain 2-PDS 942.00 (5) Papain E-64 63.80 x 10« (6) Papain H,0, 61.00 (7) Papain I o d o a c e t i c 2.3 x 10* (6) Papain o-methylsourea 1.33 x IO"4 (8) G-3P-D* T e t r a t h i o n a t e 80.80 (9) TGLU" T e t r a t h i o n a t e 8.33 (10) Reference: (1) Anderson and V a s i n i , 1970 (2) Oka and Morihara, 1986 (3) Sluyterman, 1967a (4) N i c k l i n and B a r r e t t , 1984 (5) B r o c k l e h u r s t et a l . , 1981 (6) B a r r e t t et a l . , 1982 (7) L i n et a l . , 1975 (8) Banks and Shafer, 1972 (9) P i h l and Lange, 1962 (10) Chung and F o l k , 1970 *D-glyceraldehyde 3-phosphate dehydrogenase •Transglutaminase -254-CONCLUSIONS The major o b j e c t i v e s of the present work were to i n v e s t i g a t e the e f f e c t s of chemical m o d i f i c a t i o n with t e t r a t h i o n a t e on the a c t i v i t y of papain and to c h a r a c t e r i z e the i n h i b i t i o n e f f e c t of t e t r a t h i o n a t e on the a c t i v i t y of papain i n terms of enzyme k i n e t i c s . Due t o the f a c t t h a t two methods t h a t were used i n t h i s study, i . e . , c i r c u l a r d i c h r o i s m and p r o t e o l y t i c a c t i v i t y d e t e r m i n a t i o n , had problems of high noise l e v e l s and lack of r e p e a t a b i l i t y , r e s p e c t i v e l y , o p t i m i z a t i o n of the o p e r a t i n g c o n d i t i o n s of the methods , i n order to improve t h e i r p r e c i s i o n was c a r r i e d out by means of simplex o p t i m i z a t i o n . A new o p t i m i z a t i o n approach, c o n s i s t i n g of the combined use of a c e n t r a l composite r o t a t a b l e d e s i g n and computational simplex o p t i m i z a t i o n , was a p p l i e d t o f i n d the experimental c o n d i t i o n s which y i e l d e d the h i g h e s t p r e c i s i o n f o r the p r o t e o l y t i c a c t i v i t y d e t e r m i n a t i o n of papain by using c a s e i n as a s u b s t r a t e . The optimum c o n d i t i o n s were as f o l l o w s : papain c o n c e n t r a t i o n , 0.1 mg/mL; i n c u b a t i o n time, 5.3 min and i n c u b a t i o n temperature 35<>C. Simplex c e n t r o i d o p t i m i z a t i o n was used f o r o p t i m i z a t i o n of the c o n d i t i o n s to measure the CD s p e c t r a of papain. A f t e r a t o t a l of 18 experiments ( v e r t i c e s ) , the f o l l o w i n g optimum c o n d i t i o n s were obtained: papain c o n c e n t r a t i o n , 0.23 mg/mL; band width, 1.6 nm; time const a n t , 2 sec, and f o r the value of the product of time -255-constant times the scan r a t e , 0.8 nm. A s i g n i f i c a n t improvement i n the r e p e a t a b i l i t y and s i g n a l to noise r a t i o of the CD scan of papain was achieved. The r e s u l t s of the two f r a c t i o n a l f a c t o r i a l experiments i n d i c a t e d t h a t the r e a c t i o n of papain with t e t r a t h i o n a t e caused a complete i n h i b i t i o n of the p r o t e o l y t i c a c t i v i t y of the enzyme. T h i s i n h i b i t i o n or i n a c t i v a t i o n was not a f f e c t e d by changes i n the molar r a t i o of papain to t e t r a t h i o n a t e , pH, temperature and time of the i n a c t i v a t i o n r e a c t i o n . In order to o b t a i n complete r e a c t i v a t i o n of the p r o t e o l y t i c a c t i v i t y , the i n h i b i t i o n r e a c t i o n should be c a r r i e d out at pH 6.8, 22<>C and the r e a c t i v a t i o n r e a c t i o n with 20 mM c y s t e i n e . The r e s u l t s of the UV-absorbance, near-UV CD and far-UV CD s p e c t r a l a n a l y s i s i n d i c a t e d t h a t no major changes i n secondary or t e r t i a r y s t r u c t u r e occurred i n papain upon chemical m o d i f i c a t i o n with t e t r a t h i o n a t e . The r e v e r s i b i l i t y of b l o c k i n g the o n l y c y s t e i n e r e s i d u e of papain by t e t r a t h i o n a t e was confirmed by d e t e r m i n a t i o n of s u l f h y d r y l groups and pH-fluorescence p r o f i l e s of the m o d i f i e d papain. Decreases i n f l u o r e s c e n c e i n t e n s i t y of the modified papain from t h a t of the n a t i v e papain suggests t h a t a t high molar r a t i o s of t e t r a t h i o n a t e to papain f o r chemical m o d i f i c a t i o n , some tryptophan r e s i d u e s were a l s o m o d i f i e d . P r e l i m i n a r y experiments showed that when a papain s o l u t i o n was heated to 60°C i n the presence of 0-mercaptoethanol and t e t r a t h i o n a t e , the enzyme p r e c i p i t a t e d . Using response s u r f a c e methodology, experiments were c a r r i e d out to f i n d the c o n d i t i o n s -256-producing the l a r g e s t q u a n t i t y of p r e c i p i t a t e . A d d i t i o n of 0-mercaptoethanol and t e t r a t h i o n a t e at l e v e l s of 100 and 50 mM, r e s p e c t i v e l y , p r e c i p i t a t e d 90% of the o r i g i n a l l y s o l u b l e p r o t e i n . S o l u b i l i t y measurements and e l e c t r o p h o r e s i s of the i n s o l u b l e p r o t e i n demonstrated the p o s s i b i l i t y of i n t e r - m o l e c u l a r d i s u l f i d e bond formation. The l a s t p a r t of t h i s t h e s i s i n c l u d e d a s e r i e s of enzyme k i n e t i c s experiments. The s u b s t r a t e used throughout these experiments was carbobenzoxyglycine p - n i t r o p h e n y l e s t e r . The standard e r r o r s of the Km and Vmax values f o r the papain-c a t a l y s e d h y d r o l y s i s of the p - n i t r o p h e n y l d e r i v a t i v e were not reduced when i n i t i a l v e l o c i t i e s were c a l c u l a t e d using the l i n e a r i z a t i o n approach of Durance et a l . (1986), as compared to values c a l c u l a t e d using a f i x e d time method. However, the l i n e a r i z a t i o n method gave equations t h a t r e l a t e d the change i n v e l o c i t y with time f o r each i n i t i a l s u b s t r a t e c o n c e n t r a t i o n . Those r e l a t i o n s h i p s were found to be u s e f u l i n the d e t e r m i n a t i o n of the k i n e t i c s of i n a c t i v a t i o n of papain by t e t r a t h i o n a t e i n the presence of the s u b s t r a t e . T e t r a t h i o n a t e was found to a c t as a c o m p e t i t i v e i n h i b i t o r assuming e i t h e r r e v e r s i b l e or i r r e v e r s i b l e i n h i b i t i o n k i n e t i c s . T e t r a t h i o n a t e r e a c t e d very q u i c k l y with papain, causing complete i n a c t i v a t i o n w i t h i n 5 to 10 min at l e v e l s of 1-3 moles of t e t r a t h i o n a t e / m o l e of enzyme. -257-The second order r a t e constant f o r i n a c t i v a t i o n papain by t e t r a t h i o n a t e was c a l c u l a t e d to be 16,919 M"lsec which i n d i c a t e s q u i t e a high r e a c t i o n r a t e . -258-REFERENCES Abel, E, 1907. K i n e t i c s and c a t a l y s i s o£ the hydrogen peroxide-t h i o s u l f a t e r e a c t i o n . Monatsh. 28:1239. Abe,K., Kondo, H., and A r a i , S. 1987. P u r i f i c a t i o n and c h a r a c t e r i z a t i o n of a r i c e c y s t e i n e p r o t e i n a s e i n h i b i t o r . A g r i c . B i o l . Chem. 51:2763. 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Academic Press, Orlando, FL. -272-Tsou, C.L., Zhao, K.Y., L i e n , Y.N., and Ho, Y.S. 1982. E f f e c t of m o d i f i c a t i o n of cysteine-149 on the l i g a n d b i n d i n g p r o p e r t i e s of D - g l y c e r a l d e h i d e 3-phosphate dehydrogenase. In: Oxidases  redox Svst. Proc. I n t . Symp., 3rd 1979. (In Chemical A b s t r a c t (1983) 98:103333p). Tsunemasa, Y., Cho, S., and Fukumoto, Y. 1980. Immunoglobulins d e r i v a t i v e s . Ger. Otten. 3,018,901. (In Chemical A b s t r a c t (1981) 94:127358u). Umezawa, H. and Aoyagi, T. 1983. Trends i n r e s e a r c h of low molecular weight protease i n h i b i t o r s of m i c r o b i a l o r i g i n . In:  Pr o t e i n a s e I n h i b i t o r s : Medical and B i o l o g i c a l Aspects. N. Katunuma, H. Umezawa, and H. Holzer ( E d s . ) . S p r i n g e r - V e r l a g , Japan. U n d e r k o f l e r , P. 1972. Enzymes. Ch. 1. In: Handbook of Food A d d i t i v e s . Second e d i t i o n . T.E. F u r i a (Ed.), p. 90. CRC Press, C l e v e l a n d , OH. Vaidya, P.S., Verma, K.K., and R u s t a g i , K.N. 1977. Q u a l i t y c h a r a c t e r i s t i c s of papain. ISI B u l l e t i n 29(1):9. (In Food S c i . Technol. A b s t r . (1977) 9(8):8U538). V a l e n t i n e , W.N. and P a g l i a , D.E. 1983. Studies with human e r y t h r o c y t e pyruvate k i n a s e : e f f e c t of m o d i f i c a t i o n of s u l f h y d r y l groups. Br. J . Haematol. 53:385. Vanderstoep, J . 1988. Personal communication. U n i v e r s i t y of B r i t i s h Columbia, Vancouver, B.C. Canada. Vo u t s i n a s , L.P. 1978. Covalent b i n d i n g of methionine and tryptophan to soy p r o t e i n . M.Sc T h e s i s , U n i v e r s i t y of B r i t i s h Columbia, Vancouver, B.C. Canada. Warwick,P.E.and F r e i s h e i m , J.H. 1975. M o d i f i c a t i o n of the c y s t e i n e r e s i d u e o f s t r e p t o c o c c a l d i h y d r o f o l a t e r e d u c t a s e . B i o c h e m i s t r y 14:664. Weast, R.C. (Ed.). 1987. Handbook of Chemistry and P h y s i c s . 67 t h Ed. Chemical Rubber Co., C l e v e l a n d , OH. West, S. 1988. The enzyme maze: how to overcome problems i n choosing the r i g h t enzyme f o r your process. Food Technol. 42(4):98. Whitaker, J.R. 1972. P r i n c i p l e s of Enzymology f o r the Food  S c i e n c e s . p. 211-286. Marcel Dekker, New York, NY. -273-Whitaker, J.R. 1969. F i c i n - and p a p a i n - c a t a l y z e d r e a c t i o n s . Changes i n r e a c t i v i t y of the e s s e n t i a l s u l f h y d r y l group i n the presence of s u b s t r a t e s and c o m p e t i t i v e i n h i b i t o r s . B i o c h e m i s t r y 8:4591. W i l l i a m s , W.J. 1979. P o l y t h i o n a t e s . In.: Handbook of Anion  Determinations. p. 519. Butterworth, London, England. Wollmer, A., StrafBburger, W., and G l a t t e r , U. 1983. P e r s p e c t i v e s i n the c i r c u l a r d i c h r o i c a n a l y s i s of p r o t e i n main-chain conformation.In:Modern Methods i n P r o t e i n Chemistry. H. Tschesche (Ed.), p. 361. Walter de Gruyter, B e r l i n . Yada, R. 1984. A study of secondary s t r u c t u r e p r e d i c t i v e methods for p r o t e i n s and the r e l a t i o n s h i p between p h y s i c a l - c h e m i c a l p r o p e r t i e s and enzymatic a c t i v i t y of some a s p a r t y l p r o t e i n a s e s . Ph.D. T h e s i s , U n i v e r s i t y of B r i t i s h Columbia, Vancouver, B.C. Canada. Yang, J.T., Wu, C-S.C, and M a r t i n e z , H.M. 1986. C a l c u l a t i o n of p r o t e i n conformation from c i r c u l a r d i c h r o i s m . In.: Methods i n  Enzymology. V o l . 130. P a r t K. C.H.W. H i r s and S.N. Timasheff (Eds.), p. 208. Academic Press, Orlando, FL. Yoshida, T., Ono, S., and Fukumoto, Y. 1980. Immunoglobulin d e r i v a t i v e s . Ger. Offen. 3,018,901. (In Chemical A b s t r a c t (1981) 94:127358u). Z e l t n o f f , A. 1867. Quoted i n M e l l o r , J.W. 1930. T e t r a t h i o n i c a c i d and the t e t r a t h i o n a t e s . V o l . X. In: A Comprehensive T r e a t i s e  on Inorganic and T h e o r e t i c a l Chemistry, p. 610-620. Longmans, Green and Co., London, England. Zucker, S., B u t t l e , D.J., N i c k l i n , M.J.H., and B a r r e t t , A. 1985. The p r o t e o l y t i c a c t i v i t y of chymopapain, papain and papaya p r o t e i n a s e I I I . Biochim. Biophys. Acta 828:196. -274-APPENDIX Appendix 1. L i s t i n g of the IBM-BASIC v e r s i o n of the computer program used by S i e g e l et a l . (1980) to determine secondary s t r u c t u r e f r a c t i o n s from CD s p e c t r a . -275-10 REM CD ANALYSIS SIEGEL METHOD 20 DIM A$(72),D$(72),I$(72),S$(72),D(13,10) 30 FOR 1=1 TO 13:FOR J = l TO 10 :READ D ( I , J ) : NEXT J:NEXT I 4 0 REM CONVERT 50 FOR 1=1 TO 13 60 D(I,8)=D(I,8)~2 70 NEXT I 80 INPUT "NAME OF PROTEIN";1$ 9 0 INPUT "CODE";D$ 100 N=13 110 FOR 1=1 TO 13 120 PRINT D(I,1);"NM [THETHA] = " 130 INPUT T 140 D(I,4)=T 150 NEXT I 160 REM CALCULATIONS 163 LPRINT "PROTEIN ";I$ 16 4 LPRINT "CODE= ";D$ 170 FOR 1=1 TO 13 180 D ( I / 5 ) = ( D ( I / 4 ) - D ( I / 2 ) ) / D ( I / 3 ) 18 5 PRINT "WAVELENGTH " ; D ( J , 1 ) ; " [THETA] "; D ( I , 4 ) ; " FRACTION"; D (I , 5 ) 186 LPRINT "WAVELENGTH " ; D ( 1,1) ; " [THETA] "; 0 ( 1 , 4 ) ; " FRACTION";D(I,5) 190 D ( I , 6 ) = ( D ( I / 7 ) + D ( I / 8 ) ) / ( D ( I / 3 ) " 2 ) + ( ( D ( I , 4 ) - D ( I / 2 ) ) ~ 2 ) * D ( I , 9 ) / ( D ( I , 3 ) " 4 ) 200 D ( I , 6 ) = D ( I , 6 ) + 2 * ( D ( I , 4 ) - D ( I , 2 ) ) * D ( I , 1 0 ) / ( D ( I , 3 ) ~ 3 ) 210 NEXT I 230 S1=S2=S3=0 240 FOR 1=1 TO 13 250 S1=S1+ (0 ( 1 , 5 ) 7 0 ( 1 , 6 ) ) 260 S2=S2+(1/D(I,6)) 270 NEXT I 280 FOR 1=1 TO 13 290 S3=S3+((D(I,5)-S1/S2)~2) 300 NEXT I 310 X=S1/S2 320 S4=l/S2 330 S5=S3/((N-1)*N) 340 REM PRINT OUTPUT 350 PRINT 360 PRINT "PROTEIN: ";I$ 370 PRINT "AVERAGE VALUE OF HELIX (X-BAR); ";X 375 LPRINT "AVERAGE VALUE OF HELIX (X-BAR); ";X:LPRINT 380 PRINT "CORRESPONDING TO "; X*100; "% HELICAL STRUCTURE" -276-Appendix 2. L i s t i n g of an IBM-BASIC computer program which u t i l i z e s the simplex a l g o r i t h m of Morgan and Deming (1974) used f o r computational o p t i m i z a t i o n of the c o n d i t i o n s to measure the p r o t e o l y t i c a c t i v i t y of papain. -277-1 CLEAR:CLS: DIM X(10,100) /Y(200),L(10),U(10) /M(11,10) /S{10),B(10): INPUT "Max imization?(Y/N) ",X$:PRINT:INPUT "No. of f a c t o r s ? ",NN: INPUT "Maximum v e r t i c e s to compute? " , r 1 V : INPUT "Terminating d i f f e r e n c e value? ",TERM 2 INPUT "How many v e r t i c e s without p r o h i b i t - t r e s p a s s i n g ? ",ZR:PRINT: FOR 1=1 TO NN:PRINT "Factor No. " ; I : INPUT " Enter lower then upper l i m i t s ",L(I),U(I):PR I NT:NEXT I 3 LPR1NT "Terminating d i f f e r e n c e value=" ; US I NG " ft ft ft . ft ft ft ft " ; TERM : LPRINT:LPRINT "Lower and upper limits":LPRINT " LL:";: FOR J = l TO NN : LPRINT USING " ft ft ft » ft ft . ft ft ft " ; L (J ) ; : NEXT J.-LPRINT: LPRINT " UL:";:FOR K=l TO NN 4 LPRINT USING " ft ft ft ft ft ft . ft ft ft " ; U (K ) ; : NEXT K : LPRINT : LPRI NT : LPRI NT : P=(1/(NN*SQR(2)))*(NN-1+SQR(NN+1)):Q=(1/(NN*SQR(2) ) ) *(SQR(NN +1)-1) : FOR J=l TO NN:M(1,J)=L(J):NEXT J:FOR 1=2 TO NN+l:FOR J = l TO NN 5 IF I-1=J THEN M(I,J)=L(J)+P*(U(J)-L(J)) ELSE M(I,J ) =L(J)+Q*(U(J)-L(J) ) 6 NEXT J:NEXT I:FOR K=l TO NN+l:FOR J=l TO NN:S(J)=M(K,J):NEXT J : GOSUB 46:B(K)=R:FOR L=l TO NN:M(K,L)=S(L):NEXT L:NEXT K: FOR XX = 1 TO NN + l:FOR 1=1 TO NN:X(I , XX)=M(XX,I):NEXT I:NEXT XX: FOR Y = l TO NN + 1:Y(Y)=B(Y):NEXT Y 7 LPRINT TABOO) " I n i t i a l s implex": LPRINT TAB(16) "XI";: LPRINT TAB(25) MX2";:LPRINT TAB(34) "X3";:LPRINT TAB(43) "X4";: LPRINT TAB(52) "X5";:LPRINT TAB(60) "RESPONSE":XX=XX-1:Y=Y-1: FOR 1=1 TO XX:E(I)=100-(X(1,I)+X(2,I)+X(3,I)+X(4,I ) ) 8 NEXT I:FOR J=l TO XX:LPRINT "Vertex ";USING "ftftft";J;: LPRINT USING "ftftftftft.ftftft";X(l,J);X(2,J);X(3,J);X(4,J);E(J);: LPRINT TAB(56) USING "ft ft ftftft.#ftft";Y(J):NEXT J:LPRINT: QO=0:WHILE Q0=0:WORST=B(1):WL=1:FOR 1=2 TO NN+1 9 IF(X$="Y")=0 THEN 11 ELSE IF B(I)<WORST THEN WORST=B(I):WL=I 10 GOTO 12 11 IF B(I)>WORST THEN WORST=B(I):WL=I 12 NEXT I:BEST=B(1):BL=1:FOR J=2 TO NN+1: IF(X$="Y")=0 THEN 14 ELSE IF B(J)>BEST THEN BEST=B(J):BL=J 13 GOTO 15 14 IF B(J)<BEST THEN BEST=B(J):BL=J 15 NEXT J:T=0:FOR 1=1 TO NN+1:T=T+B(I):NEXT I: NXT=(T-WORST-BEST)/(NN-l):FOR K=l TO NN:S=0:FOR L=l TO NN+1: S=S+M(L,K):NEXT L:S=S-M(WL,K):N(K)=S/NN:NEXT K:C=1!: C$="(Reflection)":GOSUB 41:FOR M=l TO NN:R(M)=S(M):NEXT M:REFL=R 16 IF(X$="Y")=0 THEN 27 ELSE IF(REFL>BEST)=0 THEN 19 ELSE C=2!: C$="(Expansion)":GOSUB 41:1F(R>REFL)=0 THEN 17 ELSE FOR N=l TO NN: Q(N)=S(N):NEXT N:GOSUB 45:GOTO 18 17 FOR 1=1 TO NN:Q(I)=R(I):NEXT I:R=REFL:GOSUB 45 18 GOTO 2 6 19 IF(REFL>NXT)=0 THEN 20 ELSE FOR J=l TO NN:Q(3)=R(J):NEXT J : R=REFL:GOSUB 45:GOTO 26 20 IF(REFL>WORST)=0 THEN 24 ELSE C=.5:C$ = "(Contract i on-R)": GOSUB 41:IF(R>REFL)=0 THEN 21 ELSE FOR 1=1 TO NN:Q(I)=S(I): NEXT I:GOSUB 45:GOTO 23 21 C=.25:C$="(Massive c o n t r a c t i o n - R ) " : GOSUB 41:IF(R<REFL)=0 THEN 22 ELSE FOR K=l TO NN: Q(K)=R(K):NEXT K:R=REFL:GOSUB 45:GOTO 2 3 22 FOR L=l TO NN:Q(L)=S(L):NEXT L:GOSUB 45 23 GOTO 26 -278-24 C=-.5:C$="(Contraction-W)":GOSUB 41: IF(R>WORST)=0 THEN 25 ELSE FOR J = l TO NN : Q ( J ) = S ( J ) :NEXT J : GOSUB 45:GOTO 26 25 C=-.25:C$="(Massive contraction-W)":GOSUB 41: FOR K=l TO NN:Q(K)=S(K):NEXT K:GOSUB 45 26 GOTO 37 27 IF(REFL<BEST)=0 THEN 30 ELSE C=2!:C$="(Expansion)": GOSUB 41:IF(R<REFL)=0 THEN 28 ELSE FOR N=l TO NN: Q(N)=S(N):NEXT N:GOSUB 45:G0T0 29 28 FOR 1=1 TO NN:Q(I)=R(I):NEXT I:R=REFL:GOSUB 45 29 GOTO 37 30 IF(REFL<NXT)=0 THEN 31 ELSE FOR J = l TO NN:Q(J)=R(J):NEXT J : R=REFL:GOSUB 45:GOTO 3 7 31 IF(REFL<WORST)=0 THEN 35 ELSE C=.5:C$ = "(Contraction-R )": GOSUB 41:IF(R<REFL)=0 THEN 32 ELSE FOR 1=1 TO NN:Q(I)=S(I):NEXT I: GOSUB 45:GOTO 3 4 32 C=.25:C$="(Massive contraction-R)":GOSUB 41: IF(R>REFL)=0 THEN 33 ELSE FOR K=l TO NN:Q(K)=R(K):NEXT K:R=REFL: GOSUB 45:GOTO 3 4 33 FOR L=l TO NN:Q(L)=S(L):NEXT L:GOSUB 45 3 4 GOTO 37 35 C=-.5:C$="(Contraction-W)":GOSUB 41: IF(R<WORST)=0 THEN 36 ELSE FOR J = l TO N N : Q ( J ) = S ( J ) : NEXT J:GOSUB 45:GOTO 3 7 36 C=-.25:C$="(Massive contraction-W)":GOSUB 41:FOR K=l TO NN: Q(K)=S(K):NEXT K:GOSUB 45 37 IF(XX>MV)=0 THEN 38 ELSE GOTO 40 38 A=XX:B=XX-l:C=XX-2: IF ABS(Y(A)-Y(B))>TERM THEN T$="N" ELSE IF ABS(Y(B)-Y(C))>TERM THEN T$="N" ELSE IF ABS(Y(A)-Y(C))>TERM THEN TS="N" ELSE T$="Y" 39 Q0=T$="Y":WEND:FOR 1=1 TO NN:AV(I)=(X(I,A)+X(I,B)+ X(I,C))/3: NEXT I:BV=(Y(A)+Y(B)+Y(C) )/3:LPRINT:LPRI NT " F i n a l average values": LPRINT T A B ( l l ) USING " » ft ft ft ft ft . ft ft ft " ; AV ( 1 ) ; AV ( 2 ) ; AV ( 3 ) ; AV ( 4 ) ; : LPRINT TAB(67) USING "## ft ftftft.ttft ft "; BV 4 0 PRINT "END":END 41 FOR 1=1 TO NN:S(I)=N(I)+C*(N(I)-M(WL,I)):NEXT I: IF(XX>ZR)=0 THEN 43 ELSE FOR J=l TO NN: IF S(J)<L(J) THEN S(J)=L(J) ELSE IF S(J)>U(J) THEN S(J)=U(J) 42 NEXT J 43 GOSUB 46:Y=Y+l:XX=XX+l:Y(Y)=R:FOR J=1 TO NN:X(J, XX)=S(J):NEXT J : LPRINT "Vertex ";USING "ft ft ft ";XX;:LPRINT C$:LPRINT " ";: FOR 1=1 TO NN:LPRINT USING "ft ft ft ft ft ft . ft ft ft";X(I,XX); :NEXT I 44 LPRINT:LPRINT TAB(60) "Response";US ING "ft ft ft ft ft ft.ft ft ft";Y(Y) :RETURN 45 B(WL)=R:FOR 1=1 TO NN:M(WL,I)=Q(I):NEXT I : RETURN 46 ADI=0:FOR 1=1 TO 4:ADI=ADI+S(I):NEXT I:IF ADI>100 THEN 47 ELSE GOTO 47 FOR J=l TO 4:S(J)=S(J)*100/ADI:NEXT J 48 Z=(S(l)*-27.8002)+(S(2)*-.07831)+(S(3)*-.241365)+ ((S(1)~2)*8.734079)+((S(2)~2)*.0004975)+ ((S(3)~2)*.001815)+(S(l)*S(2)*.664167)+ (S(1)*S(3)*3.61773)+(S(2)*S(3)*.005354)+ (S(l)*S(2)*S(3)*-8.999999E-02)+2.47512:R=Z:RETURN -279-Appendix 3. L i s t i n g of the IBM-BASIC computer program used to generate the data ( g r i d ) f o r p l o t t i n g the response s u r f a c e of the e f f e c t of |3-mercaptoethanol and t e t r a t h i o n a t e on the p r e c i p i t a t i o n of papain. Note: The a c t u a l p l o t t i n g was done with the commercial software Golden Graphic System (Golden Software, Inc., Golden, C o l o r a d o ) . -280-5 REM PROGRAM FOR GENERATION O F D A T A P O I N T S FOR R S 7 REM MER=[MERCAPTOETHANOL] T E T R A = [ T E T R A T H I ONATE] 8 REM Y= % PAPAIN PRECIPITATED 10 OPEN " p r e r s .daf'FOR OUTPUT AS ff 1 20 FOR MER=0 TO 100 STEP 10 30 FOR TETRA=0 TO 100 STEP 10 35 REM EQUATION OBTAINED BY BACKWARD REGRESSION ANALYSIS 40 Y=TETRA*1.956241+MER*.481129+ (TETRA"2)*-.012221+TETRA*MER*-.00 4 0 0 9 4 5 NORMALIZATION OF DATA 5 0 MERN = ((MER-10 0)*10)/10 0: TETRAN=((TETRA-100)*10)/100 60 LPRINT USING "###.######";MER,MERN,TETRA,TETRAN,Y 65 REM SAVING THE DATA IN THE FILE 70 PRINT #1,MERN;TETRAN;Y 8 0 NEXT TETRA 9 0 NEXT MER 100 CLOSE -281-

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