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A study of secondary structure predictive methods for proteins and the relationship between physical-chemical… Yada, Rickey Yoshio 1984

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A STUDY OF SECONDARY STRUCTURE PREDICTIVE METHODS FOR PROTEINS AND THE RELATIONSHIP BETWEEN PHYSICAL-CHEMICAL PROPERTIES AND ENZYMATIC ACTIVITY OF SOME ASPARTYL PROTEINASES by RICKEY YOSHIO YADA B.Sc.(Agr.), The U n i v e r s i t y of B r i t i s h Columbia, 1977 M.Sc, The U n i v e r s i t y of B r i t i s h Columbia, 1980 THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY i n THE FACULTY OF GRADUATE STUDIES Department of Food Science We accept t h i s t h e s i s as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA May 1984 © Rickey Yoshio Yada, 1984 In p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t o f the requirements f o r an advanced degree a t the U n i v e r s i t y o f B r i t i s h Columbia, I agree t h a t the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and study. I f u r t h e r agree t h a t p e r m i s s i o n f o r e x t e n s i v e copying o f t h i s t h e s i s f o r s c h o l a r l y purposes may be granted by the head o f my department o r by h i s o r her r e p r e s e n t a t i v e s . I t i s understood t h a t copying or p u b l i c a t i o n of t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l not be allowed without my w r i t t e n p e r m i s s i o n . Department o f Food Science  The U n i v e r s i t y o f B r i t i s h Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 June 5, 1984 - i i -ABSTRACT In the f i r s t of two studies, four algorithms were assessed for t h e i r a b i l i t y to accurately determine protein secondary structure from c i r c u l a r dichroism spectral data in r e l a t i o n to X-ray data. The major-i t y of the algorithms examined showed highly s i g n i f i c a n t (P<0.001) c o r r e l a t i o n c o e f f i c i e n t s for ^ - h e l i x and B-sheet determination while the c o r r e l a t i o n c o e f f i c i e n t s using the simplex optimization algorithm with n = variable ( i n the presence of concanavalin A) were s i g n i f i c a n t at P<0.05. None of the algorithms examined showed s i g n i f i c a n t (P>0.05) c o r r e l a t i o n c o e f f i c i e n t s for 6-turn determination. S i g n i f i c a n t (P<0.05) c o r r e l a t i o n c o e f f i c i e n t s were obtained for random c o i l determination from both the simplex optimization algorithm ( i n the absence of concan-avalin A) and the algorithm of Chang et a l . (1978). The simplex optimi-zation algorithm with n = 10.4 compared favourably to the method of Chang et a l . (1978) in the absence of concanavalin A, and may serve as an a l t e r n a t i v e algorithm to solve least squares. The algorithm of Provencher and GlOckner (1981) demonstrated the greatest v e r s a t i l i t y for secondary structure determination of the four algorithms examined. In the second study, the r e l a t i o n s h i p between physical-chemical properties and the enzymatic a c t i v i t y of aspartyl proteinases was inves-t i g a t e d . Using the diagonal plot method, pepsin and chymosin showed the highest degree of primary sequence homology while active s i t e regions were highly homologous between the aspartyl proteinases examined. Secon-dary structure prediction methods indicated that chymosin, pepsin, peni-c i l l o p e p s i n and Mucor miehei proteinase had r e l a t i v e l y high proportions of B-sheet, with active s i t e aspartic acid residues located i n B-turn regions. Near-UV and far-UV CD spectral analysis indicated that changes i n spectra occurred i n the neutral to a l k a l i n e pH range. Secondary structure determination from far-UV CD spectral data demonstrated that the 8-sheet f r a c t i o n of the aspartyl proteinases decreased above pH 6.3. The m i l k - c l o t t i n g to p r o t e o l y t i c a c t i v i t y r a t i o of the aspartyl proteinases decreased with increasing pH. P r i n c i p a l components derived from various s t r u c t u r a l and i n t r i n s i c parameters allowed for the c l a s s i -f i c a t i o n of aspartyl and non-aspartyl proteinases. Regression of the m i l k - c l o t t i n g to the p r o t e o l y t i c a c t i v i t y r a t i o on the p r i n c i p a l compon-ents indicated that a high r a t i o may be p a r t i a l l y dependent on r e l a -t i v e l y high hydrophobicity, 8-sheet and low charge. - i v -TABLE OF CONTENTS Page ABSTRACT i i TABLE OF CONTENTS i v LIST OF TABLES v i i LIST OF FIGURES i x LIST OF APPENDICES x i ACKNOWLEDGEMENTS x i i PART I. Analysis of various algorithms for the determination of secondary structure from CD data INTRODUCTION 1 LITERATURE REVIEW 3 A. R e l a t i o n s h i p between o p t i c a l p r o p e r t i e s and p r o t e i n s t r u c t u r e 3 B. O p t i c a l p r o p e r t i e s of molecular groups of p r o t e i n s 4 C. P r o t e i n s t r u c t u r e 7 D. Secondary s t r u c t u r e 10 1. «-helix 10 2. 0-sheet 11 3. 3-turn 12 4. Random c o i l region 13 E. Q u a n t i t a t i v e determination of p e r i o d i c conformations of p r o t e i n s 14 F. Curve f i t t i n g a n a l y s i s of CD spectra 17 G. Simplex o p t i m i z a t i o n 19 METHODS 21 A. Algorithm of S i e g e l et a l . (1980) 21 B. Algorithm of Chang et a l . (1978) 22 C. Algorithm of Provencher and Glockner (1981) 23 D. Simplex-least squares method 25 E. C o r r e l a t i o n c o e f f i c i e n t s 26 RESULTS AND DISCUSSION 27 A. Algorithm of S i e g e l et a l . (1980) 27 B. Algorithm of Chang et a l . (1978) .• 32 C. Algorithm of Provencher and Glockner (1981) 33 D. Simplex-least squares method 34 E. L i m i t a t i o n s of CD a n a l y s i s 43 CONCLUSIONS 47 - V -Page Part II. A physical-chemical study of aspartyl proteinases as related to enzymatic activity INTRODUCTION 49 LITERATURE REVIEW 52 A. A s p a r t y l proteinases 52 1. C l a s s i f i c a t i o n and sources 52 2. G a s t r i c a s p a r t y l proteinases 53 (a) Pepsin 54 (b) Chymosin 55 3. M i c r o b i a l proteinases 57 (a) Mucor p u s i l l u s var. Lindt proteinase 58 (b) Mucor miehei proteinase 59 (c) Endothia p a r a s i t i c a proteinase 60 (d) P e n i c i l l o p e p s i n 61 (e) A s p e r g i l l u s s a i t o i proteinase 62 B. Enzymatic coagulation of milk 64 C. M u l t i v a r i a t e a n a l y s i s 65 1. P r i n c i p a l component a n a l y s i s 66 MATERIALS AND METHODS 72 A. M a t e r i a l s 72 B. Diagonal p l o t method 73 C. Secondary s t r u c t u r e p r e d i c t i o n 73 D. C i r c u l a r dichroism 74 1. Sample preparation 74 2. Far-UV spectra (190 to 240 nm) 75 3. Near-UV spectra (240 to 320 nm) 76 E. Bigelow average hydrophobicity 76 F. Hydrophobicity using f l u o r e s c e n t probes 78 1. c i s - P a r i n a r i c acid 78 2. 1-Anilino-8-naphthalene sulfonate 79 (a) Preparation of ANS 79 (b) Hydrophobicity determination 79 G. Charge r a t i o s 80 H. Zeta p o t e n t i a l 80 I. A c c e s s i b l e surface area 81 3. Determination of m i l k - c l o t t i n g a c t i v i t y 81 K. Determination of p r o t e o l y t i c a c t i v i t y 82 L. P r i n c i p a l component a n a l y s i s 83 RESULTS AND DISCUSSION ., 85 A. Diagonal p l o t s 85 B. Secondary s t r u c t u r e p r e d i c t i o n 99 C. C i r c u l a r dichroism spectra 116 1. Far-UV spectra (190 to 240 nm) 116 2. Secondary s t r u c t u r e determination (far-UV) 126 3. Near-UV spectra (240 to 320 nm) 132 - v i -Page D. Bigelow average hydrophobicity 144 E. Hydrophobicity using f l u o r e s c e n t probes 146 F. Charge r a t i o s 150 G. Zeta p o t e n t i a l 153 H. Ac c e s s i b l e surface area 156 I. M i l k - c l o t t i n g to p r o t e o l y t i c a c t i v i t y r a t i o 157 J . P r i n c i p a l component a n a l y s i s 166 CONCLUSIONS 180 REFERENCES CITED 183 APPENDIX 202 - v i i -LIST OF TABLES Page Table 1. A comparison of X-ray secondary structures to predicted secondary structures of selected pro-teins using various algorithms . 28 Table 2. Correlation coefficients between calculated secondary structure fractions and those obtained from X-ray data 30 Table 3. Operational calculations used in the simplex optimization algorithm 41 Table 4. The molecular weight, number of residues and the mean residue weight (MRW) of the various protein-ases 77 Table 5. Degree of similarity between various pairs of aspartyl proteinases based on the diagonal plot method 98 Table 6. Secondary structure determination from CD spectra for various proteinases 128 Table 7. Bigelow average hydrophobicity values (H$/\yg) obtained for various proteinases 145 Table 8. Hydrophobicity values obtained for various pro-teinases using fluorescent probes 149 Table 9. Charge ratios of various proteinases 151 Table 10. Accessible surface areas (A s) of various pro-teinases 158 Table 11. Milk-clotting to proteolytic activity ratios for various proteinases 160 Table 12. The variance explained and the cumulative propor-tion of total variance accounted for by each factor derived from principal component analysis 167 Table 13. Factor loadings for the factors whose eigenvalues exceed 1.0 168 Table 14. Regression analysis for the milk-clotting to pro-teolytic activity ratio on the principal compon-ents computed from various structural and i n t r i n -sic properties of the proteinases 176 - v i i i -Page Table 15. Regression analysis for the milk-clotting to pro-teolytic activity ratio on the principal compon-ents computed for various structural and intrinsic properties of the proteinases in the absence of Aspergillus saitoi proteinase 178 - ix -LIST OF FIGURES Page Figure 1. Possible simplex moves 39 Figure 2. Flow chart of the simplex minimization. Calcula-tion of the new vertex, V = P + K(P-W) 40 Figure 3. Outline of the procedure for principal component analysis 68 Figure 4. Diagonal plots (a and b) of the primary amino acid sequences of pepsin and chymosin 86-87 Figure 5. Diagonal plots (a, b and c) of the primary amino acid sequences of chymosin and Mucor miehei pro-teinase 88-90 Figure 6. Diagonal plots (a, b and c) of the primary amino acid sequences of pepsin and Mucor miehei pro-teinase 91-93 Figure 7. Diagonal plots (a and b) of the primary amino acid sequences of pepsin and penicillopepsin 94-95 Figure 8. Diagonal plots (a and b) of the primary amino acid sequences of penicillopepsin and chymosin 9 6 - 9 7 Figure 9. Predicted secondary structure of chymosin using the Chou and Fasman method 101 Figure 10. Predicted secondary structure of pepsin using the Chou and Fasman method 102 Figure 11. Predicted secondary structure of penicillopepsin using the Chou and Fasman method 103 Figure 12. The bulk hydrophobicity profiles (a and b) for chymosin 107-108 Figure 13. The bulk hydrophobicity profiles (a and b) for pepsin 109-110 Figure 14. The bulk hydrophobicity profiles (a and b) for penicillopepsin 111-112 Figure 15. The bulk hydrophobicity profiles (a and b) for Mucor miehei proteinase 113-114 Figure 16. The effect of pH on the far-UV CD spectra of chymosin 117 Figure 17. The effect of pH on the far-UV CD spectra of pepsin 118 - X -P a g e F i g u r e 1 8 . The e f f e c t of pH on the far-UV CD spectra o f Mucor miehei proteinase 119 F i g u r e 1 9 . The e f f e c t o f pH on the far-UV CD spectra o f Mucor p u s i l l u s proteinase 120 F i g u r e 2 0 . The e f f e c t of pH on the far-UV CD spectra of Endothia p a r a s i t i c a proteinase 121 F i g u r e 2 1 . The e f f e c t of pH on the far-UV CD spectra of A s p e r g i l l u s s a i t o i proteinase 122 F i g u r e 2 2 . The e f f e c t of pH on the far-UV CD spectra of p e n i c i l l o p e p s i n 123 F i g u r e 2 3 . The e f f e c t o f pH on the near-UV CD spectra of chymosin 133 F i g u r e 2 4 . The e f f e c t o f pH on the near-UV CD spectra o f pepsin 134 F i g u r e 2 5 . The e f f e c t of pH on the near-UV CD spectra of Mucor miehei proteinase 135 F i g u r e 2 6 . The e f f e c t of pH on the near-UV CD spectra of Mucor p u s i l l u s proteinase 136 F i g u r e 2 7 . The e f f e c t of pH on the near-UV CD spectra o f Endothia p a r a s i t i c a proteinase 137 F i g u r e 2 8 . The e f f e c t of pH on the near-UV CD spectra of A s p e r g i l l u s s a i t o i proteinase 138 F i g u r e 2 9 . S t r u c t u r e s of c i s - p a r i n a r i c acid and 1 - a n i l i n o - 8 -napthalene s u l f o n a t e 148 F i g u r e 3 0 . The e f f e c t of pH on the zeta p o t e n t i a l of various p r o t e i n a s e s 154 F i g u r e 3 1 . The p l o t of f a c t o r 2 vs f a c t o r 1 obtained from the p r i n c i p a l component a n a l y s i s of various s t r u c t u r a l and i n s t r i n s i c p r o p e r t i e s o f the pro-t e i n a s e s 170 F i g u r e 3 2 . The p l o t of f a c t o r 3 vs f a c t o r 2 obtained from the p r i n c i p a l component a n a l y s i s of various s t r u c t u r a l and i n t r i n s i c p r o p e r t i e s o f the pro-t e i n a s e s 171 F i g u r e 3 3 . Reference spectra f or ^ - h e l i x , 6-sheet, 6-turn and random c o i l f r a c t i o n s . (Adapted from Chang et a l . , 1978.) 173 - x i -L I S T OF A P P E N D I C E S P a g e A p p e n d i x 1. L i s t i n g of a Fortran IV ve r s i o n of the computer program used by S i e g e l et a l . (1980) to deter-mine secondary s t r u c t u r e f r a c t i o n s from CD spec-t r a l data 202 A p p e n d i x 2. L i s t i n g of a Fortran IV computer program s i m i l a r t o that used by Chang et a l . (1978) to determine the secondary s t r u c t u r e f r a c t i o n s from CD spec-t r a l data 205 A p p e n d i x 3. L i s t i n g of a Fortran IV computer program to gen-erate the reference e l l i p t i c i t y values f o r the various secondary s t r u c t u r e f r a c t i o n s (<*-helix, 8-sheet, B-turn and random f r a c t i o n s ) 208 A p p e n d i x 4. L i s t i n g of a Fortran IV computer program which u t i l i z e s the simplex o p t i m i z a t i o n algorithm of Morgan and Deming (1974) to determine the secon-dary 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 l data 212 A p p e n d i x 5 . L i s t i n g of a Fortran IV computer program f o r the diagonal p l o t method 222 A p p e n d i x 6. L i s t i n g of a Fortran IV computer program for the p r e d i c t i o n of secondary s t r u c t u r e using the hydrophobicity p r o f i l e method of Cid et a l . (1982) 225 A p p e n d i x 7. L i s t i n g of a Fortran IV computer program for the determination of mean residue e l l i p t i c i t y from CD s p e c t r a l data 228 A p p e n d i x 8. L i s t i n g of a Fortran IV computer program to c a l c u l a t e average hydrophobicity based on the algorithm of Bigelow (1967) 231 - x i i -ACKNOWLEDGEMENTS I would like to express my sincere gratitude to my supervisor Dr. Shuryo Nakai whose encouragement and enthusiasm were greatly appreciated throughout the course of this work. I also would like to thank the members of the committee Drs. Brent Skura and William Powrie, Department of Food Science, and Dr. David Dolphin, Department of Chemistry, for their suggestions and assistance. Thanks are also extended to the members of my family for their constant moral support throughout my years of graduate study. A special thanks to my wife, Sylvia who was an invaluable source of encouragement and inspiration during this study. Finally, I would like to dedicate this thesis to the memory of my father, Genji. The support of the Natural Sciences and Engineering Research Coun-c i l of Canada and the Leonard S. Klinck post-graduate scholarships is also acknowledged. - 1 -PART I. Analysis of various algorithms for the determination of secondary structure from CD data INTRODUCTION One of the most i n t r i g u i n g problems in protein chemistry has been the determination of protein structure. The structure of a protein i s a major factor which, along with the hydrophobic and e l e c t r o n i c para-meters, aids in the prediction of functional properties (Stuper et a l . , 1979). X-ray d i f f r a c t i o n analysis i s the most accurate means for deter-mining the three-dimensional structure of proteins, however, only a small number of proteins have been analyzed using t h i s method due to constraints of time, cost and the i n a b i l i t y of cer t a i n proteins to form suitable c r y s t a l forms. Lev i t t and Chothia (1976) proposed the description of a protein's three-dimensional structure to include the c l a s s i f i c a t i o n of globular proteins based on the predominant type of secondary structure. P t i t s y n and F i n k e l s t e i n (1980) suggested that because the majority of proteins belonging to a given class ( i . e . «-helix, B-sheet or c-helix/g-sheet) have only a li m i t e d set of d i s t i n c t topologies, many top o l o g i c a l features may be simply determined by secondary structure. Therefore, the prediction of secondary structure i s an in t e g r a l step in predicting the o v e r a l l three-dimensional structure of a protein ( P t i t s y n and F i n k e l s t e i n , 1983). Various s t a t i s t i c a l methods for the determination of secondary structure have been developed on the basis of primary sequence data or - 2 -o p t i c a l p r o p e r t i e s ( i . e . c i r c u l a r dichroism (CD) and o p t i c a l r o t a t o r y d i s p e r s i o n (ORD) s p e c t r a l d a t a ) . One of the most popular methods f o r the determination of secondary s t r u c t u r e from primary sequence data, due to i t s s i m p l i c i t y and accuracy, was proposed by Chou and Fasman (1978b). However, the primary sequences of many food-related p r o t e i n s have not been determined which l i m i t s the use of the Chou and Fasman procedure. The o p t i c a l p r o p e r t i e s of the p r o t e i n polypeptide backbone have been extremely u s e f u l for examining p r o t e i n secondary s t r u c t u r e s i n s o l u t i o n since the ORD and CD spectra of a p r o t e i n are considered to be a r e f l e c t i o n of the secondary s t r u c t u r e f r a c t i o n s (Chang et a l . , 1978; Hennessey and Johnson, 1981). In recent years the a n a l y s i s of CD data has predominated, and a v a r i e t y of t h e o r e t i c a l and e m p i r i c a l techniques have been proposed i n order to analyze the CD spectrum of p r o t e i n for the determination of secondary s t r u c t u r e . In the present study four d i f f e r e n t algorithms were compared for t h e i r a b i l i t y to acc u r a t e l y determine p r o t e i n secondary s t r u c t u r e i n r e l a t i o n to X-ray data. The methods compared were those of S i e g e l et a l . (1980), Chang et a l . (1978), Provencher and Glockner (1981) and a procedure u t i l i z i n g the simplex o p t i m i z a t i o n algorithm of Morgan and Deming (1974). The methods that u t i l i z e reference spectra for the determination of secondary s t r u c t u r e use a form of m u l t i p l e r e g r e s s i o n a n a l y s i s to solve the l e a s t squares algorithm. The simplex procedure was w r i t t e n i n attempts to u t i l i z e the simplex algorithm as an a l t e r n a -t i v e method to solve the l e a s t squares algorithm for the determination of the secondary s t r u c t u r e f r a c t i o n s . - 3 -LITERATURE REVIEW A . RELATIONSHIP BETWEEN OPTICAL PROPERTIES AND PROTEIN STRUCTURE Conformational s t u d i e s of b i o l o g i c a l macromolecules i n s o l u t i o n have been a major t o o l i n the search for r e l a t i o n s h i p s between s t r u c t u r e and f u n c t i o n . For many years there was no o b j e c t i v e method for evaluat-ing the three dimensional arrangements of molecular groups w i t h i n the macromolecular s t r u c t u r e , t h e r e f o r e , s t u d i e s of macromolecules i n s o l u -t i o n were g e n e r a l l y r e s t r i c t e d to the d e s c r i p t i o n of gross hydrodynamic p r o p e r t i e s (Beychok, 1966). During the mid 1950's, M o f f i t t (1956) and M o f f i t t and Yang (1956) e s t a b l i s h e d q u a n t i t a t i v e r e l a t i o n s h i p s between the o p t i c a l parameters of a p r o t e i n and i t s .conformation. A great stimulus was given to the study of the o p t i c a l p r o p e r t i e s of p r o t e i n s when s y n t h e t i c polypeptides became a v a i l a b l e (Bamford et a l . , 1956; K a t c h a l s k i and Sel a , 1958) which were capable of assuming the h e l i c a l conformation p r e d i c t e d by Pauling et a l . (1951) and Pauling and Corey (1951). The synthe s i s of these polypeptides g r e a t l y aided p r o t e i n chemists since the secondary s t r u c t u r e of these simpler polymers could be e s t a b l i s h e d by s e v e r a l independent p h y s i c a l methods (Jirgensons, 1969). On the b a s i s of the c o r r e l a t i o n between the o p t i c a l r o t a t o r y d i s p e r s i o n and X-ray d i f f r a c t i o n p a t t e r n i t became p o s s i b l e to describe the content of h e l i x i n p r o t e i n s and polypeptides. In recent years, o p t i c a l r o t a t o r y d i s p e r s i o n (ORD) and i t s r e l a t e d phenomenon, c i r c u l a r dichroism (CD), have been valuable t o o l s i n study-i n g the conformation and conformational changes of p r o t e i n s i n s o l u t i o n . - 4 -Both ORD and CD are manifestations of the same property of molecules having asymmetric groupings of atoms. ORD i s a d i s p e r s i o n phenomenon ( i . e . e x h i b i t i n g a d i f f e r e n c e i n r e f r a c t i v e index for r i g h t and l e f 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 ) and i s e x h i b i t e d at frequencies (wave-lengths) f a r from, as w e l l as near, those frequencies c h a r a c t e r i s t i c of the e l e c t r o n i c t r a n s i t i o n s which are responsible for the o p t i c a l a c t i v -i t y . CD i s an absorptive phenomenon ( i . e . absorbing 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 d i f f e r e n t l y ) and i s observable only at frequency i n t e r v a l s where absorption occurs. I t i s t h e r e f o r e p o s s i b l e to l o c a t e the p o s i t i o n s and signs of bands i n c i r c u l a r dichroism with more c e r t a i n t y than i s p o s s i b l e i n an ORD spectrum (Foss, 1963). Due to the i n t i m a t e nature of the r e l a t i o n s h i p , ORD and CD can be a n a l y t i c a l l y i n t e r c o n v e r t e d by the use of a mathematical expression known as the "Kronig-Kramers transform". The nature of t h i s mathemati-c a l r e l a t i o n s h i p has been dealt with by s e v e r a l authors ( M o f f i t t and Yang, 1956; Yang, 1956; M o f f i t t and Moscowitz, 1959). B. OPTICAL PROPERTIES OF MOLECULAR GROUPS OF PROTEINS The peptide group i s the fundamental chemical and s t r u c t u r a l u n i t of a l l p r o t e i n s , thus polypeptides and p r o t e i n s may be regarded as peptide polymers. Each peptide group contains at l e a s t one asymmetric centre at the ^-carbon atom, except i n the case of g l y c i n e . Although the asymmetric centres c o n t r i b u t e to the o p t i c a l p r o p e r t i e s of p r o t e i n s , the greatest i n f l u e n c e on the o p t i c a l p r o p e r t i e s of p r o t e i n s i s the s p e c i f i c arrangement of these peptide groups i n three-dimensional space ( B l o u t , 1971). - 5 -Since the o p t i c a l a c t i v i t y of any molecule i s r e l a t e d to i t s absorptive p r o p e r t i e s , i t i s necessary to i d e n t i f y the groups respon-s i b l e for the absorptive p r o p e r t i e s found i n p r o t e i n s . Donovan (1969) summarized the absorption maxima and e x t i n c t i o n c o e f f i c i e n t s for these groups. From t h i s summary i t i s apparent that most of the absorption due to peptide chains l i e s i n the u l t r a v i o l e t region of 185 to 300 nm, with the strongest absorption appearing below 230 nm. I t should also be noted that there are intense p r o t e i n absorption bands which l i e i n the vacuum u l t r a v i o l e t r e g i o n , i . e . below 185 nm. Several researchers (Brahms et a l . , 1977; Bush et a l . , 1981; Hennessey and Johnson, 1981; Hennessey et a l . , 1982) have used the vacuum UV region for CD a n a l y s i s of various p r o t e i n s to y i e l d u s e f u l i n f o r m a t i o n . Research by Okabayashi et a l . (1968) and Deker et a l . (1970) studying the r o t a t o r y p r o p e r t i e s of oligomeric peptides, has shown that under c e r t a i n c o n d i t i o n s large increases i n o p t i c a l a c t i v i t y occur when the number of peptide u n i t s i s between f i v e and twelve. I t was con-cluded that the observed increase i n o p t i c a l a c t i v i t y with added chain length was due to the formation of p e r i o d i c s t r u c t u r e s which e x h i b i t inherent o p t i c a l a c t i v i t y . Studies with homopolypeptides have demonstrated that an important o r d e r i n g i n f l u e n c e i s e x e r c i s e d by i o n i c detergents on amino a c i d residues bearing an opposite charge (Hamed et a l . , 1983). C a t i o n i c homopolypeptides r e a d i l y become ordered i n the presence of dodecyl s u l f a t e . Poly ([.-ornithine) (Grouke and Gibbs, 1967; Satake and Yang, 1973) and poly ( L - a r g i n i n e ) (McCord et a l . , 1977) form an «-helix, poly - 6 -( L - h i s t i d i n e ) adopts a pleated sheet s t r u c t u r e and poly ( L - l y s i n e ) forms e i t h e r a pleated sheet or h e l i x depending on the c o n d i t i o n s employed (Sakar and Doty, 1966; Mattice and Harrison, 1976). Work by Madison and Schellman (1970) on model amides has i n d i c a t e d t h a t when two peptide groups are held r i g i d l y with respect to one another, large increases i n o p t i c a l a c t i v i t y are observed. I t could be concluded that large r o t a t i o n s observed with p r o t e i n s are caused when the peptide groups i n p r o t e i n s are held with a f i x e d geometry, e i t h e r p e r i o d i c or a p e r i o d i c . The side chain groups of p r o t e i n s which have s i g n i f i c a n t absorp-t i o n i n the u l t r a v i o l e t region and c o n t r i b u t e to the o p t i c a l a c t i v i t y , are the aromatic side chains of tryptophan, t y r o s i n e and phenylalanine, and the d i s u l f i d e groups o f c y s t i n e residues (Townend et a l . , 1967; S t r i c k l a n d , 1974). In a d d i t i o n , small c o n t r i b u t i o n s to the o p t i c a l a c t i v i t y may r e s u l t from the presence o f carboxylate and ammonium groups. The presence o f metals i n c e r t a i n p r o t e i n s can modify the absorptive and o p t i c a l p r o p e r t i e s of the molecular groups with which the metals are associated (Nakano and Yang, 1981). In general, the c o n t r i -butions o f the above mentioned groups to the o p t i c a l spectra o f p r o t e i n s are small compared with the c o n t r i b u t i o n s of the peptide groups for two reasons: the r e l a t i v e numbers of such groupings are s m a l l ; and, the inherent o p t i c a l a c t i v i t y of these groups i s lower than that of the peptide group ( B l o u t , 1971). The o p t i c a l p r o p e r t i e s o f the aromatic amino a c i d r e s i d u e s , however, may i n some cases be used to obtain impor-tant information about the molecular conformation and i n t e r a c t i o n s o f - 7 -peptide chains of p r o t e i n s ( B l o u t , 1971). S t r i c k l a n d (1974) presented an e x c e l l e n t t r e a t i s e of the c o n t r i b u t i o n to near-UV CD spectra by aromatic groups. C. PROTEIN STRUCTURE Linderstrom-Lang and Schellmann (1959) proposed a scheme whereby pr o t e i n s could be d i s t i n g u i s h e d i n t o four l e v e l s of s t r u c t u r a l organiza-t i o n , namely the primary, secondary, t e r t i a r y and quaternary s t r u c -t u r e s . These terms r e f e r to the amino acid sequence, the regular arrangements of the polypeptide backbone, the three-dimensional s t r u c -ture of the gl o b u l a r p r o t e i n , and the s t r u c t u r e s of aggregates of gl o b u l a r p r o t e i n s , r e s p e c t i v e l y . Anfinsen et a l . (1961) and Anfinsen (1973) i n r e n a t u r a t i o n experiments with ribonuclease, showed that the primary amino a c i d sequence contained a l l the s t r u c t u r a l i n f o r m a t i o n necessary f or p r o t e i n conformation, and postulated that the r e l a t i o n s h i p between the l e v e l s of s t r u c t u r a l o r g a n i z a t i o n was dependent upon one another with elements at a lower l e v e l determining the elements of higher l e v e l s . Undoubtedly, the t e r t i a r y s t r u c t u r e 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 and polypeptides i s one of the main f a c t o r s for the high degree of s p e c i f i c i t y of r e a c t i o n s seen i n vivo (Fasman, 1980). X-ray d i f f r a c t i o n a n a l y s i s i s the best method for the determina-t i o n of t e r t i a r y s t r u c t u r e , however, many pr o t e i n s such as hi s t o n e s , membrane and ribosomal p r o t e i n s as w e l l as many food r e l a t e d p r o t e i n s do not y i e l d s u i t a b l e c r y s t a l forms (Chou and Fasman, 1978b; Nakai, 1983). In the absence of three-dimensional s t r u c t u r a l i n f o r m a t i o n , however, one - 8 -can s t i l l l e a r n much about the nature of the r e l a t i o n s h i p between s t r u c -ture and f u n c t i o n by examining lower l e v e l s of s t r u c t u r e . Since e s s e n t i a l l y a l l information concerning s t r u c t u r e and func-t i o n of a p r o t e i n r e s i d e i n the primary s t r u c t u r e , a study of homology or s i m i l a r i t y between p r o t e i n s may be important from the viewpoint of comparative biochemistry. I f homologies e x i s t between two p r o t e i n sequences over a number of amino a c i d r e s i d u e s , a s t r u c t u r a l and func-t i o n a l c o r r e l a t i o n may e x i s t between these two p r o t e i n s (Kubota et a l . , 1981). Gibbs and Mclntyre (1970) proposed the "diagonal-match" method i n which the two primary amino a c i d sequences are recorded along adja-cent sides of a rectangular matrix. Within the body of the rectangle every match i s recorded. The method allows for ra p i d and simple detec-t i o n of r e p e t i t i o n s i n the amino a c i d sequences of various p r o t e i n s . Beynon (1982) wrote a program i n BASIC for the Apple® computer to c a r r y out such a task. Various attempts have been made to p r e d i c t secondary s t r u c t u r e from primary amino a c i d sequence (Guzzo, 1965; Kotelchuk and Scheraga, 1969; P t i t s y n and F i n k e l s t e i n , 1970; Nagano, 1973; Kabat and Wu, 1973a and 1973b; Chou and Fasman, 1978a and 1978b; Gamier et a l . , 1978; Palau et a l . , 1982). In t h i s connection, one of the most promising methods has been the one proposed by Chou and Fasman (1978b). The method i s simple and easy to use and i s reported to have reasonable accuracy when compared to r e s u l t s obtained from X-ray data. However, some of the ru l e s set f o r t h by the authors are q u a l i t a t i v e rather than q u a n t i t a t i v e , and are thus open to i n t e r p r e t a t i o n . As a r e s u l t various authors - 9 -(Burgess and Scheraga, 1975; Gamier et a l . , 1976) have had only l i m i t e d success using the Chou and Fasman method. Pham (1981) wrote a computer program for the Chou and Fasman (1978b) method i n attempts to c l a r i f y some of the ambiguities i n that method and found that the r e s u l t s were i n good agreement with X-ray data. The importance of hydrophobic forces i n p r o t e i n s t r u c t u r e as w e l l as i n p r o t e i n - p r o t e i n i n t e r a c t i o n has been w e l l documented i n the l i t e r -ature (Kauzmann, 1959; Tanford, 1962; Bigelow, 1967; Jones, 1975; Ponnuswamy et a l . , 1980). Several methods have been suggested for the p r e d i c t i o n of secondary s t r u c t u r e based on hydrophobicity. Rose (1978) proposed a method whereby 8-turns i n various g l o b u l a r p r o t e i n s were determined using a "hydrophobicity p r o f i l e " technique. In t h i s method the free energy of t r a n s f e r for each amino a c i d , as defined by Nozaki and Tanford (1971), i n the primary sequence i s p l o t t e d against residue number; 8-turns were i d e n t i f i e d i n the p r o f i l e as being regions of low energy. Rose and Roy (1980) suggested a procedure where p r o t e i n - p r o t e i n contact density about each residue was p l o t t e d against residue number. Contact density i s defined as the "number of p r o t e i n atoms other than hydrogen w i t h i n a sphere of radius r about each ^-carbon but excluding i n t r a - r e s i d u e atoms". The method was used to p r e d i c t n u c l e a t i o n s i t e s of secondary s t r u c t u r e . In a s i m i l a r approach, Kyte and D o o l i t t l e (1982) wrote a computer program "that p r o g r e s s i v e l y evaluates the h y d r o p h i l i c i t y and hydropho-b i c i t y of a p r o t e i n along i t s amino acid sequence" using a "hydropathy" - 10 -s c a l e . "Hydropathy" i s derived from an amalgamation of experimental observations derived from the l i t e r a t u r e . Cid et a l . (1982) suggested a simple method which allows for the p r e d i c t i o n of secondary s t r u c t u r e of pr o t e i n s based on the examination of patterns e x h i b i t e d when the "bulk hydrophobicity" (Ponnuswamy et a l . , 1980) of each amino ac i d residue i n the primary amino ac i d sequence i s p l o t t e d against residue number. P t i t s y n and F i n k e l s t e i n (1983) derived an algorithm which took i n t o account both l o c a l i n t e r a c t i o n s i n s i d e each chain region and long-range i n t e r a c t i o n s between d i f f e r e n t regions of the p r o t e i n molecule. This algorithm was then used as a means for p r o t e i n secondary s t r u c t u r e p r e d i c t i o n . Results from " b l i n d " p r e d i c t i o n s (made before the X-ray s t r u c t u r e became a v a i l a b l e ) were l a t e r found to compare favourably to r e s u l t s obtained from X-ray a n a l y s i s . D. SECONDARY STRUCTURE Secondary s t r u c t u r e s are regular arrangements of the backbone of the polypeptide chain without reference to side chain types or conforma-t i o n s , and are s t a b i l i z e d by hydrogen bonds between peptide amide and carbonyl groups (IUPAC-IUB Commission, 1970; Schulz and Schirmer, 1978). Four b a s i c s t r u c t u r e s comprise the secondary s t r u c t u r e f r a c t i o n : these being the " - h e l i x , B-sheet, B-turn and random c o i l . 1. " - h e l i x The " - h e l i x i s the most s t a b l e secondary s t r u c t u r e found i n pr o t e i n s and was f i r s t described by Pauling et a l . (1951). In t h i s - 11 -s t r u c t u r e there are 3.6 amino ac i d residues per t u r n . The a l k y l (R) groups of the amino acids extend outward from the backbone of the h e l i x such that the spacing per residue i s approximately 1.5A. A s i n g l e turn of the h e l i x extends 5.4A along the a x i s . The " - h e l i c a l arrangement of the peptide chain i s favoured since i t permits the formation of i n t r a -chain hydrogen bonds between successive turns of the h e l i x ; hydrogen bonds form between the CO group of residue n and NH group of residue n+4. Since each peptide bond of the polypeptide chain p a r t i c i p a t e s i n hydrogen bonding, the " - h e l i x i s maximally hydrogen bonded. With natur-a l l y o c c u r r i n g L-amino a c i d s , e i t h e r r i g h t - or left-handed h e l i c a l c o i l s can occur, however, the right-handed h e l i x i s s i g n i f i c a n t l y more s t a b l e (Lehninger, 1970). In a l l n a t u r a l l y o c c u r r i n g p r o t e i n s examined so f a r , the " - h e l i x has been right-handed. 2. B-sheet Concomitantly with the " - h e l i x , P a uling and Corey (1951) postu-l a t e d that p a r a l l e l and a n t i - p a r a l l e l 6-sheets are s u i t a b l e regular hydrogen bonded s t r u c t u r e s for polypeptide chains. In t h i s s t r u c t u r e the peptide backbone forms a zig-zag p a t t e r n with the R groups of the amino acids extending above and below the peptide c h a i n . A l l peptide bonds are a v a i l a b l e for hydrogen bonding thus a l l o w i n g for maximum c r o s s - l i n k i n g between adjacent peptide chains and r e s u l t i n g i n high s t a b i l i t y (Pham, 1981). Two d i s t i n c t i v e patterns of hydrogen bonding can occur with B-strands depending on t h e i r i n t e r a c t i o n i n a p a r a l l e l or a n t i - p a r a l l e l - 12 -o r i e n t a t i o n . The a n t i - p a r a l l e l sheet has hydrogen bonds which are perpendicular to the strands, and has narrowly spaced bond p a i r s a l t e r -n a t i ng with widely spaced p a i r s . P a r a l l e l sheet has evenly spaced hydrogen bonds which angle across between the strands (Richardson, 1981). A pure p a r a l l e l sheet, a pure a n t i - p a r a l l e l sheet, or a mixed sheet with some strand p a i r s p a r a l l e l and some a n t i - p a r a l l e l can be formed. However, mixed sheets are not r e a d i l y formed since s l i g h t l y d i f f e r e n t peptide o r i e n t a t i o n s are required for the two types of hydro-gen bonding (Richardson, 1977). P a r a l l e l 6-sheets and the p a r a l l e l p o r t i o n s of mixed sheets are g e n e r a l l y buried w i t h i n the p r o t e i n s t r u c t u r e with ^ - h e l i c e s p r o t e c t i n g them on both s i d e s . On the other hand, a n t i - p a r a l l e l sheets t y p i c a l l y have one side exposed to the solvent and the other side buried which r e s u l t s i n an a l t e r n a t i o n of s i d e chain hydrophobicity i n the amino acid sequence. 3. B-turn B-turns ( a l s o known as reverse t u r n s , B-bends, h a i r p i n bends, 3^ 0 bends, etc.) are s t r u c t u r a l features of peptides and p r o t e i n s i n v o l v i n g four consecutive amino a c i d residues and are located i n regions where the peptide chain f o l d s back on i t s e l f by 180°. An i n t r a m o l e c u l a r hydrogen bond i s formed between the C=0 of residue n and the N-H of residue n+3. About one t h i r d of the residues i n g l o b u l a r p r o t e i n s may be i n v o l v e d i n B-turns (Crawford et a l . , 1973; Zimmerman and Scheraga, 1977). Since these s t r u c t u r e s are thought to c o n s t i t u t e a s u b s t a n t i a l p r o p o r t i o n of the surface residues of p r o t e i n s , i t i s l i k e l y that - 13 -c e r t a i n B-turns provide r e c o g n i t i o n s i t e s for the i n i t i a t i o n of various f u n c t i o n a l r e a c t i o n s (Smith and Pease, 1980). Venkatachalam (1968) f i r s t recognized B-turns from t h e o r e t i c a l conformational a n a l y s i s and described three types based on d i h e d r a l angles. In a d d i t i o n , Lewis et a l . (1973) defined f i v e a d d i t i o n a l types of B-turns. From these eight types of 8-turns i t was p o s s i b l e to describe a l l cases (with the excep-t i o n of the ^ - h e l i x ) where the ^-carbons of residues n and n+3 are l e s s than 7A apart (Chou and Fasman, 1977). A. Random c o i l r e g ion When d e s c r i b i n g p r o t e i n secondary s t r u c t u r e for the purpose of d e s c r i p t i o n , p r e d i c t i o n , spectroscopic c h a r a c t e r i z a t i o n , e t c . , the c l a s s i f i c a t i o n s are ^ - h e l i x , B-sheet, B-turn and random c o i l . Random c o i l i s defined as being those p o r t i o n s of secondary s t r u c t u r e that do not f a l l i n t o the f i r s t three c l a s s i f i c a t i o n s (Richardson, 1981). Although random i s used as an a d j e c t i v e i n a s s o c i a t i o n with c o i l i t has been shown from X-ray c r y s t a l l o g r a p h i c s t u d i e s that some p o r t i o n s described as random c o i l are not random or dis o r d e r e d . These regions have been shown to be "highly organized and f i r m l y held i n place as the repeating secondary s t r u c t u r e s - they are simply harder to d e s c r i b e " (Richardson, 1981). In a d d i t i o n to regions of random c o i l that are h i g h l y organized, there are areas i n p r o t e i n s that are h i g h l y d i s -ordered. These areas are e i t h e r e n t i r e l y absent on e l e c t r o n density maps or appear l e s s dense and more spread out than other regions of the p r o t e i n . - 14 -E . QUANTITATIVE DETERMINATION OF PERIODIC CONFORMATIONS OF PROTEINS Once the r e l a t i o n s h i p between the o p t i c a l parameters of the " - h e l i x of pr o t e i n s and polypeptides and the data from X-ray d i f f r a c t i o n a n a l y s i s had been e s t a b l i s h e d , a l o g i c a l progression was then to attempt to use ORD and CD data to make q u a n t i t a t i v e estimates of secondary s t r u c t u r e f r a c t i o n s ( i . e . " - h e l i x , B-sheet, B-turn and random c o i l ) . I t i s now ge n e r a l l y accepted that the ORD and CD spectra are a d i r e c t r e f l e c t i o n of p r o t e i n secondary s t r u c t u r e (Hennessey and Johnson, 1981). Various attempts using a v a r i e t y of t h e o r e t i c a l and e m p i r i c a l techniques have been used to q u a n t i t a t e ORD and more r e c e n t l y , CD data. Ear l y attempts were made by G r e e n f i e l d et a l . (1967) to f i t experimental ORD spectra of p r o t e i n s to reference ORD curves of p o l y ( L - l y s i n e ) , c o n taining varying amounts of " - h e l i x , B-structure and random c o i l segments. Results i n d i c a t e d that the reference curves were u s e f u l i n p r e d i c t i n g gross polypeptide and p r o t e i n s t r u c t u r e , however, i n t e r p r e t a -t i o n of the ORD data became d i f f i c u l t when the polypeptide or p r o t e i n contained aromatic groups, d i s u l f i d e bridges or p r o s t h e t i c chromophores. Magar (1968) proposed the L 2 norm method ( l e a s t squares) for minimizing the variance between observed and computed ORD curves f or the determination of " - h e l i x , 6-form and random c o i l . The inherent problems of ORD curve a n a l y s i s , coupled with advances i n technology has r e s u l t e d i n the a n a l y s i s of CD spectra for the deter-mination of secondary s t r u c t u r e becoming prevalent. G r e e n f i e l d and Fasman (1969) used c i r c u l a r dichroism curves of p o l y ( L - l y s i n e ) - 15 -c o n t a i n i n g varying amounts of " - h e l i x , 8-sheet and random c o i l segments to determine p r o t e i n secondary s t r u c t u r e . CD curves of s e v e r a l p r o t e i n s (e.g. myoglobin, lysozyme, etc.) whose three-dimensional s t r u c t u r e s had been determined from X-ray data were f i t t e d by a l i n e a r combination of the three reference s t r u c t u r e s . The computed curves were found u s e f u l i n p r e d i c t i n g p r o t e i n s t r u c t u r e . I f the p r o t e i n i n question possessed a high degree of secondary s t r u c t u r e , the agreement between the c a l c u l a t e d and the X-ray d i f f r a c t i o n determined s t r u c t u r e was extremely good, however, i f the p r o t e i n was l a r g e l y non-regular the r e s u l t s were l e s s s a t i s f a c t o r y . In the method of i s o d i c h r o i c p o i n t s ( i . e . the point of c r o s s i n g of two of the three CD s p e c t r a ) , Myer (1970) estimated the f r a c t i o n s of the three forms i n p r o t e i n s from the CD measurements at three wavelengths corresponding to the three i s o d i c h r o i c p o i n t s . P o l y ( L - l y s i n e ) was again used as a model for the three reference forms. I t was found that the method y i e l d e d s a t i s f a c t o r y r e s u l t s for various p r o t e i n s when compared to other procedures, for example G r e e n f i e l d et a l . (1967) and Magar (1968), however, i n comparison to X-ray d i f f r a c t i o n data the r e s u l t s obtained were f a i r . The use of s y n t h e t i c polypeptides such as poly ( L - l y s i n e ) to estimate secondary s t r u c t u r e f r a c t i o n s i n g l o b u l a r p r o t e i n s has lead to s e v e r a l c r i t i c i s m s . Synthetic polypeptides do not resemble r e a l p r o t e i n s whose s t r u c t u r a l elements d i f f e r from i d e a l models; h e l i c a l polypeptides of high molecular weight such as depro-tonated p o l y ( L - l y s i n e ) are u n l i k e short h e l i c a l segments i n a p r o t e i n whose CD spectrum i s chain-length dependent (Chen et a l . , 1972). In a d d i t i o n , the conformations assumed by the 6-polypeptides may have a - 16 -d i f f e r e n t CD spectrum and/or magnitude under d i f f e r e n t c o n d i t i o n s ( G r e e n f i e l d and Fasman, 1969; L i and Spector, 1969; Chang et a l . , 1978). Saxena and Wetlaufer (1971), i n order to avoid the aforementioned problems, used the CD spectra of three p r o t e i n s ( r i b o n u c l e a s e , lysozyme and myoglobin) of known secondary s t r u c t u r e to derive the reference e l l i p t i c i t i e s f or the three s t r u c t u r a l f r a c t i o n s ( a - h e l i x , B-structure and random). The method gave r e s u l t s that were comparable to d i f f r a c -t i o n estimates, and were as good as, or b e t t e r than, analyses of p r o t e i n CD s p e c t r a based on p o l y l y s i n e . S i m i l a r s t u d i e s were c a r r i e d out by Chen and Yang (1971) and Chen et a l . (1972) where f i v e reference p r o t e i n s (myoglobin, lysozyme, l a c t a t e dehydrogenase, papain and r i b o -nuclease) were used to c a l c u l a t e the reference e l l i p t i c i t i e s of «-helix, 8-sheet and random c o i l at the d i f f e r e n t wavelengths by a l e a s t squares method. In a subsequent paper, Chen et a l . (1974) increased the number of reference p r o t e i n s to eight and introduced an expression to account for the chain-length dependence of h e l i c e s , which r e p o r t e d l y lead to "a more reasonable determination of f r a c t i o n s of h e l i x " . Woody and Tinoco (1967) found that the o p t i c a l p r o p e r t i e s of h e l i c e s were dependent on the chain-length of the h e l i x . E a r l y s t u d i e s regarding the a n a l y s i s of CD spectra considered only the ^ - h e l i x , 8-sheet and random c o i l f r a c t i o n s . Chang et a l . (1978) modified the algorithm of Chen et a l . (1974) to i n c l u d e the B-turn f r a c t i o n and increased the p r o t e i n reference base to f i f t e e n . A s i m i l a r approach was used by B o l o t i n a et a l . (1980) where f i v e p r o t e i n s were used as a reference base. In attempts to e x t r a c t more informa t i o n from - 17 -the CD spectrum, B o l o t i n a et a l . (1979) defined new CD reference spectra to i n c l u d e both p a r a l l e l and a n t i - p a r a l l e l 8-sheet as w e l l as " - h e l i x , B-turn and random c o i l . S a t i s f a c t o r y r e s u l t s were obtained when the c a l c u l a t e d secondary s t r u c t u r e f r a c t i o n s were compared to data obtained from X-ray d i f f r a c t i o n a n a l y s i s . F. CURVE FITTING ANALYSIS OF CD SPECTRA In attempts to determine the i n d i v i d u a l secondary s t r u c t u r e f r a c -t i o n s through the a n a l y s i s of CD s p e c t r a , a v a r i e t y of techniques have been used. E a r l y s t u d i e s ( G r e e n f i e l d and Fasman, 1969) v i s u a l l y com-pared the experimental curve with s y n t h e t i c curves c o n t a i n i n g various amounts of " - h e l i x , 8-sheet and random c o i l , however, only approxima-t i o n s could be made using t h i s technique. A mathematical approach was taken i n subsequent s t u d i e s where most researchers (Saxena and Wetlau-f e r , 1971; Barela and D a r n a l l , 1974; Grosse et a l . , 1974; Bannister and Bannister, 1974; Chang et a l . , 1978; Brahms and Brahms, 1979; B o l o t i n a et a l . , 1979; S i e g e l et a l . , 1980) used l e a s t squares a n a l y s i s to solve the reference e l l i p t i c i t i e s of the secondary s t r u c t u r e f r a c t i o n s and to determine the secondary s t r u c t u r e f r a c t i o n s . In a d i f f e r e n t approach Baker and Isenberg (1976) presented a method whereby the c i r c u l a r dichroism spectra was analyzed by employing i n t e g r a l s over the data. The " - h e l i x , 8-sheet and random c o i l contents of the p r o t e i n s were c a l c u l a t e d from such i n t e g r a l s . I t was shown that the analyzed " - h e l i x content was u s u a l l y r e l i a b l e , B-sheet somewhat l e s s r e l i a b l e and the random c o i l values were l e a s t r e l i a b l e when compared to X-ray data. - 18 -Hennessey and Johnson (1981) presented a method for p r e d i c t i n g secondary s t r u c t u r e from the CD spectrum where eight types of secondary s t r u c t u r e were considered: h e l i x ; p a r a l l e l and a n t i - p a r a l l e l 8-strand; types I, I I and I I I 8-turn; a l l other 8-turns combined; and "other" s t r u c t u r e s . The method was based on the eigenvector technique of m u l t i -component matrix a n a l y s i s ( L l o y d , 1969) using a set of f i f t e e n p r o t e i n CD spectra and one polypeptide (poly(L-glutamic a c i d ) ) CD spectrum over the range of 178 and 260 nm i n order to generate a set of orthogonal CD sp e c t r a for use as a reference b a s i s . The eight types of s t r u c t u r e were determined from the b a s i s CD spectra as a f u n c t i o n of the p r o t e i n CD spectrum. Results compared favourably with X-ray data. Provencher and Glockner (1981) developed a method i n which the CD spectrum of a p r o t e i n was analyzed d i r e c t l y as a l i n e a r combination of the c i r c u l a r dichroism s p e c t r a (190 to 240 nm) of s i x t e e n p r o t e i n s whose secondary s t r u c t u r e s were determined from X-ray data. The technique avoids the problem of t r y i n g to determine reference CD spectra charac-t e r i s t i c of each of the i n d i v i d u a l secondary s t r u c t u r e f r a c t i o n s as i s required when using l e a s t squares f i t t i n g a n a l y s i s . The method i s based on a simple constrained s t a t i s t i c a l r e g u l a r i z a t i o n and employs quadratic programming. When the c a l c u l a t e d " - h e l i x and B-sheet f r a c t i o n s of the various p r o t e i n s analyzed were compared to X-ray d i f f r a c t i o n r e s u l t s , accuracy was very good. Less accurate r e s u l t s were obtained f o r B-turn and remainder f r a c t i o n s . - 19 -G. SIMPLEX OPTIMIZATION P r e s e n t l y , one of the main goals of research i s to develop and e s t a b l i s h mathematical methods to e f f i c i e n t l y search for optimum e x p e r i -mental c o n d i t i o n s (Nakai, 1982). One of the most popular methods for f i n d i n g these c o n d i t i o n s i s the s e q u e n t i a l simplex method of Morgan and Deming (1974). The usefulness of t h i s technique l i e s i n i t s a b i l i t y to handle s e v e r a l v a r i a b l e s at a time i n a s t r a i g h t forward manner. This m u l t i f a c t o r technique was o r i g i n a l l y developed by Spendley et a l . (1962) as an a l t e r n a t i v e to the time consuming and complex e v o l u t i o n a r y opera-t i o n (EVOP) method of Box (1957). Nelder and Mead (1965) modified the technique to allow for the r a p i d search of an optimal response and Morgan and Deming (1974) l a t e r r e f i n e d the procedure to allow for even f a s t e r searches. The simplex o p t i m i z a t i o n method has been 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 and s e v e r a l reviews have been published regarding i t s a p p l i c a t i o n i n t h i s f i e l d (Deming and Parker, 1978; Kowalski, 1980; Frank and Kowalski, 1982). However, very few a p p l i c a t i o n s of the simplex method have been c i t e d with regards to food science. Dols and Armbrecht (1976) were one of the f i r s t to propose and o u t l i n e the simplex o p t i m i z a t i o n procedure for a p p l i c a t i o n to food r e l a t e d problems, van de Voort et a l . (1979) applied the simplex o p t i m i z a t i o n technique to c a l c u l a t e the molecular weights and r e l a t i v e c o ncentration of the i n d i v i d u a l p r o t e i n s i n a mixture of three food r e l a t e d p r o t e i n s using sedimentation e q u i l i b r i u m data. Results obtained were i n good agreement with the t h e o r e t i c a l values. F u j i i and Nakai (1980) used - 20 -simplex o p t i m i z a t i o n to transform food r e l a t e d data f or l i n e a r i z a t i o n and obtained good r e s u l t s when c a l c u l a t e d and model curves were com-pared. Li-Chan et a l . (1979) using simplex o p t i m i z a t i o n , optimized the c o n d i t i o n s f or the covalent attachment of l y s i n e to wheat g l u t e n . Recently, Nakai (1982) c a r r i e d out an extensive survey of various o p t i m i z a t i o n techniques f or use i n food product and process development and proposed that a form of the simplex o p t i m i z a t i o n technique (modified super-simplex algorithm) could be very u s e f u l i n o p t i m i z i n g food pro-cessing and a n a l y s i s . METHODS The CD spectra of s i x t e e n p r o t e i n s of known secondary s t r u c t u r e (as determined by X-ray a n a l y s i s ) were used i n the comparison of various algorithms f or determination of secondary s t r u c t u r e f r a c t i o n s . The algorithms included those of S i e g e l et a l . (1980), Chang et a l . (1978), Provencher and GlOckner (1981) and a technique which u t i l i z e s the simplex algorithm as proposed by Morgan and Deming (1974). The CD spectra of the f o l l o w i n g p r o t e i n s were obtained from the CONTIN computer program of Provencher (1980) f or use as the reference data base: adenylate kinase, "-chymotrypsin, carboxypeptidase A, concanavalin A, cytochrome C, e l a s t a s e , i n s u l i n , l a c t a t e dehydrogenase, lysozyme, myoglobin, nuclease, papain, parvalbumin, ribonuclease A, ribonuclease S and t r y p s i n i n h i b i t o r . A . ALGORITHM OF SIEGEL ET AL. (1980) The method of S i e g e l et a l . (1980) allows f or the 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 an uncharacterized p r o t e i n . The computer program was t r a n s l a t e d from DEC ( D i g i t a l Equipment Corporation) standard BASIC to For t r a n IV and appears i n Appendix 1. Thirteen mean residue e l l i p t i c i t i e s at wavelengths between 210 and 240 nm ( i . e . 210, 213, 216, 218, 220, 222, 224, 227, 229, 231, 234, 237 and 240 nm) were entered i n t o the program to determine the proportion of h e l i x . The amount of B-sheet i s c a l c u l a t e d i n d i r e c t l y , as i t i s l i n e a r l y dependent on the qua n t i t y of " - h e l i x present. No 8-turns or unordered f r a c t i o n s - 22 -can be c a l c u l a t e d using the method. The f o l l o w i n g equations were used for the c a l c u l a t i o n of " - h e l i x and B-sheet f r a c t i o n s : f =  C± Eq. 1 H 9H(X) f D = -0.729 f u + 0.583 Eq. 2 P n where f u = f r a c t i o n of " - h e l i x n [©..] = observed mean residue e l l i p t i c i t y A. C. = c o r r e c t i o n f a c t o r A. f Q = f r a c t i o n of B-sheet P $u(\\ = mean residue e l l i p t i c i t y of a h y p o t h e t i c a l p r o t e i n that H U ; i s 100% h e l i x . B. ALGORITHM OF CHANG ET AL. (1978) Chang et a l . (1978) used a form of m u l t i p l e r e g r e s s i o n to solve the l e a s t squares i n order to determine the secondary s t r u c t u r e f r a c t i o n s . This method i s based on the assumption that the mean residue e l l i p t i c i t y [0] at any wavelength i s the sum of the s t r u c t u r a l elements ( " - h e l i x , B-sheet, B - t u r n and unordered) of the p r o t e i n molecule. In order to c a l c u l a t e the secondary s t r u c t u r e f r a c t i o n s the f o l l o w i n g equation (Eq. 3) was used: [ E ] X = V6^1-1^ + F B C 0 ] B + f t [ 0 ] t + f R [ 0 ] R Et>* 3 The [0] values on the r i g h t hand side of the equation represent the reference values f or h e l i x (H), B-sheet ( 6 ) , B - t u r n ( t ) and unordered form (R), r e s p e c t i v e l y . The f values correspond to the f r a c t i o n s of the - 23 -s t r u c t u r a l elements. The chain length dependence of the «-helix was accounted for by the i n c l u s i o n of k which i s a wavelength dependent constant, and n, the average number of amino acid residues per h e l i c a l segment. Since the program of Chang et a l . (1978) was not a v a i l a b l e , a computer program was w r i t t e n i n Fortran IV using subroutines from the UBC Matrix (1979) manual to solve the l e a s t squares problem (Appendix 2). The method implemented was one which solved overdetermined systems ( i . e . more equations than unknowns) and was b a s i c a l l y a m u l t i p l e regres-s i o n method which allowed for l i n e a r c o n s t r a i n t s . This program u t i l i z e d the reference e l l i p t i c i t i e s f or the various secondary s t r u c t u r e f r a c -t i o n s obtained from J . T. Yang (unpublished data). P r e l i m i n a r y r e s u l t s i n d i c a t e d that the secondary s t r u c t u r e f r a c t i o n s obtained using the computer program were nearly i d e n t i c a l to those reported by Chang et a l . (1978) and t h e r e f o r e , the l a t t e r r e s u l t s were used for comparative purposes i n the present study. C. ALGORITHM OF PROVENCHER AND GLOCKNER (1981) The computer program of Provencher (1980) which implements the method of Provencher and GlBckner (1981) was adapted to the Amdahl 470 V/8 computer and tes t e d with sample data to ensure that the program was running c o r r e c t l y . In t h e i r method Provencher and Glockner (1981) determined the secondary s t r u c t u r e f r a c t i o n s of each p r o t e i n a f t e r removing that p r o t e i n ' s CD spectrum from the reference data base ( i . e . " b l i n d a n a l y s i s " ) . Attempts to d u p l i c a t e the r e s u l t s of Provencher and GlHckner (1981) by m o d i f i c a t i o n of the data base were not pursued, due - 24 -to the complexity of the computer program. Therefore, the secondary s t r u c t u r e f r a c t i o n values as reported by the authors were used i n the comparison with those determined by X-ray data. The f o l l o w i n g equations were used f o r the c a l c u l a t i o n of the various secondary s t r u c t u r e f r a c t i o n s : N y ( \ ) = .ZY y.RAX) Eq. 4 j=1 J J N N N y Y 1 £ [ y ( V ) - y n h e H ( \ J ] + a E (Y-- — ) = minimum Eq. 6 k=1 j=1 J N N f E f. = 1 f. > 0 Eq. 7 i=1 i i - M where y(X) = mean residue e l l i p t i c i t y = wavelength k Y• = a p r o p o r t i o n a l i t y f a c t o r J N = the number of p r o t e i n s used i n the data base Y K R.(\) = mean residue e l l i p t i c i t y of p r o t e i n j at wavelength X <3 f. = f r a c t i o n of secondary s t r u c t u r e l ' = f r a c t i o n of secondary s t r u c t u r e i i n p r o t e i n j Nj- = number of secondary s t r u c t u r e c l a s s e s N = number of measured mean residue e l l i p t i c i t i e s y tt - r e g u l a r i z e r - 25 -D. SIMPLEX-LEAST SQUARES METHOD The f i n a l method examined used the simplex algorithm of Morgan and Deming (1974) to determine secondary s t r u c t u r e f r a c t i o n s i n order to o b t a i n a l e a s t squares s o l u t i o n for Eq. 8. The technique was the r e f o r e termed the s i m p l e x - l e a s t squares method. min = Eq. 8 The r i g h t hand side of Eq. 3 (Chang et a l . , 1978) was incorporated i n t o Eq. 8 for the l e a s t squares c a l c u l a t i o n . The reference e l l i p t i c i t i e s f o r the various secondary s t r u c t u r e f r a c t i o n s , as w e l l as the wavelength dependent f a c t o r (k) at 1 nm i n t e r v a l s from 190 to 240 nm were obtained from J . T. Yang (unpublished data). Since the h e l i x reference e l l i p t i -c i t y used by Chang et a l . (1978) i s dependent on both k and n, a program was w r i t t e n which c a l c u l a t e s the h e l i x reference e l l i p t i c i t i e s for v a r i -ous n values and appears i n Appendix 3. Two versions of the s i m p l e x - l e a s t squares method were used i n the study: one i n which n was held constant at 10.4 and the other where n was v a r i e d according to the p r o t e i n examined. The value of 10.4 i s an average based on 18 p r o t e i n s of known secondary s t r u c t u r e (Chang et a l . , 1978). In order to c a l c u l a t e the secondary s t r u c t u r e f r a c t i o n s , 51 mean residue e l l i p t i c i t i e s (190 to 240 nm) were entered i n t o the program. A l i s t i n g of the program i s found i n Appendix 4. - 26 -E . CORRELATION COEFFICIENTS In order to assess the accuracy of the algorithms the p r o p o r t i o n of secondary s t r u c t u r e f r a c t i o n s determined by each method was regressed against the proportions determined from X-ray d i f f r a c t i o n a n a l y s i s (obtained from Chang et a l . , 1978). The l i n e a r r e g r e s s i o n program for the Monroe 1880 Programmable C a l c u l a t o r ( L i t t o n Business Systems Inc., Orange, NJ) was used to c a l c u l a t e the c o r r e l a t i o n c o e f f i c i e n t ( r ) . The r value was then compared to a t a b l e of c r i t i c a l values (Zar, 1974) to t e s t for s i g n i f i c a n c e . - 27 -RESULTS AND DISCUSSION The r e s u l t s of the secondary s t r u c t u r e determination using the four algorithms are compared i n Table 1 to the X-ray d i f f r a c t i o n data reported by Chang et a l . (1978). A. ALGORITHM OF SIEGEL ET AL. (1980) The method of S i e g e l et a l . (1980) was the most l i m i t e d of the algorithms examined for use i n s t r u c t u r e - f u n c t i o n s t u d i e s s i n c e only " - h e l i x and B-sheet f r a c t i o n s were determined. In comparison, four secondary s t r u c t u r e f r a c t i o n s ( " - h e l i x , B-sheet, B-turn and unordered) were determined by the other algorithms. Examination of the equations u t i l i z e d by S i e g e l et a l . (1980) (Eq. 1 and 2) i n d i c a t e s that the " - h e l i x content of the t e s t p r o t e i n i s c a l c u l a t e d d i r e c t l y from the CD spectra while the B-sheet f r a c t i o n i s c a l c u l a t e d i n d i r e c t l y and i s a f u n c t i o n of the " - h e l i x content. Equation 1 was derived by determining the f i t of the e l l i p t i c i t i e s of 16 reference p r o t e i n s to t h e i r X-ray s t r u c t u r e s at 13 s e l e c t e d wavelengths using m u l t i p l e l i n e a r r e g r e s s i o n a n a l y s i s . However, the authors reported that only the mean residue e l l i p t i c i t y (9H(^)) s i g n i f i c a n t l y (P<0.01) described the observed e l l i p t i c i t y . The r e l a t i o n s h i p between " - h e l i x and 8-sheet (Eq. 2) was formulated by r e g r e s s i n g the f r a c t i o n of h e l i x on the f r a c t i o n of B-sheet using 60 p r o t e i n s as a data base. No i n d i c a t i o n was given by the authors as to the nature of these p r o t e i n s . Although only e l l i p t i c i t i e s from 210 nm to 240 nm were entered i n t o the algorithm, the method showed s u r p r i s i n g l y good accuracy for T a b l e 1. A comparison of X-ray secondary s t r u c t u r e s to predicted secondary s t r u c t u r e s of s e l e c t e d p r o t e i n s using various algorithms. P r o t e i n H e l i x B-sheet B-turn Random 1 2 3 4 5 6 y 1 2 3 4 5 6 1 3 4 5 6 1 3 4 5 6 Myoglobin .79 1.09 .80 .86 .78 .72 .00 .00 .00 .00 .05 .11 .05 .02 .00 .02 .03 .16 .18 .14 .16 .14 Lysozyme .41 .41 .32 .45 .31 .33 .16 .29 .29 .21 .31 .33 .23 .08 .26 .07 .04 .20 .31 .08 .32 .31 Ribonuclease A .23 .28 .21 .26 .22 .22 .40 .38 .39 .44 .36 .35 .13 .10 .11 .12 .11 .17 .13 .19 .31 .31 Papain .28 .46 .29 .27 .28 .28 .14 .25 .00 .05 .01 .01 .17 .15 .31 .15 .15 .41 .56 .36 .56 .56 Lactate dehydrogenase s .45 .53 .45 .40 .41 .42 .24 .20 .18 .22 .29 .21 .06 .13 .13 .08 .12 .25 .24 .26 .23 .25 "-chymotrypsin .09 .14 .05 .09 .06 .09 .34 .48 .53 .29 .50 .43 .34 .02 .22 .03 .05 .23. .40 .40 .41 .44 Concanavalin A .02 .15 .25 .08 .19 .62 .51 .47 .46 .41 .61 .00 .09 .20 .15 .12 .24 .38 .09 .36 .08 .14 Cytochrome C .39 .42 .44 .33 ..43 .48 .00 .27 .00 .09 .00 .00 .24 .28 .17 .28 .24 .37 .28 .41 .29 .28 Nuclease .24 .39 .30 .32 .23 .22 .15 .30 .21 .25 .38 .39 .18 .12 .14 .05 .05 .43 .37 .29 .35 .34 I n s u l i n .51 .43 .46 .49 .40 .43 .24 .27 .22 .23 .37 .34 .12 .19 .27 .12 .12 .13 .13 .00 .11 .10 Parvalbumin .62 .59 .49 .58 .38 .37 .05 .15 .00 .00 .32 .35 .17 .26 .00 .12 .11 .16 .25 .42 .19 .18 Carboxypeptidase A .37 .31 .45 .43 .45 .41 .15 .36 .00 .15 .00 .06 .26 .37 .25 .36 .35 .22 .18 .16 .19 .19 Trypsin i n h i b i t o r .28 .29 .07 .21 .08 .09 .33 .37 .32 .28 .29 .27 .03 .00 .23 .02 .02 .36 .61 .29 .61 .61 Adenylate kinase .54 .54 .46 .44 .46 .45 .12 .19 .26 .15 .26 .29 .19 .08 .20 .08 .07 .15 .20 .21 .20 .19 Ribonuclease S .26 .28 .24 .25 .19 .19 .44 .38 .33 .37 .46 .46 .13 .14 .16 .08 .07 .17 .29 .23 .27 .27 Elas t a s e .07 .03 .00 .04 .00 .01 .52 .56 .46 .49 .46 .44 .26 .07 .14 .08 .08 .15 .47 .32 .46 .47 Methods: 1 = X-ray (Chang et a l . , 1978). 2 = S i e g e l et a l . (1980). 3 = Chang et a l . (1978). 4 = Provencher and Glbckner (1981). 5 = Simplex-least squares n = 10.4. 6 = Simplex-least squares n = v a r i a b l e . - 29 -both " - h e l i x and B-sheet f r a c t i o n s as i n d i c a t e d by the s i g n i f i c a n t cor-r e l a t i o n c o e f f i c i e n t s (P<0.001) (Table 2). These r e s u l t s were obtained d e s p i t e the f a c t that a number of CD phenomena occur below 210 nm, i n c l u d i n g : the 193 nm maximum of " - h e l i x , the 198 nm maximum of 6-sheet and the 202 nm maximum and the 190 nm minimum of the 8-turn. Several authors (Brahms et a l . , 1977; Brahms and Brahms, 1980; Hennessey and Johnson, 1981) using vacuum u l t r a v i o l e t CD have observed new c h a r a c t e r -i s t i c bands f o r the secondary s t r u c t u r e f r a c t i o n s . The i n c l u s i o n of these bands i n t o algorithms which analyze CD data may improve the deter-mination of p r o t e i n secondary s t r u c t u r e . In order to examine the v e r s a t i l i t y of each of the methods examined, c o r r e l a t i o n c o e f f i c i e n t s were c a l c u l a t e d i n the presence and absence of concanavalin A. I f a method can a c c u r a t e l y determine secon-dary s t r u c t u r e f r a c t i o n s , regardless of the p r o t e i n examined, then the c o r r e l a t i o n c o e f f i c i e n t should not be g r e a t l y a f f e c t e d by removing that p r o t e i n from the a n a l y s i s . Concanavalin A i s known to give anomalous secondary s t r u c t u r e r e s u l t s when CD s p e c t r a l data are analyzed by some methods (Chang et a l . , 1978; B o l o t i n a et a l . , 1979). Removal of secon-dary s t r u c t u r e values that deviate l a r g e l y from X-ray data, as may be the case with concanavalin A, should increase the c o r r e l a t i o n c o e f f i -c i e n t ; i d e a l l y , the secondary s t r u c t u r e f r a c t i o n p r e d i c t e d should equal that determined by X-ray d i f f r a c t i o n a n a l y s i s . Using the method of S i e g e l et a l . (1980) the c o r r e l a t i o n c o e f f i c i e n t s for " - h e l i x and B-sheet were not g r e a t l y a f f e c t e d by the removal of concanavalin A. Therefore, the method showed good a b i l i t y to determine " - h e l i x and B-sheet f r a c t i o n s (Table 1). - 30 -Table 2. C o r r e l a t i o n c o e f f i c i e n t s between c a l c u l a t e d secondary s t r u c -t u r e - f r a c t i o n s and those obtained from X-ray data. Methods Secondary s t r u c t u r e s H e l i x 8-sheet B-turn Random Si e g e l et a l . (1980) .1 + con A - con A 1 Chang et a l . (1978) + con A - con A Provencher and Glockner (1981) + con A - con A Simplex-least squares n = 10.4 + con A - con A Simplex-least squares n = v a r i a b l e + con A - con A .9139 3 .8305 3 N.A.2 ,9158 a .8116 3 N.A. .8833 .9299* .9650' .9634£ .8799c .9080* .5858' .9025* .8526 .8329* .9377' .9346* .7718 •7173fc .3697 .6602h .2126 .2629 .3030 .2967 .3450 .3611 .2027 .3236 N.A. N.A. .5260C .5571C .4944 .4572 .3950 ,5994c .4317 .5808C ' C o r r e l a t i o n c o e f f i c i e n t s c a l c u l a t e d i n the presence of concanavalin A (+ con A) and i n the absence of concanavalin A (- con A). "N.A. = not a p p l i c a b l e . ' S i g n i f i c a n t at P<0.001. S i g n i f i c a n t at P<0.01. S i g n i f i c a n t at P<0.05. - 31 -It i s i n t e r e s t i n g to note, however, that the " - h e l i x f r a c t i o n of myoglobin p r e d i c t e d by the method of S i e g e l et a l . (1980) d i f f e r e d markedly from the X-ray data (Table 1). P r o t e i n s such as myoglobin which are predominantly h e l i c a l i n nature ( L e v i t t and Chothia, 1976) may r e q u i r e the i n c l u s i o n of the 193 nm band i n the a n a l y s i s of CD s p e c t r a l data for the determination of " - h e l i x . Although some of the CD spectra used by both S i e g e l et a l . (1980) and Chang et a l . (1978) to generate the reference e l l i p t i c i t i e s were obtained from Yang (e.g. myoglobin, lysozyme, ribonuclease A, e t c . ) , the secondary s t r u c t u r e f r a c t i o n s used to generate the reference e l l i p t i c i -t i e s d i f f e r e d f or some of the p r o t e i n s . For example, for myoglobin S i e g e l et a l . (1980) used 87.6 as the percentage of h e l i x , while Chang et a l . (1978) used 79.0. S i e g e l et a l . (1980) obtained the secondary s t r u c t u r e f r a c t i o n s f or the various p r o t e i n s from the data of L e v i t t and Greer (1977), whereas Chang et a l . (1978) obtained the secondary s t r u c -t ures from the X-ray d i f f r a c t i o n l i t e r a t u r e . The d i f f e r e n c e s i n the secondary s t r u c t u r e f r a c t i o n s used by the two groups may e x p l a i n why S i e g e l et a l . (1980) were only able to f i n d a s i g n i f i c a n t r e l a t i o n s h i p between observed e l l i p t i c i t y and h e l i x . However, the methods (e.g. Chang et a l . , 1978; B o l o t i n a et a l . , 1980) which assume that the observed e l l i p t i c i t y i s a l i n e a r f u n c t i o n of the secondary s t r u c t u r e s have not used m u l t i p l e r e g r e s s i o n a n a l y s i s to examine the p o s s i b i l i t y that only c e r t a i n secondary s t r u c t u r e f r a c t i o n s s i g n i f i c a n t l y describe the observed e l l i p t i c i t y . In view of the s i g n i f i c a n t c o r r e l a t i o n s f o r both h e l i x and 8-sheet f r a c t i o n s , i t would nevertheless appear that the - 32 -use of a l i m i t e d number of wavelengths between 210 and 240 nm, as proposed by S i e g e l et a l . (1980), i s s u f f i c i e n t to determine these two secondary s t r u c t u r e f r a c t i o n s for the p r o t e i n s examined. B. ALGORITHM OF CHANG ET AL. (1978) The algorithm of Chang et a l . (1978) showed r e l a t i v e l y good a b i l i t y to determine secondary s t r u c t u r e f r a c t i o n s for the p r o t e i n s examined with the exception of concanavalin A. X-ray a n a l y s i s has shown concanavalin A to contain 2 percent " - h e l i x while the method of Chang et a l . (1978) p r e d i c t e d 25 percent (Table 1). The discrepancy noted for concanavalin A may stem from the choice of reference spectra of the various s t r u c t u r e f r a c t i o n s . According to Chen et a l . (1974) inappro-p r i a t e reference spectra for the secondary s t r u c t u r e s used to examine a p r o t e i n may r e s u l t i n i n c o r r e c t s t r u c t u r e determinations. The choice of reference spectra i s d i f f i c u l t due to the number of s t r u c t u r a l v a r i a n t s fo r each of the secondary s t r u c t u r e f r a c t i o n s w i t h i n the p r o t e i n mole-c u l e . The reference spectrum for each f r a c t i o n becomes "at best a s t a t i s t i c a l average of numerous v a r i a n t s " (Chang et a l . , 1978), so that for example, the h e l i x f r a c t i o n represents " - h e l i x as w e l l as 3 1 0 - h e l i x . Examination of the c o r r e l a t i o n c o e f f i c i e n t s (Table 2) i n the presence and absence of concanavalin A i n d i c a t e d that the method could determine " - h e l i x and 8-sheet f r a c t i o n s with a high degree of accuracy, i n r e l a t i o n to X-ray data. A r e l a t i v e l y accurate determination of the unordered f r a c t i o n s was also obtained. The 8-turn f r a c t i o n s were the l e a s t c o r r e l a t e d (P>0.05) of the secondary s t r u c t u r e f r a c t i o n s . The - 33 -B-turn f r a c t i o n , although c o n s i s t i n g of only four r e s i d u e s , has a large number of s t r u c t u r a l v a r i a n t s (Venkatachalam, 1968; Lewis et a l . , 1973). This makes the B-turn reference e l l i p t i c i t y d i f f i c u l t to charac-t e r i z e (Chen et a l . , 1974). In order to improve the accuracy of methods based on reference spectra f or the various secondary s t r u c t u r e f r a c t i o n s , one could i n -crease the number of reference p r o t e i n s , but more im p o r t a n t l y , increase the conformational d i v e r s i t y of the reference p r o t e i n s . However, Grosse et a l . (1974) postulated that a combination of these two f a c t o r s would be most favourable. C. ALGORITHM OF PROVENCHER AND GLOCKNER (1981) U n l i k e the other methods examined i n t h i s study which used a form of l e a s t squares to solve for the s t r u c t u r e f r a c t i o n s , the algorithm of Provencher and Glbckner (1981) analyzed the CD spectrum of a p r o t e i n d i r e c t l y as a l i n e a r combination of the CD spectra (from 190 to 240 nm) of the s i x t e e n p r o t e i n s i n order to determine the secondary s t r u c t u r e f r a c t i o n s . The authors stated that using such a method would avoid the problem of d e f i n i n g s i n g l e reference e l l i p t i c i t i e s f o r each secondary s t r u c t u r e . Highly s i g n i f i c a n t c o r r e l a t i o n c o e f f i c i e n t s (P<0.001) were found for both h e l i x and 8-sheet f r a c t i o n s (Table 2) and were the highest among the methods t e s t e d . The B-turn and unordered f r a c t i o n s , however, were not s i g n i f i c a n t l y (P>0.05) c o r r e l a t e d to X-ray data, p o s s i b l y due to the l i m i t e d v a r i e t y of reference p r o t e i n spectra i n the data base. - 34 -I t was the contention of the authors that the accuracy of the method would increase as the v a r i e t y of the reference p r o t e i n s i n c r e a s e s . As seen from Table 2, the c o r r e l a t i o n c o e f f i c i e n t s generated for each of the secondary s t r u c t u r e f r a c t i o n s were not a f f e c t e d to a large extent by the removal of concanavalin A from the a n a l y s i s . Thus the method was able 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 concanavalin A with a reasonable amount of accuracy (Table 1) which may i n d i c a t e the versa-t i l i t y of t h i s algorithm. The method of Provencher and Glockner (1981) was the most s t a t i s -t i c a l l y unbiased of the algorithms examined since a " b l i n d a n a l y s i s " was c a r r i e d out by the authors where the p r o t e i n being examined was removed from the reference spectra p r i o r to secondary s t r u c t u r e determination. P r e l i m i n a r y r e s u l t s obtained i n the present study revealed that secon-dary s t r u c t u r e determinations from the CD spectrum of a p r o t e i n , while keeping that p r o t e i n i n the reference data base, were nearly i d e n t i c a l to r e s u l t s from X-ray a n a l y s i s . This would be expected since the method b a s i c a l l y matches the CD spectrum being analyzed to one i n the reference data base. For example, when the CD spectrum of concanavalin A was analyzed the method pre d i c t e d 0.03 h e l i x , 0.48 8-sheet, 0.10 8-turn and 0.39 unordered f r a c t i o n , while from X-ray 0.02 h e l i x , 0.51 8-sheet, 0.09 B-turn and 0.38 unordered f r a c t i o n s were determined. D. SIMPLEX-LEAST SQUARES METHOD The f i n a l method was one i n which the simplex o p t i m i z a t i o n a l g o r -ithm of Morgan and Deming (1974) was used to minimize the l e a s t squares - 35 -d i f f e r e n c e between the observed and c a l c u l a t e d mean residue e l l i p t i c i -t i e s (Eq. 8 ). Numerous papers have been published regarding the deter-mination of secondary s t r u c t u r e from CD data with the majo r i t y using l e a s t squares to solve for the s t r u c t u r e f r a c t i o n s . The l e a s t squares problem has i n turn been solved with m u l t i p l e r e g r e s s i o n techniques (Chang et a l . , 1978; B o l o t i n a et a l . 1980). The simplex o p t i m i z a t i o n algorithm was i n v e s t i g a t e d as an a l t e r n a -t i v e method for s o l v i n g the l e a s t squares problem. The method i s an e m p i r i c a l i t e r a t i v e s trategy i n which the c a l c u l a t i o n s are simple and the d e c i s i o n s are formalized (Morgan and Deming, 1974), and no assump-t i o n s are made about the mathematical f u n c t i o n i n which simplex i s to be employed (Leggett, 1977). The concept of the simplex algorithm can be more e a s i l y understood with the f o l l o w i n g d i s c u s s i o n . A simplex i s represented by a geometric f i g u r e defined by one more dimension than the number of v a r i a b l e s to be determined (Deming and Parker, 1978). Since h e l i x , 8-sheet, B-turn and unordered f r a c t i o n s were to be determined, the simplex i n t h i s study would be represented by a pentagon. The i n i t i a l step i n the simplex algorithm i s the generation of the s t a r t i n g simplex matrix which has the dimensions n+1 by n i n standard matrix n o t a t i o n (where n i s the number of o r i g i n a l v a r i a b l e s ) ; the matrix i s represented by the f o l l o w i n g , where rows represent v e r t i c e s and columns represent secondary s t r u c t u r e f r a c t i o n s : 0 0 0 0 p q q q q p q q q q p q q q q p - 36 -where 0 = lower boundary value 1 {(n-1) + /n+T} Eq. 9 P = n/2 1 {/nTi - 1} Eq. 10 q = n/2 The matrix thus defines the pro p o r t i o n of each secondary s t r u c t u r e f r a c t i o n to be entered i n t o Eq. 8. In the o r i g i n a l procedure set f o r t h by Spendley et a l . (1962) the range for the v a r i a b l e s p and q used i n the s t a r t i n g simplex were t r a n s -formed to achieve a range from zero to one. When d e a l i n g with secondary s t r u c t u r e f r a c t i o n s , however, t h i s c r i t e r i o n i s already met since the proportion of any one f r a c t i o n cannot be l e s s than zero or greater than one i n r e l a t i o n to the t o t a l s t r u c t u r e of a p r o t e i n . Normalization of the data generated i n the simplex was done i n order to meet the con-s t r a i n t that the sum of the secondary s t r u c t u r e f r a c t i o n s would t o t a l one. This c o n s t r a i n t i s based on the assumption that the CD spectrum of a p r o t e i n represents the sum of the independent c o n t r i b u t i o n s of n types of secondary s t r u c t u r e (Brahms and Brahms, 1979; Hennessey and Johnson, 1981). The mean residue e l l i p t i c i t y at each wavelength i s represented by a l i n e a r combination of n reference spectra S^, which are assumed to be r e p r e s e n t a t i v e of the secondary s t r u c t u r e s : Eq. 11 where n i n most cases equals 4, representing h e l i x , 8-sheet, B-turn and unordered f r a c t i o n s . - 37 -To obtain the normalized value, the secondary s t r u c t u r e f r a c t i o n s fo r each vertex were t o t a l l e d and then the i n d i v i d u a l secondary s t r u c t u r e f r a c t i o n s f or that vertex were d i v i d e d i n t o the t o t a l (Eq. 12 and 13). In t h i s way the sum of the i n d i v i d u a l secondary s t r u c t u r e f r a c t i o n s f or each vertex would equal one. X = I f . for i=1 to 4 Eq. 12 i=1 1 f. = f./X Eq. 13 where X = sum of the secondary s t r u c t u r e f r a c t i o n s f. = secondary s t r u c t u r e f r a c t i o n l f^ = normalized secondary s t r u c t u r e f r a c t i o n The normalized values are entered i n t o Eq. 8. The squared d i f f e r e n c e s between the observed mean residue e l l i p t i c i t y and the c a l c u l a t e d mean residue e l l i p t i c i t y using the normalized secondary s t r u c t u r e f r a c t i o n s at each vertex are then summed over the wavelength range (190 to 240 nm). The responses at the f i v e v e r t i c e s are compared to i d e n t i f y the worst (W) and best (B) values (Figure 1). Since a minimization of Eq. 8 i s r e q u i r e d , the worst vertex would be one which generated a large squared d i f f e r e n c e value and conversely, the best vertex would be one which generated a small value. The pentagon used i n t h i s study may a c t u a l l y be condensed to the t r i a n g l e BNW, as i l l u s t r a t e d i n Figure 1, by having N (next-to-worst vertex) represented by the average of the responses excluding the best and worst responses. - 38 -W - WORST VERTEX B - BEST VERTEX N - NEXT-TO - WORST VERTEX P - CENTROID OF N-B VERTICES C r - CONTRACTION OF R C w - CONTRACTION OF W R - REFLECTION VERTEX E - EXPANSION VERTEX Figure 1. P o s s i b l e simplex moves. - 39 -Once the best and worst responses are i d e n t i f i e d , operations such as r e f l e c t i o n s , expansions or various c o n t r a c t i o n s are c a r r i e d out i n order to r e p o s i t i o n the simplex on the response surface. The f i r s t operation i s the generation of a r e f l e c t i o n or the mirror image of the worst l o c a t i o n which i s c a l c u l a t e d according to the f o l l o w i n g r e l a t i o n s h i p : R e f l e c t i o n = "P + 1.0 (F-W) Eq. 14 — — M, where P i s the c e n t r o i d p o i n t , P = / n M = the sum of a l l f r a c t i o n s w i t h i n a simplex except that f r a c t i o n i d e n t i f i e d with the worst l o c a t i o n . The r e f l e c t i o n value for each s t r u c t u r e f r a c t i o n i s entered i n t o Eq. 8 i n order to c a l c u l a t e the response ( i . e . the squared d i f f e r e n c e ) . A comparison of the responses ( i . e . the i n i t i a l simplex plus the r e f l e c -t i o n ) i s then conducted and the appropriate operation taken according to the r u l e s set f o r t h by Morgan and Deming (1974) as o u t l i n e d i n Figure 2. The formulae for the various operations are given i n Table 3. The major p r i n c i p l e of the simplex algorithm i s to replace those c o n d i t i o n s which e l i c i t the worst response; once completed, a new simplex i s formed and the procedure s t a r t i n g with i d e n t i f i c a t i o n of the best and worst response l o c a t i o n s i s i n i t i a t e d again. The procedure i s repeated u n t i l the responses e l i c i t e d by the simplex do not d i f f e r by more than some predetermined value. Results from Table 2 i n d i c a t e d that when n was anchored at 10.4 the c o r r e l a t i o n c o e f f i c i e n t s f o r the various secondary s t r u c t u r e f r a c t i o n s were g e n e r a l l y higher than when n was v a r i e d , the only excep-t i o n being the unordered f r a c t i o n with n = 10.4 i n the presence of - 40 -R R REFLECTION , K = 1.0 E EXPANSION , (( = 2.0 C r CONTRACTION OF R , K = 0.5 C w CONTRACTION OF W , K=-0.5 C r i MASSIVE CONTRACTION OF R, K = 0.25 C w i MASSIVE CONTRACTION OF W, K = -0.25 Y RESPONSE Figure 2. Flow chart of the simplex m i n i m i z a t i o n . C a l c u l a t i o n of the new vertex, V = P + K ( P - W ) . - 41 -Table 3. Operational c a l c u l a t i o n s used i n the simplex o p t i m i z a t i o n a l g o r i t h m . R e f l e c t i o n (R) Eq. 14 R = F + 1.O(F-W) Expansion (E) Eq. 15 E = F + 2.0CT-W) R e f l e c t i o n c o n t r a c t i o n (C ) Eq. 16 r C = F + 0.5(F-W) r Worst c o n t r a c t i o n (C ) Eq. 17 w C = F - 0.5(F-W) w i Massive r e f l e c t i o n c o n t r a c t i o n (C ) Eq. 18 r C ' = F + 0.25(F-W) r i Massive worst c o n t r a c t i o n (C ) Eq. 19 w C ' = F - 0.25(F-W) w - 42 -concanavalin A which was s l i g h t l y lower than n = v a r i a b l e (+ con A). Examination of the r e s u l t s from Table 1, however, i n d i c a t e d that with the exception of concanavalin A, the s t r u c t u r e s p r e d i c t e d d i d not d i f f e r g r e a t l y with n = 10.4 and n = v a r i a b l e . The c o r r e l a t i o n c o e f f i c i e n t s c a l c u l a t e d i n the absence of concanavalin A ( f o r both n = 10.4 and n = v a r i a b l e ) were very s i m i l a r to one another (the h e l i x , B-sheet and unordered f r a c t i o n s were s i g n i f i c a n t at the P<0.001, P<0.001 and P<0.05 l e v e l s , r e s p e c t i v e l y ) . As with the other methods examined, no s i g n i f i -cant c o r r e l a t i o n c o e f f i c i e n t s (P>0.05) were obtained for the B-turn f r a c t i o n s . The e f f e c t on the c o r r e l a t i o n c o e f f i c i e n t s of removing values that deviate l a r g e l y from one another was c l e a r l y i l l u s t r a t e d i n the case where n i s v a r i e d . The r values for a l l the f r a c t i o n s increased with the removal of concanavalin A which was poorly estimated by the simplex method. Provencher and GlOckner (1981) found that the i n c l u s i o n of an e m p i r i c a l chain-length dependent f a c t o r (n) for the h e l i x f r a c t i o n i n t o t h e i r algorithm decreased the accuracy of t h e i r r e s u l t s , despite the fact that other authors (Woody and Tinoco, 1967; Madison and Schellman, 1972) have found that the o p t i c a l a c t i v i t y of a h e l i c a l polypeptide i s chain-length dependent. In general, the simplex method with n anchored at 10.4 gave r e -s u l t s comparable to those of Chang et a l . (1978) (where n was also set at 10.4). Since both the Chang et a l . (1978) and the simplex method used the same reference p r o t e i n s p e c t r a , any d i f f e r e n c e i n r e s u l t s for the secondary s t r u c t u r e f r a c t i o n s for the various p r o t e i n s would be - 43 -a t t r i b u t e d to the method used to solve l e a s t squares. Since the r e s u l t s were comparable t h i s would i n d i c a t e that the simplex o p t i m i z a t i o n a l g o r -ithm could be used as an a l t e r n a t i v e method to m u l t i p l e r e g r e s s i o n i n s o l v i n g l e a s t squares. The simplex method may have produced more accur-ate r e s u l t s i f d i f f e r e n t reference spectra had been generated which were more re p r e s e n t a t i v e of the major s t r u c t u r a l c l a s s e s . In order to gener-ate new reference spectra one would have to increase the number of p r o t e i n s examined and increase the v a r i e t y of s t r u c t u r e s included w i t h i n a c l a s s , as p r e v i o u s l y discussed. O v e r a l l , the simplex method showed poor v e r s a t i l i t y ( l a r g e changes i n r values with the removal of concanavalin A from the r e g r e s s i o n c a l -c u l a t i o n ) . The change i n r values was l e s s extensive when n = 10.4 as compared to n = v a r i a b l e . E . LIMITATIONS OF CD ANALYSIS No method to date has been able to determine the secondary s t r u c -ture f r a c t i o n s of p r o t e i n s examined ( i . e . " b l i n d a n a l y s i s " ) with one hundred percent accuracy, which would imply that some information neces-sary for understanding the r e l a t i o n s h i p between the CD spectrum and secondary s t r u c t u r e f r a c t i o n s i s l a c k i n g , or that some of the assump-t i o n s being made are i n c o r r e c t . For example, i t i s assumed that the CD s p e c t r a l range examined (190 to 240 nm) y i e l d s a l l the necessary i n f o r -mation to determine p r o t e i n secondary s t r u c t u r e . The use of vacuum u l t r a v i o l e t CD may y i e l d some i n t e r e s t i n g information that was p r e v i -ously l a c k i n g . Brahms and Brahms (1980) extended the a n a l y s i s down - 44 -to 165 nm and found that the B-turn was c h a r a c t e r i z e d by the presence of strong bands i n the 182 to 189 nm region, as w e l l as those bands ob-served at 202 and 225 nm. In the case of " - h e l i x , the authors observed that i n a d d i t i o n to the w e l l known 222 nm and 210 nm minima and the 193 nm maxima, there was also a shoulder at 174 nm. The i n c l u s i o n of these new amide t r a n s i t i o n s could lead to a more d e t a i l e d c h a r a c t e r i z a t i o n of the c i r c u l a r dichroism of p r o t e i n s . In a d d i t i o n to the absence of the various types of s t r u c t u r e s w i t h i n the major s t r u c t u r a l c l a s s e s , a l l algorithms to date have neg-l e c t e d the e f f e c t of non-peptide chromophores below 250 nm. Although g e n e r a l l y disregarded due to t h e i r small c o n t r i b u t i o n to the t o t a l o p t i -c a l a c t i v i t y , c i r c u l a r dichroism bands a r i s i n g from aromatic side chains and d i s u l f i d e l i n k a g e s may be important for the CD a n a l y s i s of some p r o t e i n s . P r o t e i n s such as a v i d i n and a c i d DNase are known to have strong c i r c u l a r dichroism bands i n the region of 225 to 250 nm (Sears and Beychok, 1973). One of the major assumptions made i n most of the recent algorithms used to analyze CD data, i s that the CD spectrum r e s u l t s from the con-t r i b u t i o n of the o p t i c a l a c t i v i t i e s of the four major c l a s s e s of secon-dary s t r u c t u r e ( i . e . h e l i x , B-sheet, 8-turn and unordered f r a c t i o n s ) . Although t h i s assumption has produced some f a i r l y accurate r e s u l t s , e s p e c i a l l y i n the determination of h e l i c a l and B-sheet f r a c t i o n s , i t may be overly s i m p l i s t i c i n l i g h t of the various types of s t r u c t u r e that e x i s t w i t h i n each major c l a s s of secondary s t r u c t u r e . Due to the i n h e r -ent v a r i a b i l i t y of s t r u c t u r e i n p r o t e i n s i t i s u n l i k e l y that s i n g l e - 45 -reference s p e c t r a , which represent the four major s t r u c t u r e s , can be used to c a l c u l a t e the secondary s t r u c t u r e s of a l l p r o t e i n s c o r r e c t l y and thus d i s c r e p a n c i e s between X-ray and CD r e s u l t s would be expected. Hennessey and Johnson (1981) presented a method i n which eight types of secondary s t r u c t u r e were considered: h e l i x ; p a r a l l e l and a n t i -p a r a l l e l B-strands; types I, II and I I I B-turn; a l l other B-turns com-bined; and other s t r u c t u r e s . The authors found that there was good c o r r e l a t i o n between the X-ray s t r u c t u r e s and the s t r u c t u r e s obtained from the a n a l y s i s of the CD spectrum, although a t o t a l agreement for a l l p r o t e i n s was s t i l l not achieved. The underlying assumption of a l l CD st u d i e s i s that the s t r u c t u r e determined for a p r o t e i n from X-ray data i s the same as that determined by c i r c u l a r dichroism a n a l y s i s although the p r o t e i n i s i n two d i f f e r e n t s t a t e s : s o l i d ( c r y s t a l ) s t a t e f or X-ray d i f f r a c t i o n and an aqueous s o l u t i o n f or CD. Chen et a l . (1972) s t a t e d that t h i s assumption may be reasonable since p r o t e i n c r y s t a l s prepared for X-ray d i f f r a c t i o n a n a l y s i s contain considerable amounts of water. Drenth (1981) reported that 50 percent of the i n t e r s t i t i a l spaces be-tween p r o t e i n molecules i n good s i z e c r y s t a l s are occupied by water molecules. However, one cannot discount the p o s s i b i l i t y that changes i n p r o t e i n conformation do occur when c r y s t a l s are d i s s o l v e d i n an aqueous s o l u t i o n . Such changes i n p r o t e i n conformation may c o n t r i b u t e to the lack of t o t a l agreement between CD and X-ray r e s u l t s . Closer agreement between X-ray and CD data may also r e s u l t from an improvement i n the accuracy of the CD data. Hennessey and Johnson (1982) i d e n t i f i e d and examined the types of e r r o r s that were associated - 46 -with the a n a l y s i s of p r o t e i n c i r c u l a r dichroism spectra for secondary s t r u c t u r e determination. Three e r r o r s were r e l a t e d to the operation of the spectropolarimeter and included wavelength s y n c h r o n i z a t i o n , s p e c t r a l bandwidth and scan speed; three a d d i t i o n a l e r r o r s were experimental and included i n t e n s i t y adjustments and two sources of b a s e l i n e s h i f t . Since the CD s p e c t r a l data used i n t h i s study were obtained from Provencher (1980), any e r r o r i n the CD data would be c o n s i s t e n t for a l l the a l g o r -ithms examined. In methods which assume that the observed e l l i p t i c i t y i s a l i n e a r f u n c t i o n of the secondary s t r u c t u r e f r a c t i o n s , the p o s s i b i l i t y e x i s t s that i n t e r a c t i o n s may occur between the various secondary s t r u c t u r e f r a c t i o n s . The i n i t i a l model t r e a t s each secondary s t r u c t u r e as a separate e n t i t y , however, t h i s may be i n e r r o r i f such i n t e r a c t i o n s e x i s t . Gayle and Bennett (1978) examined the consequences of model departures on the r e s o l u t i o n of multicomponent spectra and found that i n t e r a c t i o n s may be present among the independent v a r i a b l e s , thus y i e l d -i n g i n c o r r e c t r e s u l t s . - 47 -CONCLUSIONS The o b j e c t i v e of the present study was to compare four d i f f e r e n t algorithms for t h e i r a b i l i t y to accu r a t e l y determine p r o t e i n secondary s t r u c t u r e i n r e l a t i o n to X-ray data. The majority of methods examined showed h i g h l y s i g n i f i c a n t (P<0.001) c o r r e l a t i o n c o e f f i c i e n t s i n r e l a t i o n to " - h e l i x determination when compared to X-ray data; the simplex o p t i -mization method for n = v a r i a b l e i n the presence of concanavalin A was s i g n i f i c a n t at the P<0.05 l e v e l . The methods of Chang et a l . (1978), S i e g e l et a l . (1980) and Provencher and Glockner (1981) al s o showed hi g h l y s i g n i f i c a n t (P<0.001) c o r r e l a t i o n c o e f f i c i e n t s for B-sheet deter-mination, although these c o e f f i c i e n t s were s l i g h t l y lower than those determined for " - h e l i x . The c o r r e l a t i o n c o e f f i c i e n t s f or B-sheet obtained for the simplex o p t i m i z a t i o n method were s i g n i f i c a n t at the P<0.05 l e v e l with the exception of r for n = v a r i a b l e (+ con A) which was found to be not s i g n i f i c a n t (P>0.05). None of the algorithms examined were able to produce s i g n i f i c a n t c o r r e l a t i o n c o e f f i c i e n t s (P>0.05) when the B - t u r n f r a c t i o n was determined. Random c o i l determin-a t i o n r e s u l t e d i n s i g n i f i c a n t (P<0.05) c o r r e l a t i o n c o e f f i c i e n t s from the simplex o p t i m i z a t i o n method i n the absence of concanavalin A and from the method of Chang et a l . (1978). In the absence of concanavalin A, the simplex o p t i m i z a t i o n method with n = 10.4 compared favourably to the method of Chang et a l . (1978) and thus could serve as an a l t e r n a t e algorithm to solve l e a s t squares. Of the four methods examined, the method of Provencher and GlOckner (1981) showed the l e a s t change i n the - 48 -c o r r e l a t i o n c o e f f i c i e n t s when concanavalin A was removed from the analy-s i s , thereby demonstrating the greatest v e r s a t i l i t y for secondary s t r u c -ture determination. - 49 -PART II. A physical-chemical study of aspartyl proteinases as related to enzymatic activity INTRODUCT ION The c l o t t i n g of milk by p r o t e o l y t i c enzymes during the cheese-making process represents one of the oldest operations i n food tech-nology. References to cheese have been found on Sumerian cuneiform t a b l e t s dating from 4000 B.C. (Hofmann, 1974). T r a d i t i o n a l l y , chymosin (rennin) a p r o t e o l y t i c enzyme prepared from c a l f stomach, has been used for cheese-making; however, due to increases i n cheese consumption and shortages of chymosin, a l t e r n a t i v e enzymes have been examined to replace chymosin. These replacements have included such proteinases as porcine pepsin, chicken pepsin and enzymes produced from m i c r o b i a l sources (Kay and V a l l e r , 1981). The proteinases which are s u c c e s s f u l l y used for cheese-making belong to the c l a s s of enzymes known as a s p a r t y l p r o t e i n a s e s . These enzymes are c h a r a c t e r i z e d by the presence of two a s p a r t i c a c i d residues i n the a c t i v e s i t e as w e l l as having a general optimal a c t i v i t y i n the pH range of 1.5 to 5.0, depending on the p a r t i c u l a r enzyme and substrate combination being studied (Voynick and Fruton, 1971; D a l g l e i s h , 1982). In a d d i t i o n , sequence and s t r u c t u r a l homology have been demonstrated for a number of the a s p a r t y l proteinases (Foltmann and Pedersen, 1976; Hsu et a l . , 1977). The success of an enzyme used i n cheese-making l i e s not only i n i t s a b i l i t y to c l o t m ilk, but i n the r e l a t i o n s h i p between m i l k - c l o t t i n g a b i l i t y and general p r o t e o l y t i c a b i l i t y ( D a l g l e i s h , 1982). M i l k -- 50 -c l o t t i n g a b i l i t y i s defined as the s p e c i f i c h y d r o l y s i s of the P h e 1 Q 5 -MetiQ6 bond of K-casein necessary for the i n i t i a t i o n of m i l k - c l o t t i n g ( D a l g l e i s h , 1982). M i l k - c l o t t i n g enzymes s u i t a b l e for cheese-making should combine high c l o t t i n g a c t i v i t y with low p r o t e o l y t i c a c t i v i t y ( V i s s e r , 1981). The enzymes t r y p s i n and papain, which are representa-t i v e of s e r i n e and s u l f h y d r y l p r o teinases, r e s p e c t i v e l y , are able to c l o t m i lk, but due to t h e i r high general p r o t e o l y t i c a b i l i t y the c l o t i s r a p i d l y degraded. Even w i t h i n the a s p a r t y l proteinase c l a s s , the enzymes show d i s s i m i l a r i t y i n t h e i r a c t i o n . Chymosin has been demonstrated to have a high m i l k - c l o t t i n g to p r o t e o l y t i c a c t i v i t y r a t i o , whereas porcine pepsin has a low r a t i o . This d i f f e r e n c e i n a c t i v i t y may be the r e s u l t of s u b t l e conformational d i f f e r e n c e s ( V i s s e r , 1981). The a b i l i t y to q u a n t i t a t e r e l a t i o n s h i p s between s t r u c t u r e and f u n c t i o n of a molecule i s a problem that has c o n t i n u a l l y perplexed s c i e n t i s t s . It i s g e n e r a l l y accepted that the f u n c t i o n of a p r o t e i n cannot be understood u n t i l i t s s t r u c t u r e i s known (Barry et a l . , 1974). Hydrophobic, e l e c t r o s t a t i c and s t e r i c forces are three parameters that a f f e c t s t r u c t u r e and may be used to p r e d i c t f u n c t i o n (Stuper et a l . , 1979). Although previous researchers have measured various p h y s i c a l and s t r u c t u r a l p r o p e r t i e s of some a s p a r t y l p r o t e i n a s e s , no attempts have been made to q u a n t i t a t e the nature of the r e l a t i o n s h i p between s t r u c t u r e and f u n c t i o n . The major o b j e c t i v e s of the present research were as f o l l o w s : f i r s t l y , to measure various s t r u c t u r a l and i n t r i n s i c proper-t i e s of some a s p a r t y l , as w e l l as some non-aspartyl p r o t e i n a s e s ; second-l y , to attempt to c l a s s i f y the proteinases based on these p r o p e r t i e s - 51 -using m u l t i v a r i a t e s t a t i s t i c a l techniques (e.g. p r i n c i p a l component a n a l y s i s ) ; and t h i r d l y , to regress the m i l k - c l o t t i n g to p r o t e o l y t i c a c t i v i t y r a t i o on the p r i n c i p a l components generated from the s t r u c t u r a l and i n t r i n s i c parameters i n order to i d e n t i f y parameters important f o r t h i s f u n c t i o n a l property. By i d e n t i f y i n g these parameters i t may be p o s s i b l e i n the future to modify enzymes to achieve a de s i r e d f u n c t i o n i n food processing. - 52 -LITERATURE REVIEW A. ASPARTYL PROTEINASES 1. C l a s s i f i c a t i o n and sources The majo r i t y of the known proteinases have been c l a s s i f i e d i n t o four d i s t i n c t c l a s s e s according to the nature of the groups involved i n the c a t a l y t i c process ( H a r t l e y , 1960; Voynick and Fruton, 1971). These four enzyme c l a s s e s i n c l u d e the s e r i n e , s u l f h y d r y l , m e t a l l o - and aspar-t y l p r o t e i n a s e s . The s e r i n e proteinases such as chymotrypsin, t r y p s i n , e l a s t a s e , s u b t i l i s i n , e t c . are c h a r a c t e r i z e d by the presence of r e a c t i v e s e r y l and h i s t i d y l residues at the c a t a l y t i c s i t e . In the s u l f h y d r y l proteinases such as papain, f i c i n and bromelain, a c y s t e i n y l and a h i s -t i d y l residue are involved i n c a t a l y s i s . The metalloproteinases r e q u i r e a metal i on for a c t i v i t y and a v a r i e t y of metal ions are associated with t h i s c l a s s of enzymes. Such a s s o c i a t i o n s can include i r o n (e.g. c y t o -chrome oxidase, c a t a l a s e , peroxidase), copper ( l y s y l o x i d a s e ) , z i n c (NADP-linked dehydrogenases, carboxypeptidase) and manganese (arginase) (Lehninger, 1982). The a s p a r t y l p r o t e i n a s e s , o r i g n a l l y known as the acid p r o t e i n a s e s , g e n e r a l l y have maximal a c t i v i t y i n the acid pH range and have one or more c a t a l y t i c a l l y important carboxyl groups i n t h e i r a c t i v e centres. I t was suggested that the name ac i d proteinases be changed to a s p a r t y l proteinases i n keeping with the enzyme c l a s s i f i c a t i o n scheme based on the nature of the s p e c i f i c group(s) l o c a t e d at the a c t i v e s i t e (Hofmann, 1974; Foltmann and Pedersen, 1976; V i s s e r , 1981). This sug-g e s t i o n i s v a l i d i f one considers the example of r e n i n , an endopeptidase - 53 -found i n the kidney, which has an optimal pH of about 6 although i t s c a t a l y t i c residues and mechanism belong to the a s p a r t y l proteinases (Hofmann, 1974). Several c a t a l y t i c features are shared by the a s p a r t y l p r o t e i n a s e s , such as i n h i b i t i o n by p e p s t a t i n (a p e n t a p e p t i d e - l i k e compound i s o l a t e d from Streptomyces) as w e l l as i n h i b i t i o n by the a c t i v e centre i n a c t i v a t o r s d i a z o a c e t y l - n o r l e u c i n e methylester (DAN) and 1,2 epoxy-3-(p-nitrophenoxy) propane (EPNP). I n h i b i t i o n by DAN and EPNP has demonstrated that two c a t a l y t i c a l l y important a s p a r t i c a c i d residues are present i n most of these enzymes (Tang, 1976 and 1979). The a s p a r t y l proteinases have been i s o l a t e d from a wide v a r i e t y of sources i n c l u d i n g : g a s t r i c sources (e.g. pepsin, chymosin, g a s t r i c s i n ) ; m i c r o b i a l sources (e.g. proteinases from Mucor miehei and Mucor p u s i l - l u s ) ; lysosomes (e.g. cathepsin D and E); kidney ( r e n i n ) ; protozoan (e.g. proteinase from Tetrahymena p y r i f o r m i s ) ; as w e l l as plant sources (proteinase from Sorghum v u l g a r e ) . 2. G a s t r i c a s p a r t y l proteinases Probably the best known and most w e l l c h a r a c t e r i z e d of the a s p a r t y l proteinases are those i s o l a t e d from g a s t r i c sources such as the enzymes pepsin and chymosin. It i s g e n e r a l l y accepted that the g a s t r i c p r o teinases may be c l a s s i f i e d i n t o three main groups, pepsin (EC 3.4.23.1), g a s t r i c s i n (EC 3.4.23.3) and chymosin (EC 3.4.23.4) ( F o l t -mann, 1981). Only the two more important enzymes, pepsin and chymosin, w i l l be discussed here. - 54 -(a) Pepsin The term pepsin i s used for the g a s t r i c proteinases formed from the i n a c t i v e zymogen pepsinogen. This conversion of i n a c t i v e pepsinogen to a c t i v e pepsin occurs as a r e s u l t of l i m i t e d p r o t e o l y s i s that cleaves f o r t y - e i g h t residues from the N-terminal end of pepsinogen. Although m u l t i p l e forms of pepsin e x i s t , the name pepsin i s g e n e r a l l y used i n the l i t e r a t u r e i n reference to porcine pepsin A, the most e x t e n s i v e l y studied form of the pepsins. The primary amino ac i d sequence of porcine pepsin has been deter-mined and c o n s i s t s of 327 amino ac i d residues (Tang et a l . , 1973; Moravek and Kostka, 1974). Examination of t h i s primary sequence i n d i -cates that pepsin i s unusual because of i t s e x c e p t i o n a l l y low content of b a s i c amino a c i d s . The sequence contains one l y s i n e , one h i s t i d i n e and two a r g i n i n e r e s i d u e s . The X-ray c r y s t a l s t r u c t u r e of pepsin has been described at 2.7A r e s o l u t i o n (Andreeva et a l . , 1976). In t h i s study i t was found that the molecule c o n s i s t e d of two lobes separated by a c l e f t , and the a c t i v e s i t e a s p a r t i c a c i d residues (Asp32 and A s p 2 i s ) were located w i t h i n the c l e f t . I t was also found that a maj o r i t y of the residues were in v o l v e d i n the 6-sheet conformation. The p r o t e o l y t i c a b i l i t y of pepsin has been w e l l s t u d i e d . The enzyme has broad side chain s p e c i f i c i t y and can cleave many types of peptide bonds. In general, the s e n s i t i v e bonds are present i n dipeptide u n i t s c o n t a i n i n g at l e a s t one hydrophobic amino acid residue such as phenylalanine, t y r o s i n e , l e u c i n e or methionine; however, a number of - 55 -exceptions to t h i s g e n e r a l i z a t i o n have been noted (e.g. s c i s s i o n of Glu-Asn bond i n the A chain of bovine i n s u l i n ; Glu-Lys and Asp-Pro bonds i n the 8-chain of human hemoglobin A) ( H i l l , 1965; Fruton, 1970). Porcine pepsin i s i r r e v e r s i b l y i n a c t i v a t e d at pH values above 6.0 (Fruton, 1970). I t has been suggested that hydrogen bonds associated with the carboxyl groups are broken at pH values greater than 6 ( E d e l -hoch, 1958a). Upon a l k a l i n e denaturation, changes i n v i s c o s i t y have been observed which would i n d i c a t e that the p r o t e i n i s unfolded to a l i n e a r p o l y e l e c t r o l y t e (Edelhoch, 1957). Below pH 6.0 pepsin may be s t a b l i z e d by hydrophobic bonding since i t i s unaffected by heating to 60°C or by treatment with 4M urea or 3M guanidium c h l o r i d e (Perlmann, 1959; Blumenfeld et a l . , 1960). (b) Chymosin Chymosin i s the predominant m i l k - c l o t t i n g enzyme from the fo u r t h stomach of the c a l f (Foltmann, 1966) and.is found i n the form of rennet, the crude e x t r a c t from the c a l f stomach. The f i r s t attempts to i s o l a t e the e nzymatically a c t i v e component of rennet were published i n 1840 by Deschamps (as c i t e d i n Foltmann, 1966), at which time Deschamps named the enzyme chymosin. I t was suggested by Foltmann (1971) that the name chymosin be used for the pure enzyme, instead of the name rennin, to avoid confusion with the p r o t e o l y t i c enzyme renin i s o l a t e d from the kidney. Chymosin, l i k e most g a s t r i c p r o t e i n a s e s , i s i n i t i a l l y secreted as an i n a c t i v e precursor, and i n t h i s s t a t e i s c a l l e d prochymosin. The conversion of prochymosin to the a c t i v e enzyme takes place through a - 56 -l i m i t e d p r o t e o l y s i s which removes forty-two residues from the N-terminal segment of the peptide chain (Foltmann, 1981). This l i m i t e d p r o t e o l y s i s r e s u l t s i n a molecular weight reduction of approximately 14 percent (Foltmann et a l . , 1977). The primary amino a c i d sequence of chymosin has been determined and contains 323 amino acid residues (Foltmann et a l . , 1977). Attempts to prepare c r y s t a l s appropriate for X-ray d i f f r a c t i o n a n a l y s i s have been unsuccessful (Bunn et a l . , 1.971; Jenkins et a l . , 1976), t h e r e f o r e , no exact secondary or t e r t i a r y s t r u c t u r a l information i s a v a i l a b l e . Compared to the other a s p a r t y l proteinases, chymosin i s d i s t i n -guished by having a high m i l k - c l o t t i n g to p r o t e o l y t i c a c t i v i t y r a t i o , a property that has been f u l l y e x p l o i t e d i n cheese-making. Like pepsin, chymosin i s h i g h l y s p e c i f i c for peptide bonds adjacent to and p r e f e r a b l y between a p a i r of hydrophobic residues (Fox, 1981). However, s t u d i e s with various substrates ( o x i d i z e d B-chain of i n s u l i n and ribonuclease) have i n d i c a t e d that chymosin i s much more l i m i t e d i n i t s p r o t e o l y t i c p r o p e r t i e s (Bang-Jensen et a l . , 1964). The r a p i d and s p e c i f i c p r o t e o l y -t i c cleavage of K-casein by chymosin during the i n i t i a l step of the m i l k - c l o t t i n g process has been the subject of numerous i n v e s t i g a t i o n s (Bang-Jensen et a l . , 1964; de Koning, 1968; J o l l e s et a l . , 1968; Raymond et a l . , 1973; V i s s e r et a l . , 1976 and 1977; Raap et a l . , 1983). Studies ( V i s s e r et a l . , 1976 and 1977) with s y n t h e t i c peptide substrates for chymosin have i n d i c a t e d that the residues around the s e n s i t i v e Phe^ 0b~ Metioe bond of K - c a s e i n , as w e l l as chain length are important f a c t o r s for h y d r o l y s i s of K - c a s e i n . On the b a s i s of these r e s u l t s and r e s u l t s - 57 -obtained with pepsin (Raymond and B r i c a s , 1979), i t was concluded that chymosin made higher demands upon the s t r u c t u r e of i t s substrate than pepsin, which was there f o r e r e s p o n s i b l e f or i t s (chymosin) l i m i t e d p r o t e o l y t i c behaviour ( V i s s e r , 1981). Foltmann (1959) i n v e s t i g a t e d the s t a b i l i t y of the enzymatic a c t i v i t y of chymosin as a f u n c t i o n of pH and found that s t a b i l i t y was good between pH 5.8 and 6.3. At pH values above 6.5 enzymatic a c t i v i t y decreased r a p i d l y with increasng pH. Mickelson and Ernstrom (1963) reported s i m i l a r r e s u l t s i n that maximum s t a b i l i t y of chymosin was main-tained from pH 4.6 to 6.5. Schober et a l . (1960) found that s o l u t i o n s of chymosin were i n a c t i v a t e d at pH 7.0. The i n a c t i v a t i o n was accom-panied by an increase i n ninhydrin r e a c t i o n , which was concluded by the authors to be the r e s u l t of a u t o l y s i s . Foltmann (1966) suggested that the a u t o l y t i c decomposition of chymosin seen at pH 7.0 was the r e s u l t of the chymosin molecule u n f o l d i n g or rearranging " i n such a way that as a substrate i t i s e a s i l y a c c e s s i b l e to the s l i g h t p r o t e o l y t i c a c t i v i t y which i s present even at pH 7.0". Andren and de Koning (1982) examined the e f f e c t of temperature and pH on the m i l k - c l o t t i n g a b i l i t y of chymo-s i n and found that as pH and/or temperature were increased, c l o t t i n g a c t i v i t y decreased. 3. M i c r o b i a l p roteinases In recent years the supply of chymosin has decreased while the demand for cheese has increased. A c c ordingly, there has been an a c t i v e search f o r enzymes with high m i l k - c l o t t i n g and low p r o t e o l y t i c - 58 -a c t i v i t i e s which could be used as s u b s t i t u t e s for chymosin (Fox, 1969; Kay and V a l l e r , 1981). Recently, a number of m i c r o b i a l proteinases have been used commercially for the production of cheese. These enzymes, l i k e the g a s t r i c enzymes pepsin and chymosin, are c l a s s i f i e d as a s p a r t y l p r o t e i n a s e s . U n l i k e the g a s t r i c proteinases which are i n i t i a l l y secreted as zymogens, no zymogens for m i c r o b i a l a s p a r t y l proteinases have been found (Foltmann and Pedersen, 1976). (a) Mucor p u s i l l u s var. Lindt proteinase Arima et a l . (1967) i s o l a t e d a m i l k - c l o t t i n g proteinase from Mucor  p u s i l l u s var. Lindt a f t e r an extensive search of approximately 800 s t r a i n s of micro-organisms. Iwasaki et a l . (1967a) found that the enzyme had a m i l k - c l o t t i n g to p r o t e o l y t i c a c t i v i t y r a t i o s i m i l a r to that of chymosin which made i t s u i t a b l e as a chymosin s u b s t i t u t e . No primary amino ac i d sequence data has been published for Mucor  p u s i l l u s p r oteinase; however, based on amino acid composition data, the enzyme has between 277 and 281 residues (Arima et a l . , 1970). D-galac-tose and D-glucosamine have also been found to be associated with the proteinase (Etoh et a l . , 1979). Yu et a l . (1970) examined various s y n t h e t i c peptides i n order to determine the h y d r o l y t i c s p e c i f i c i t y of the proteinase and found that the s p e c i f i c i t y was s i m i l a r to that of pepsin and chymosin. Peptide u n i t s c o n t a i n i n g at l e a s t one hydrophobic amino ac i d residue were found to be the most s u s c e p t i b l e to h y d r o l y s i s . Iwasaki et a l . (1967a and b) found that at pH values greater than 6.5 there was a r a p i d drop i n both m i l k - c l o t t i n g and p r o t e o l y t i c - 59 -a c t i v i t y . This l o s s of a c t i v i t y at pH values near n e u t r a l i t y i s c o n s i s -tent with that observed for other a s p a r t y l proteinases (Kay and V a l l e r , 1981). (b) Mucor miehei proteinase In 1970, Ottesen and R i c k e r t (1970a) published a paper on the i s o l a t i o n and p a r t i a l c h a r a c t e r i z a t i o n of an ac i d protease from the fungus Mucor miehei. The authors found that l i k e the proteinase produced from Mucor p u s i l l u s var. L i n d t , Mucor miehei proteinase was also able to c l o t milk and t h e r e f o r e , resembled pepsin and chymosin. From i n h i b i t i o n s t u d i e s using DAN and EPNP i t was concluded that M. miehei proteinase belonged to the a s p a r t y l proteinases ( R i c k e r t and McBride-Warren, 1977). Compositional a n a l y s i s has shown that M. miehei proteinase i s a g l y c o p r o t e i n with about 370 amino ac i d residues (Ottesen and R i c k e r t , 1970a and 1970b; R i c k e r t and E l l i o t t , 1973; R i c k e r t and McBride-Warren, 1974). The proteinase has approximately 6 percent carbohydrate which i s composed of hexosamine and n e u t r a l hexoses (Ottesen and R i c k e r t , 1970a and 1970b). Bech and Foltmann (1981) r e c e n t l y published a p a r t i a l primary s t r u c t u r e of _M. miehei proteinase and found that a large number of amino acids i n the N-terminal domain of the molecule were s i m i l a r to that of c a l f chymosin. The researchers also found that amino ac i d residues that would be expected to p a r t i c i p a t e i n the c a t a l y t i c mechanism were found i n the same i n v a r i a n t p o s i t i o n s as for other members of the a s p a r t y l pro t e i n a s e s . - 60 -R i c k e r t (1970), using the B-chain of o x i d i z e d i n s u l i n as a sub-s t r a t e , found that only those bonds i n v o l v i n g aromatic amino a c i d r e s i -dues were hydrolyzed by the pro t e i n a s e . Sternberg (1972) working with s y n t h e t i c s ubstrates also found that peptide bonds having an aromatic amino a c i d (on the N-terminal side of the peptide bond) were hydrolyzed by Mucor miehei p r o t e i n a s e . Enzymatic a c t i v i t y f or the proteinase i s maximal between pH 3.0 and pH 6.0; outside t h i s range a c t i v i t y i s l o s t at a r a p i d rate (Ottesen and R i c k e r t , 1970a). (c) Endothia p a r a s i t i c a proteinase A chymosin-like proteinase i s produced by the fungus Endothia  p a r a s i t i c a (Tarn and Whitaker, 1972). This proteinase c a t a l y z e s both a s p e c i f i c p r o t e o l y s i s l e a d i n g to the c l o t t i n g of milk, as w e l l as a general p r o t e o l y s i s of p r o t e i n s (Whitaker, 1970). No primary amino a c i d sequence data for _E. p a r a s i t i c a proteinase has been published to date although Whitaker (1970) found that the mole-cul e contains approximately 330 amino a c i d r e s i d u e s . P r e l i m i n a r y work on the X-ray c r y s t a l l o g r a p h i c data of the proteinase was c a r r i e d out by Moews and Bunn (1970) and was l a t e r r e f i n e d by Jenkins et a l . (1975) and Jenkins et a l . (1976). Jenkins et a l . (1976) described the s t r u c t u r e of the proteinase as c o n s i s t i n g of two lobes which were separated by "a deep and extensive c l e f t " which formed a hydrophobic pocket. Within the c l e f t the two a c t i v e s i t e a s p a r t i c a c i d residues were found and were i n c l o s e proximity to one another. As with pepsin and p e n i c i l l o p e p s i n , whose c r y s t a l s t r u c t u r e s - 61 -have been determined, X-ray data i n d i c a t e d that a large proportion of the secondary s t r u c t u r e c o n s i s t s of B-sheet s t r u c t u r e s (Jenkins et a l . 1975; Jenkins et a l . , 1976). This observation was confirmed by the c i r c u l a r dichroism a n a l y s i s of the proteinase where three d i f f e r e n t algorithms were used to analyze the CD spectra (Jenkins et a l . , 1976). H y d r o l y s i s of the o x i d i z e d B-chain of i n s u l i n showed that E_. p a r a s i t i c a proteinase had a s p e c i f i c i t y f or the hydrophobic regions s i m i l a r to that of pepsin, although peptide maps of d i g e s t s of the o x i d i z e d B-chain of i n s u l i n by _E. p a r a s i t i c a proteinase and chymosin showed more extensive p r o t e o l y s i s by the former (Whitaker, 1970; Williams et a l . , 1972). E_. p a r a s i t i c a proteinase shows maximum s t a b i l i t y at pH 3.8 to 4.5. Below pH 2.5 l o s s i n a c t i v i t y i s associated with an increase i n ninh y d r i n r e a c t i v e groups which may be a t t r i b u t e d to a u t o l y s i s . Above pH 6.5 a c t i v i t y i s l o s t r a p i d l y and above pH 8.0 a c t i v i t y i s l o s t almost instantaneously; no increase i n ninhydrin r e a c t i v e groups i s associated with t h i s l o s s of a c t i v i t y (Whitaker, 1970). (d) P e n i c i l l o p e p s i n P e n c i l l o p e p s i n i s the acid proteinase produced by the mold P e n i - c i l l i u m j a n t h i n e l l u m at pH values l e s s than 4. The enzyme i s produced once m y c e l i a l growth has ceased and s p o r u l a t i o n has begun (Sodek and Hofmann, 1970b). The primary amino a c i d sequence for the proteinase has been determined (Hsu et a l . , 1977; James and S i e l e c k i , 1983) and c o n s i s t s of - 62 -323 amino a c i d r e s i d u e s . The amino a c i d composition i s somewhat compar-able to that of pepsin i n that the number of the various residues ( i . e . h y d r o p h i l i c , hydrophobic) are s i m i l a r . X-ray d i f f r a c t i o n a n a l y s i s at 1.8A r e s o l u t i o n (James and S i e l e c k i , 1983) has shown that the polypep-t i d e f o l d s i n t o 2 hydrophobic domains with a c l e f t separating the two. As with pepsin and E_. p a r a s i t i c a p r o t e i n a s e , the a c t i v e s i t e a s p a r t i c a c i d residues were found i n the c l e f t with each domain c o n t r i b u t i n g one a s p a r t y l r e s i d u e . Secondary s t r u c t u r e a n a l y s i s of the proteinase based on the X-ray a n a l y s i s i n d i c a t e d that more than two-thirds of the r e s i -dues (218/323) were in v o l v e d i n p a r a l l e l or a n t i - p a r a l l e l B-sheets (Sodek and Hofmann, 1970c; James and S i e l e c k i , 1983). Using the o x i d i z e d 8-chain of i n s u l i n as a s u b s t r a t e , p e n i c i l l o -pepsin showed a h y d r o l y t i c s p e c i f i c i t y s i m i l a r to pepsin (Sodek and Hofmann, 1970a; Mains et a l . , 1971). Peptide bonds which had a hydro-phobic amino a c i d i n the P^' p o s i t i o n (as defined by Berger and Schech-t e r , 1970) were p r e f e r e n t i a l l y cleaved by p e n i c i l l o p e p s i n (Mains et a l . , 1971). To date no information p e r t a i n i n g to m i l k - c l o t t i n g a c t i v i t y has been reported for t h i s p r o t e i n a s e . pH s t a b i l i t y s t u d i e s (Sodek and Hofmann, 1970b) have shown that maximum s t a b i l i t y i s between pH 2.2 and 6.6; beyond t h i s range there i s a r a p i d drop i n a c t i v i t y . The s t a b i l i t y , however, i s both time and temperature dependent. (e) A s p e r g i l l u s s a i t o i proteinase Yoshida (1956) i s o l a t e d a proteinase from the mold A s p e r g i l l u s - 63 -s a i t o i which was capable of a c t i v a t i n g trypsinogen and chymotrypsinogen A at a c i d i c pH values. No primary amino a c i d sequence data for t h i s proteinase has yet been published, however, from the amino a c i d composition data i t i s estimated that the proteinase contains between 283 and 289 amino acid r e s i d u e s . The proteinase contains a high amount of the hydroxy amino acids s e r i n e and threonine (Ichishima and Yoshida, 1965). Conformational s t u d i e s using o p t i c a l r o t a t o r y d i s p e r s i o n (ORD) have i n d i c a t e d that A_. s a i t o i proteinase contains l i t t l e i f any «-helix (Ichishima and Yoshida, 1966a and 1967). In the i n f r a r e d spectrum of the deuterium-exchanged proteinase an amide I band was l o c a t e d at 1632 cm - 1, which would suggest that the enzyme contains a n t i - p a r a l l e l B-structure (Ichishima and Yoshida, 1966a). L i t t l e i s known about the h y d r o l y t i c s p e c i f i c i t y of A_. s a i t o i p roteinase other than the a c t i v a t i o n of trypsinogen by cleavage of a l y s i n e - i s o l e u c i n e bond, r e s u l t i n g i n an a c t i v e enzyme (Gabeloteau and Desnuelle, 1960). In work with s y n t h e t i c s u b s t r a t e s , i t was found that A s p e r g i l l u s s a i t o i proteinase had h y d r o l y t i c p r o p e r t i e s that were "unique among the w e l l known pr o t e i n a s e s " (Yoshida and Nagasawa, 1956b). Fukumoto et a l . (1967) examined the m i l k - c l o t t i n g a b i l i t y of v a r i o u s a s p a r t y l proteinases and found that A s p e r g i l l u s s a i t o i p r o t e i n -ase was i n a c t i v e . The proteinase shows good s t a b i l i t y i n the pH range of 2 to 6, however, beyond t h i s range a c t i v i t y i s r a p i d l y l o s t (Yoshida and Nagasawa, 1956a; Ichishima, 1970). - 64 -B. ENZYMATIC COAGULATON OF MILK The enzymatic coagulation or c l o t t i n g of milk by c e r t a i n p r o t e i n -ases i s a process that may be d i v i d e d i n t o three stages: primary, secondary and t e r t i a r y stages. During the primary stage the bond between Phe^o^-Met-j^g °^ K-casein i n the casein m i c e l l e i s s p e c i f i c a l l y attacked to y i e l d two peptides: glycomacropeptide and para-K-casein. These two peptides have very d i f f e r e n t p r o p e r t i e s . Glycomacropeptide or caseinomacropeptide, which inc l u d e s residues 106 to 169, i s h y d r o p h i l i c and s o l u b l e and w i l l d i f f u s e away from the m i c e l l e subsequent to K-c a s e i n attack. Para-K-casein, on the other hand, which i n c l u d e s residues 1 to 105, i s very hydrophobic and remains associated with the m i c e l l e . The progressive h y d r o l y s i s of K-casein during the primary stage r e s u l t s i n a l t e r a t i o n s of casein m i c e l l e p r o p e r t i e s and subse-quently leads to aggregation. The aggregation of casein m i c e l l e s i s the secondary stage of the m i l k - c l o t t i n g process. The primary and secondary stages of m i l k - c l o t t i n g are not r e a d i l y d i s t i n g u i s h a b l e since the aggre-gation of the casein m i c e l l e s i s i n i t i a t e d before the complete hydroly-s i s of K-casein by the m i l k - c l o t t i n g proteinases (Mackinlay and Wake, 1971; D a l g l e i s h , 1982). The t e r t i a r y stage of the m i l k - c l o t t i n g process i s the l e a s t c l e a r l y defined of the three stages, however, i t in c l u d e s s y n e r e s i s , the expulsion of water by the curd a r i s i n g from a s t r u c t u r a l rearrangement a f t e r c l o t formation, and n o n - s p e c i f i c p r o t e o l y s i s of the various casein components i n the c l o t ( D a l g l e i s h , 1982). Although the s a t i s f a c t o r y c l o t t i n g of milk i s dependent on the cleavage of K-casein at or very near the Phe^ 05-Met^o6 bond, there i s - 65 -al s o a need for a more general i n t e r a c t i o n between enzyme and sub-s t r a t e . This i n t e r a c t i o n i s necessary to promote the c o r r e c t binding which allows for the h y d r o l y s i s of K - c a s e i n at the appropriate s i t e ( D a l g l e i s h , 1982). Most enzymes which s u c c e s s f u l l y c l o t milk belong to the a s p a r t y l p r o t e i n a s e s , however, the s p e c i f i c h y d r o l y s i s of K - c a s e i n by these enzymes i s not e n t i r e l y a t t r i b u t a b l e to the p a r t i c u l a r s p e c i f i -c i t y of the enzymes themselves ( D a l g l e i s h , 1982). Studies ( H i l l , 1969; Trout and Fruton, 1969; Schattenkerk and K e r l i n g , 1973; Powers et a l . , 1976; V i s s e r et a l . , 1976; Visser et a l . , 1980) concerning the i n t e r a c -t i o n of enzymes and peptide substrates resembling the P h e 1 0 5 - M e t 1 0 6 area of K - c a s e i n have shown that composition, sequence and length of the substrate are important determinants regarding enzyme-substrate i n t e r -a c t i o n p r i o r to K - c a s e i n h y d r o l y s i s . C. MULTIVARIATE ANALYSIS One of the primary goals of s c i e n t i f i c research i s the c o l l e c t i o n of data. It i s now p o s s i b l e with modern technology to generate l a r g e amounts of data wherein s e v e r a l parameters are measured f or a s i n g l e sample. However, once these data are c o l l e c t e d one must be able to c r i -t i c a l l y evaluate the data so that u s e f u l information can be e x t r a c t e d . The use of m u l t i v a r i a t e data a n a l y s i s techniques allows f or the e f f i -c i e n t s i m p l i f i c a t i o n and i n t e r p r e t a t i o n of many d i f f e r e n t v a r i a b l e s simultaneously such that maximum information and minimum noise are obtained (Derde and Massart, 1982). Gower (1982) defined m u l t i v a r i a t e a n a l y s i s as those s t a t i s t i c a l methods concerned with e i t h e r the a n a l y s i s - 66 -of data on many v a r i a b l e s or the d i s p l a y of r e s u l t s i n many dimensions, or both. By using m u l t i v a r i a t e techniques the conclusions made are more p r e c i s e than those obtained from the i n d i v i d u a l input data since the e f f e c t of random noise i s decreased; t h i s i s s i m i l a r to averaging over r e p l i c a t e measurements (Martens, 1982). M u l t i v a r i a t e a n a l y s i s tech-niques have been 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, medicine and the s o c i a l s c iences. Recently, a number of papers have al s o appeared where the use of such techniques (e.g. stepwise l i n e a r d i s c r i m i n a n t a n a l y s i s , p r i n c i p a l component a n a l y s i s , etc.) have been ap p l i e d to food research. Several authors (Kowalski, 1980; Frank and Kowalski, 1982; Martens and Russwarm, 1982) have reviewed the use of m u l t i v a r i a t e analy-s i s techniques i n food research. 1. P r i n c i p a l component a n a l y s i s P r i n c i p a l component a n a l y s i s (PCA) i s a data transformation tech-nique that i s concerned with the t o t a l variance of the v a r i a b l e s ( D a u l t r y , 1976). This technique reduces the number of o r i g i n a l v a r i -ables to a smaller number of new v a r i a b l e s or components that e x p l a i n as much of the observed variance as i n the o r i g i n a l v a r i a b l e s . The i n t e n t i s to e x p l a i n the maximum amount of observed systematic v a r i a t i o n i n the data with the fewest p o s s i b l e components. The p r i n c i p a l components derived are mutually uncorrelated and are weighted according to the amount of t o t a l variance they represent, such that the f i r s t component has the l a r g e s t v a r i a n c e , the second component accounts for as much of the remaining variance as p o s s i b l e while being uncorrelated with the f i r s t , and so on ( D a u l t r y , 1976; Hoffman and Young, 1982). - 67 -There are four main steps i n p r i n c i p a l component a n a l y s i s which in c l u d e the f o l l o w i n g : f i r s t l y , the c o r r e l a t i o n or covariance matrix i s generated; secondly, component loadings are obtained; t h i r d l y , the component scores are computed; and f o u r t h l y , two dimensional p l o t s of v a r i o u s p r i n c i p a l component p a i r s are constructed. A basic o u t l i n e of the procedure i s given i n Figure 3. Data for N objects ( i = 1 to N) for which R v a r i a b l e s ( j = 1 to R) are measured can be represented by a data matrix [ 0 ] . The measurements for each v a r i a b l e are f i r s t standardized (matrix [A]) so that no s i n g l e v a r i a b l e dominates the a n a l y s i s (Martens, 1982). A c o r r e l a t i o n matrix [B] i s then c a l c u l a t e d for the R v a r i -a b l e s . Eigenvalues and eigenvectors for the c o r r e l a t i o n matrix are then determined. The eigenvalues generated are an i n d i c a t i o n of the amount of variance i n the data that i s accounted for by the p r i n c i p a l component associated with that eigenvalue. G e n e r a l l y , only p r i n c i p a l components with eigenvalues greater than or equal to one are used i n the a n a l y s i s . In order to ob t a i n the component loading matrix [ L ] , the eigenvector matrix [E] i s m u l t i p l i e d by the square root of the eigenvalues 1 / [A] 2 . The component loadings describe the c o r r e l a t i o n between each v a r i a b l e and each component. Squaring the elements of the compon-ent loading matrix produces the proportion of the variance that each v a r i a b l e c o n t r i b u t e s to each component. V a r i a b l e s that have high l o a d -ings on a component tend to be h i g h l y c o r r e l a t e d to one another while v a r i a b l e s which do not have s i m i l a r loadings are l e s s h i g h l y c o r r e l a -ted. In order to c a l c u l a t e the p r i n c i p a l component scores for each object i i n the various p r i n c i p a l components, the vector of the - 68 -F i g u r e 3. Outline of the procedure for p r i n c i p a l component a n a l y s i s . [0] X n X12 x 1 R X 2 i • O r i g i n a l data matrix [0] Objects i = 1 to N V a r i a b l e s j = 1 to R *N1 N^R V [A] = [B] S n s12 ••• s1R S 2 i • S N1 SNR V C n C 1 2 • • • c l R C 2 1 • Standardized data matrix [A] Objects i = 1 to N V a r i a b l e s j = 1 to R C o r r e l a t i o n matrix [B] C R1 CRR Solve Eigenvalue matrix [A] and Eigenvector matrix [E] Component loading matrix [L] [L] = [E] * [A] / 2 (continued) - 69 -ure 3. (continued) P C n P C 1 2 ••• P C 1 R PC 2 1 • [L] = • • • PC i •••PC V P C n 2 P C 1 2 2 ••• P C 1 R 2 P C 2 i • • Explained variance matrix [V] [V] = • • • • p c R 1 2 ... P C R R 2 P r i n c i p a l component scores matrix [Y] [Y] = [A] * [E] The loadings for each p r i n c i p a l component are represented by the column vectors of the matrix. P C S n PCS 1 2 ... P C S l R [Y] = PC5 2 1 • PCS N i P C S N R The p r i n c i p a l component scores (PCS) for each object i i n each p r i n c i p a l component j are repre-sented by the values i n each column vector. Two dimensional p l o t s of the various p r i n c i p a l components - 70 -standardized measurements for the various R v a r i a b l e s for object i are m u l t i p l i e d by the eigenvector matrix [ E ] . Once the component scores (matrix [Y]) have been c a l c u l a t e d , two dimensional p l o t s of various p a i r s of p r i n c i p a l components may be p l o t t e d i n order to c h a r a c t e r i z e the various N o b j e c t s . Since two dimensional p l o t s are an i n t e g r a l part of p r i n c i p a l component a n a l y s i s (PCA), PCA i s r e f e r r e d to as a l i n e a r d i s p l a y method (Derde and Massart, 1982). Recently, a number of papers have appeared i n the l i t e r a t u r e regarding the use of PCA i n food r e l a t e d problems. In 1979, Aishima published a s e r i e s of papers (Aishima, 1979a; 1979b; 1979c; Aishima et a l . , 1979) i n which p r i n c i p a l component a n a l y s i s was used as an objec-t i v e means for the d i f f e r e n t i a t i o n of soy sauces based on gas chromato-graphic p r o f i l e s . Kwan and Kowalski (1980) used p r i n c i p a l component a n a l y s i s on both o b j e c t i v e chemical measurements and s u b j e c t i v e sensory e v a l u a t i o n data from various Pinot Noir wines. The authors found that i t was p o s s i b l e to c o r r e l a t e the p r i n c i p a l components generated from the two groups of measurements (chemical and sensory), and reported that t h i s type of approach would e v e n t u a l l y lead to "a b e t t e r understanding of the d i r e c t stimulus-response mechanism". Martens (1982) published a paper o u t l i n i n g the p r i n c i p l e s of p r i n c i p a l component a n a l y s i s and then c i t e d an example of i t s a p p l i c a t i o n which involved A f r i c a n f i n g e r m i l l e t samples grown under various growth c o n d i t i o n s . Sixteen amino acids were measured i n the m i l l e t samples and by p l o t t i n g the f i r s t two p r i n c i p a l components i t was p o s s i b l e to show the m i l l e t ' s response to i n c r e a s i n g nitrogen f e r t i l i z a t i o n . Wold et a l . (1982) used p r i n c i p a l component - 71 -a n a l y s i s for the a n a l y s i s of gas chromatographic (GC) data from f r e s h and stored swedes (rutabagas) and found i t was p o s s i b l e to separate the two based on t h e i r GC p r o f i l e s . Other a p p l i c a t i o n s of p r i n c i p a l compon-ent a n a l y s i s as r e l a t e d to food research and data a n a l y s i s have been compiled by Martens and Russwurm (1982). - 72 -MATERIALS AND METHODS A- MATERIALS Proteinases used i n t h i s study were obtained from a v a r i e t y of sources. Porcine pepsin (pepsin A, EC 3.4.23.1) two times c r y s t a l l i z e d and chymosin ( r e n n i n , EC 3.4.23.4) were obtained from Sigma Chemical Co. ( S t . Lou i s , MO). Mucor miehei proteinase and Endothia p a r a s i t i c a proteinase were generous g i f t s from P f i z e r Canada ( K i r k l a n d , PQ). Mucor  p u s i l l u s var. Lindt proteinase was a generous g i f t from Dr. S. Iwasaki (Meito Sangyo Co., L t d . , Tokyo, Japan). A s p e r g i l l u s s a i t o i proteinase was obtained from Calbiochem (San Diego, CA). C r y s t a l s of p e n i c i l l o -pepsin were k i n d l y s u p plied by Dr. T. Hofmann ( U n i v e r s i t y of Toronto, Toronto, ON). The non-aspartyl p r o t e i n a s e s , namely papain (two times c r y s t a l l i z e d ) , t r y p s i n (two times c r y s t a l l i z e d from bovine pancreas) and "-chymotrypsin (three times c r y s t a l l i z e d from bovine pancreas) were obtained from Sigma Chemical Co. ( S t . Louis, MO). The p u r i t y of the proteinases was determined using the SDS polyacrylamide g e l e l e c t r o p h o r -e s i s method of Laemmli (1970). A l l proteinases used i n the present study were shown to be at l e a s t 80 percent pure. The f l u o r e s c e n t probe c i s - p a r i n a r i c a c i d (9,11,13,15-cis, t r a n s , t r a n s , c i s - o c t a d e c a t e t r a e n o i c acid) was purchased from Molecular Probe L t d . ( Junction C i t y , OR). The sodium s a l t of 1-anilino-8-napthalene s u l f o n i c a c i d was obtained from Eastman Kodak (Rochester, NY). The p r o t e i n c a r r i e r 3,3'-dimethylbiphenyl used f o r zeta p o t e n t i a l - 73 -measurements was a product of A l d r i c h Chemical Co., Inc. (Milwaukee, WI). Fluorescamine was purchased from Chemical Dynamics Corp. (South P l a i n f i e l d , NJ). Unless otherwise s t a t e d , reagent grade chemicals were used throughout the study. Glass d i s t i l l e d , deionized water was used i n the preparation of a l l s o l u t i o n s and b u f f e r s . B. DIAGONAL PLOT METHOD In order to determine primary amino ac i d sequence homologies between pr o t e i n a s e s , the diagonal p l o t method of Beynon (1982) was used. B r i e f l y , i n t h i s method the two sequences are aligned along the normal axes and a point i s drawn i n the enclosed area where two residues are i d e n t i c a l . A computer program was w r i t t e n i n Fortran IV to perform the comparison of primary amino ac i d sequences of two proteinases (Appendix 5). C. SECONDARY STRUCTURE PREDICTION For those proteinases with known primary amino ac i d sequences, secondary s t r u c t u r e s were pre d i c t e d using a computerized v e r s i o n of the Chou and Fasman method (1978b) as modified by Pham (1981) which was run on an Amdahl 470 V/8 computer. The primary sequence was converted i n t o a numerical sequence which was then entered i n t o four separate computer programs to determine regions of " - h e l i x , B-sheet, B-turn and overlap regions ( i . e . regions where " - h e l i x and B-sheet regions o v e r l a p ) . A p r e d i c t e d secondary s t r u c t u r e was drawn on the b a s i s of the computer output. - 74 -In a d d i t i o n to the Chou and Fasman method (1978b), secondary s t r u c t u r e was also p r e d i c t e d using the hydrophobicity p r o f i l e method of Cid et a l . (1982). A computer program was w r i t t e n i n Fortran IV for t h i s method (Appendix 6 ) . The primary amino a c i d sequence was again d i g i t i z e d for use i n the computer program. D. CIRCULAR DICHROISM CD spectra were measured using a JASCO J-500A spectropolarimeter (Japan Spectroscopic 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 a two-point c a l i b r a t i o n technique at wavelengths 290.5 and 192.5 nm using a 600 mg/L s o l u t i o n of d-10-camphorsulfonic a c i d (Chen and Yang, 1977). The corresponding 0 1 molar e l l i p t i c i t i e s , [0], are 7800 and -15600 deg cm dmol - , respec-t i v e l y . 1 . Sample pr e p a r a t i o n Proteinases were d i s s o l v e d i n e i t h e r phosphate or acetate b u f f e r (r/2 = 0.01) depending on the pH of the assay. Acetate b u f f e r was used for pH values 5.0, 5.3 and 5.8, while phosphate b u f f e r was used for pH values 6.3, 7.0 and 8.0. The p r o t e i n s o l u t i o n s were then f i l t e r e d through a 0.45 urn Millex-HA f i l t e r ( M i l l i p o r e Corporation, Bedford, MA) p r i o r to CD a n a l y s i s . Analyses were c a r r i e d out w i t h i n 1.0 h of sample p r e p a r a t i o n . - 75 -2. Far-UV s p e c t r a (190 to 240 nm) Between 190 and 240 nm a 1.0 mm c e l l was used with a p r o t e i n con-c e n t r a t i o n of approximately 0.1 mg/mL i n the appropriate b u f f e r . The s p e c t r a l data were reported i n terms of [0], the molar e l l i p t i c i t y per residue (mean residue e l l i p t i c i t y ) . The molar e l l i p t i c i t i e s were not corrected for the r e f r a c t i v e index f a c t o r . In order to obtain [0] data the s i g n a l from the p h o t o m u l t i p l i e r of the spectropolarimeter was d i g i -t i z e d i n t o an analogue reading v i a a d i g i t a l voltmeter (Schlumberger-S o l a r t r o n A220). The s i g n a l was then t r a n s f e r r e d to a Texas Instruments S i l e n t 700 data a c q u i s i t i o n t e r m i n a l v i a a data t r a n s f e r u n i t (Schlum-berger - S o l a r t r o n ) and a hard copy of the data was obtained. 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 for each p r o t e i n sample was obtained by running the appropriate bu f f e r under the i d e n t i c a l c o n d i t i o n s used for the sample. The average d i g i t i z e d s i g n a l at 1 nm i n t e r v a l s from 240 to 190 nm was entered i n t o a computer program (Appendix 7) which allowed for b a s e l i n e c o r r e c t i o n and subsequently converted the data i n t o [0] based on the f o l l o w i n g equation: [0] 0 * MRW E q . 20 MRW, 10 * d * c where X wavelength 0 observed e l l i p t i c i t y i n degrees MRW mean residue weight c concentration i n gm/mL d pathlength i n cm - 76 -The values for MRW were c a l c u l a t e d from the r a t i o of molecular weight to t o t a l number of residues i n the p r o t e i n . The values for molecular weight, t o t a l number of residues and MRW are summarized i n Table 4. Secondary s t r u c t u r e f r a c t i o n s f or each of the p r o t e i n samples were determined using the constrained r e g u l a r i z a t i o n procedure of Provencher and GlOckner (1981) by entering the [0] at 1 nm i n t e r v a l s (from 240 to 190 nm) i n t o the computer program. 3. Near-UV s p e c t r a (240 to 320 nm) Between 240 and 320 nm a 10.0 mm 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 approximately 1.0 mg/mL i n the appropriate b u f f e r . The CD spectra were expressed i n terms of Ae ( 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 ) . The Ae was c a l c u l a t e d according to the f o l l o w i n g equation: re 1 * N A M R W C O-l Ae = Eq. 21 3300 where N = t o t a l number of residues i n the p r o t e i n . The use of [0]MRW i n the near-UV i s not co r r e c t since CD i n t e n s i t y i s a f u n c t i o n of only a few amino a c i d side chains rather than a l l the amino acids i n the p r o t e i n ( S t r i c k l a n d , 1974). E. BIGELOW AVERAGE HYDROPHOBICITY Bigelow (1967) formulated an equation (Eq. 22) to c a l c u l a t e aver-age hydrophobicity (HS/wrj) based on the free energies of t r a n s f e r of amino a c i d side chains as p r e v i o u s l y c a l c u l a t e d by Tanford (1962). - 77 -Table 4. The molecular weight, number of residues and the mean residue weight (MRW) of the various p r o t e i n a s e s . Proteinase Molecular Weight Number of Residues Mean Residue Weight (MRW) Chymosin 30700 a 323 b 96 Pepsin 35000° 327 b 107 Mucor miehei proteinase 38000 d 369 e 103 Mucor p u s i l l u s proteinase 30600 f 281 f 109 Endothia p a r a s i t i c a proteinase 37500 9 328^ 114 A s p e r g i l l u s s a i t o i proteinase 34500 h 289 1 119 P e n i c i l l o p e p s i n 32000^ 323 k 99 Papain 20900 1 212 m 99 Trypsin 24000° 223° 108 "-chymotrypsin 21600 P 214 m 101 a Foltmann (1966). b Foltmann (1981). c Fruton (1970). d Ottesen and R i c k e r t (1970b). e Bech and Foltmann (1981). f Arima et a l . (1970). 9 Whitaker (1970). n Hayashi et a l . (1967). 1 Ichishima and Yoshida (1966b). J Sodek and Hofmann (1970a). k Hsu et a l . (1977). 1 Smith and Kimmel (1960). m Chang et a l . (1978). n Walsh and Neurath (1964). 0 Cunningham et a l . (1953). P Sober (1970). - 78 -^AVG = — where H3? = free energy of t r a n s f e r n = number of amino a c i d r e s i d u e s . The c a l c u l a t i o n was computerized and a l i s t i n g of the program appears i n Appendix 8. F. HYDROPHOBICITY USING FLUORESCENT PROBES P r o t e i n hydrophobicity was determined using the f l u o r e s c e n t probes c i s - p a r i n a r i c a c i d and the magnesium s a l t of 1-anilino-8-napthalene s u l f o n i c a c i d . 1. c i s - P a r i n a r i c a c i d The method of Kato and Nakai (1980) was employed with s l i g h t modi-f i c a t i o n s f or the determination of hydrophobicity using c i s - p a r i n a r i c acid (CPA). P r o t e i n s o l u t i o n s ranging i n concentrations from 0.002% to 0.01% (w/v) i n 0.01 M phosphate b u f f e r pH 6.3 were used f or the assay. Ten \xL of c i s - p a r i n a r i c acid (3.6 x 1 0 - 3 M i n absolute ethanol c o n t a i n -ing 10 ug/mL b u t y l a t e d hydroxyanisole to prevent o x i d a t i o n ) were added to a 2 mL a l i q u o t of each sample which was then r a p i d l y vortexed using a Vortex mixer (Thermolyne Corp., Dubuque, I A ) . The r e l a t i v e fluorescence i n t e n s i t i e s of the s o l u t i o n s were measured with an Aminco Bowman spec-tr o f l u o r o m e t e r No. 4-8202 (American Instrument Co., Inc., S i l v e r Spring, MD) at an e x c i t a t i o n wavelength of 325 nm and an emission wavelength of 420 nm. P r i o r to the sample readings the instrument was c a l i b r a t e d with a mixture of 2 mL n-decane and 10. uL c i s - p a r i n a r i c a c i d s o l u t i o n to Eq. 22 - 79 -o b t a i n a fluorescence i n t e n s i t y reading of 30 percent. "Blank" readings were also obtained for p r o t e i n s o l u t i o n s that d i d not c o n t a i n any f l u o r e s c e n t probe. The fluorescence of these samples was subtracted from the appropriate samples c o n t a i n i n g f l u o r e s c e n t probe to give the c o r r e c t e d fluorescence values. 2. 1-Anilino-8-napthalene s u l f o n a t e (a) P r e p a r a t i o n of ANS Preparation of 1-anilino-8-napthalene s u l f o n a t e (ANS) was c a r r i e d out according to the method of Weber and Young (1964) with s l i g h t modi-f i c a t i o n s . The magnesium s a l t of 1-anilino-8-napthalene s u l f o n i c a c i d was prepared from the sodium s a l t of ANS by p r e c i p i t a t i o n with saturated magnesium acetate. The product was then r e c r y s t a l l i z e d three times from water a f t e r f i l t r a t i o n of the hot s o l u t i o n s through a c t i v a t e d charcoal and Hyflo Super-Cel (3ohns-Manville, Etobicoke, ON). (b) Hydrophobicity determination The determination of hydrophobicity using 1-anilino-8-napthalene s u l f o n a t e was c a r r i e d out employing the method of Hayakawa and Nakai (1983). P r o t e i n concentrations of the same range and at the same pH as those used for CPA were u t i l i z e d . Ten uL of 8.0 x 10~ 3 M ANS i n 0.01 M phosphate b u f f e r pH 6.3 was added to 2 mL a l i q u o t s of each sample and vortexed immediately. R e l a t i v e f l u o r e s c e n t i n t e n s i t i e s were measured at an e x c i t a t i o n wavelength of 390 nm and an emission wavelength of 470 nm. The fluorometer was c a l i b r a t e d with a mixture of 2 mL methanol - 80 -(HPLC grade) and 10 uL ANS s o l u t i o n to obtain a fluorescence i n t e n s i t y reading of 30 percent. Blank readings and corrected fluorescence values were obtained as for the c i s - p a r i n a r i c a c i d study. For both f l u o r e s c e n t probes (CPA and ANS) p r o t e i n hydrophobicity was measured as the i n i t i a l slope of the curve of percent r e l a t i v e fluorescence versus percent p r o t e i n . Duplicate determinations were made fo r each probe. G. CHARGE RATIOS Based on amino a c i d composition data various r a t i o s were c a l c u -l a t e d for each p r o t e i n a s e . These included the number of charged groups (Asp, Glu, Lys, H i s , Arg) to t o t a l number of amino acid r e s i d u e s , the number of a c i d i c groups (Asp, Glu) to t o t a l number, the number of ba s i c groups (Lys, H i s , Arg) to t o t a l number, and the number of a c i d i c groups to number of basic groups. H. ZETA POTENTIAL The zeta p o t e n t i a l measurements of the various proteinases at d i f f e r e n t pH values were measured according to the method of Hayakawa and Nakai (1983). Five mL of a 0.05% (w/v) p r o t e i n s o l u t i o n and 0.15 mL 3,3'-dimethylbiphenyl were e m u l s i f i e d with a P o l y t r o n mixer (Brinkmann Instruments, Rexdale, ON) at 2200 rpm for 20 s. A 0.5 mL a l i q u o t of the emulsion was then p i p e t t e d i n t o 40 mL of the appropriate b u f f e r . The b u f f e r s used were s i m i l a r to those prepared i n Section D-1. The zeta p o t e n t i a l reading of the s o l u t i o n was then obtained from a Laser Zee - 81 -Meter Model 501 (Pen Kern, Inc., Bedford H i l l s , NY) and correct e d for temperature as suggested by the manufacturer. A l l measurements were done i n d u p l i c a t e . I . ACCESSIBLE SURFACE AREA The a c c e s s i b l e surface area (ASA) of each of the proteinases was c a l c u l a t e d based on t h e i r molecular weights, according to the algorithm (Eq. 23) of 3anin (1976): ASA = 11.1 M 7 3 Eq. 23 where M = molecular weight of the proteinase. J . DETERMINATION OF MILK-CLOTTING ACTIVITY The m i l k - c l o t t i n g a c t i v i t y of the various proteinases was determined according to the method of Berridge (1952) as modified by Iwasaki et a l . (1967a). Spray-dried skim-milk powder was used as the substrate and was stored desiccated at 4°C. Twelve percent s o l u t i o n s (w/v) of skim-milk powder i n 0.01 M C a C l 2 were adjusted to various pH values with 0.1 N HC1 or 0.1 N NaOH (Iwasaki et a l . , 1967a) and allowed to e q u i l i b r a t e f or 1 h at room temperature. A ten mL a l i q u o t of r e c o n s t i t u t e d skim-milk was dispensed i n t o a 18 x 150 mm t e s t tube and incubated i n a shaking water bath at 30°C for 10 min. The proteinase was d i l u t e d with the appropriate b u f f e r to obtain a m i l k - c l o t t i n g a c t i v i t y of 4 to 5 min. A 1.0 mL a l i q u o t of the d i l u t e d proteinase s o l u t i o n , pre-incubated at 30°C, was p i p e t t e d i n t o the skim-milk - 82 -s o l u t i o n and mixed by i n v e r s i o n while simultaneously beginning to measure c l o t t i n g time. The i n v e r s i o n was repeated an a d d i t i o n a l two times to ensure complete mixing and the t e s t tube was then returned to the shaking water bath. About 30 s before the expected end-point ( c l o t formation) a glass rod was placed i n t o the milk s o l u t i o n and then r a i s e d about 4 cm above the l e v e l of the milk, keeping the lower end of the rod i n contact with the side of the t e s t tube to allow a stream of milk to run from the rod. C l o t t i n g time was taken as the moment when the milk f i l m broke i n t o v i s i b l e p a r t i c l e s . Berridge (1945) defined one uni t of m i l k - c l o t t i n g a c t i v i t y as the amount of proteinase which c l o t s 10 mL of r e c o n s t i t u t e d skim-milk i n 100 s at 30°C. The s p e c i f i c a c t i v i t y was expressed as m i l k - c l o t t i n g a c t i v i t y per mg p r o t e i n . In order to a c t i v a t e papain f or m i l k - c l o t t i n g measurement, the proteinase s o l u t i o n was prepared i n the presence of 0.05 M cy s t e i n e and 0.02 M EDTA (Arnon, 1970). K. DETERMINATION OF PROTEOLYTIC ACTIVITY The a b i l i t y of the proteinases to hydrolyze sodium caseinate was determined using a method described by Green (1972) with s l i g h t m o d i f i -c a t i o n s . The substrate s o l u t i o n contained 1% (w/v) sodium caseinate i n e i t h e r 0.1 M acetate or 0.1 M phosphate buf f e r depending on the pH. Acetate b u f f e r s were used f or pH 5.0, 5.3 and 5.8, while phosphate b u f f e r s were used f or pH 6.3, 7.0 and 8.0. To 2.5 mL of s u b s t r a t e , pre-incubated at 35°C for 10 min, 0.5 mL of enzyme was added and the mixture incubated at 35°C for 10 min; 2.5 mL of 6.6% (w/v) t r i c h l o r o -- 83 -a c e t i c a c i d (TCA) was then added to give a f i n a l TCA concentration of 3?o (Ma, 1979). The r e s u l t i n g p r e c i p i t a t e was removed by f i l t r a t i o n through Whatman No. 2 paper. The f i l t r a t e was then analyzed for free amino groups according to the method of Kwan et a l . (1983) with s l i g h t m o d i f i -c a t i o n s . To 0.1 mL of f i l t r a t e , 0.3 mL of 1 M K^HPO^ was f i r s t added, followed by 0.15 mL of 0.03?o (w/v) fluorescamine i n acetone which was added r a p i d l y and d i r e c t l y to the s o l u t i o n and mixed immediately using a Vortex mixer. A volume of 3.0 mL of d i s t i l l e d - d e i o n i z e d water was added to the r e a c t i o n mixture and the mixture vortexed once again. The fluorescence of the f i n a l mixture was measured with an Aminco Bowman 4-8202 spectrofluorometer using an e x c i t a t i o n wavelength of 395 nm and an emission wavelength of 480 nm. Blanks were prepared f o l l o w i n g the same procedure except that TCA was added to the substrate p r i o r to the ad d i t i o n of the enzyme s o l u t i o n . A standard curve was constructed using t y r o s i n e . P r o t e o l y t i c a c t i v i t y was defined as the amount of t y r o s i n e released per mg of enzyme. Papain was a c t i v a t e d as p r e v i o u s l y described fo r m i l k - c l o t t i n g . L. PRINCIPAL COMPONENT ANALYSIS Data c o l l e c t e d for the various proteinases was subjected to p r i n -c i p a l component a n a l y s i s using the BMDP:4M program (Frane et a l . , 1981) and run on an Amdahl 470 V/8 computer. Sixteen v a r i a b l e s were inc l u d e d : molar e l l i p t i c i t y values at 10 wavelengths from the CD spectra (190, 193, 198, 200, 202, 210, 213, 222, 224 and 225 nm), zeta p o t e n t i a l , a c i d i c / b a s i c amino acid r a t i o , Bigelow average hydrophobicity, access-i b l e surface area, CPA hydrophobicity and ANS hydrophobicity. The ten - 84 -wavelengths were s e l e c t e d on the ba s i s of c h a r a c t e r i s t i c wavelengths r e p r e s e n t a t i v e of each of the secondary s t r u c t u r e f r a c t i o n s (Chang et a l . , 1978). The " - h e l i x has a c h a r a c t e r i s t i c double minimum at 222 and 210 nm and a maximum at 193 nm. The 8-sheet has a minimum at 213 nm and a maximum at 198 nm while the B-turn has two maxima at 224 and 202 nm with a strong negative band below 190 nm. The unordered or random c o i l s t r u c t u r e shows a strong negative band near 200 nm and another negative band around 225 nm. The m i l k - c l o t t i n g to p r o t e o l y t i c a c t i v i t y r a t i o was then regressed on the p r i n c i p a l components derived from the s i x t e e n v a r i a b l e s using the BMDP: 4R program ( r e g r e s s i o n using p r i n c i p a l components). - 85 -RESULTS AND DISCUSSION A . DIAGONAL PLOTS In the diagonal p l o t method the primary sequences of the two p r o t e i n s i n question are aligned along the two normal axes with the N-terminal amino acids s t a r t i n g at the o r i g i n . The method i s simple and h i g h l y v i s u a l and allows for a r a p i d comparison of two primary amino acids sequences (Gibbs and Mclntyre, 1970). A complete i d e n t i t y i n sequence would r e s u l t i n a diagonal l i n e d i s s e c t i n g the p l o t from corner to corner (Beynon, 1982). The diagonal p l o t s for comparison of primary amino ac i d sequences between pepsin and chymosin are presented i n Figures 4a - 4b, between chymosin and Mucor miehei proteinase i n Figures 5a - 5c, between pepsin and M. miehei proteinase i n Figures 6a - 6c, between pepsin and p e n i c i l l o p e p s i n i n F i g u r s 7a - 7b and between chymo-s i n and p e n i c i l l o p e p s i n i n Figures 8a - 8b. Examination of the p l o t s i n d i c a t e d that there were d i f f e r e n t degrees of homology between the p a i r s of a s p a r t y l proteinases compared, as demonstrated by the various degrees of diagonal completeness. Over-a l l , however, the homologies were r e l a t i v e l y high. From the p l o t between pepsin and chymosin (Figures 4a - 4b) the compilation of the number of a s t e r i s k s along the diagonal i n comparison to the t o t a l number of residues i n d i c a t e d that approximately 60 percent of the t o t a l amino aci d residues were i d e n t i c a l i n the two sequences (Table 5 ) . S i m i l a r comparisons between the other proteinases revealed s u b s t a n t i a l l y lower homology values with approximately 24 to 27 percent of the t o t a l Figure 4a. Diagonal p l o t of the primary amino acid sequences of pepsin and chymosin for residues 1 to 85 and 85 to 170. 260 230" 220-210-• t • • • l • • a • • • * • • • aaa • « a • • • • • mm • • a i a a aa a aa a a a a a aaa • •a a • „ B ACTIVE, S! SITET a a a mm a a a •a a • a i a a a a * a a • • • a a aaa • mm a mm m a m a 335 325-} 315 305 (0 a. ui °- 295-I 285 i 275 265 170 180 190 200 210 220 231) 240 250 260 CHYMOSIN 255 e a a a a a • a a a 255 265 275 285 295 305 315 325 335 CHYMOSIN Figure 4b. Diagonal p l o t of the primary amino ac i d sequences of pepsin and chymosin for residues 170 255 and 255 to 327. • •• • I * B • • "•«.." ACTI,?E SITE —1 1 1 1 1 1 1 1 10 20 30 40 50 60 70 80 MUCOR MIEHEI PROTEINASE 160-1 150 HOH 130 z CO o 2 120 > x o 110-j 100 90-1 80 m mm • • mm I 1 1 1 " I 1 1 1 1 80 90 100 110 120 130 HO 150 160 MUCOR MIEHEI PROTEINASE oo co Figure 5a. Diagonal p l o t of the primary amino acid sequences of chymosin and Mucor miehei proteinase for residues 1 to 80 and 80 to 155. 225 215 H 205 I 195 o z >-o 185-175 165-155-U  • • a a a a mm mm m mm "."ACTIVE • " SITE • • • • • mm m m m a a a (X) 145 155 165 175 185 195 2ol 215 225 MUCOR MIEHEI PROTEINASE Figure 5b. Diagonal plot of the primary amino acid sequences of chymosin and Mucor miehei proteinase for residues 145 to 225. 285-, 275-265-z to I 255-> x o 245 235-1 a • • m ww • • m * • • • 330 320 in S 310-1 > x u 300 225 i t—* 1 1 • i — i 1 225 235 245 255 265 275 285 MUCOR MIEHEI PROTEINASE O 290-1——T 1 1 1 290 300 310 320 330 MUCOR MIEHEI PROTEINASE Figure 5c. Diagonal plot of the primary amino acid sequences of chymosin and Mucor miehei proteinase for residues 225 to 285 and for residues 290 to 330. CO Q. Ill & 80-70-60-50-40-30-j-20-10-0-• • KM • • ACTIVE SITE 160 150 140 130 2 120 111 a 110 100 90 ~10 20 3 0 4 0 5 0 60 70 80 MUCOR MIEHEI PROTEINASE 80 * n mm • m nm m m mm m * • * m * m T " 1 1 1 1 1 80 90 100 110 120 130 140 MUCOR MIEHEI PROTEINASE 150 160 i Figure 6a. Diagonal p l o t of the primary amino acid sequences of pepsin and Mucor miehei proteinase for residues 1 to 80 and 80 to 155. 225 215 205 195 a. u i 0. 175 165 155 145 ' "ACTIVE " • SITE m m * mm mm m m w mm m m m mm m w m m \0 ho • m m m m • * » • U5 155 165 175 185 195 205 2t5 225 MUCOR MIEHEI PROTEINASE Figure 6b. Diagonal p l o t of the primary amino acid sequences of pepsin and Mucor miehei proteinase for residues 145 to 225. 2951 285-275-£ 265-1 ui a 255 245 235-1 225 • • mm mm a n mm* m mmm m mmm m mmm m man a aaa 330 320-1 2 310 Ul a. 300 290 • • i t * 225 235 245 255 265 275 285 295 290 300 310 320 330 MUCOR MIEHEI PROTEINASE MUCOR MIEHEI PROTEINASE \0 Figure 6 c . Diagonal p l o t of the primary amino acid sequences of pepsin and Mucor miehei proteinase for residues 225 to 290 and 290 to 330. 90-80-70-60-50-(0 a. HI °- 40-30-20-10-175 m m • m m m •ACTIVE .SITE "* * I • '« • * m m 165-::" : a • • • * m * • 155-• • 145 135-1 "lO 20 30 40 50 60 70 80 90 PENICILLOPEPSIN (A a. in °- 125 115 105 95-j 85 mm m • • m w m mm m mm u mm m mm mm m m mm mm mm m mm m mm m m m mm m 8 5 9 5 105 115 125 135 145 155 PENICILLOPEPSIN 165 175 S O Figure 7a. Diagonal p l o t of the primary amino acid sequences of pepsin and p e n i c i l l o p e p s i n for residues 1 to 85 and 85 to 170. 260-250-240-230-" i z 2 2 ° " CO a. ui a 210-1. 200-190-180-1 t * • • • mm m m mm nm m mm mm • « • • • » • * SITE • mm m mm mm mm m mm m m 170-• m m mm mm mm « 345 335-1 325 315-1 305 w a ui °- 295-1 285 275-265-170 180 190 200 210 220 230 240 250 260 PENICILLOPEPSIN 255 • • • * • mm mmm mmm m mmm mmm mmm mmm m mmm mmm m mm mmm mmm m mmm mmm 255 265 275 285 29S 305 315 325 335 345 PENICILLOPEPSIN Figure 7b. Diagonal p l o t of the primary amino acid sequences of pepsin and p e n i c i l l o p e p s i n f or residues 170 to 255 and 225 to 340. 90-80-70-60-50-40-30-20-10-• • m m • a a m mm m m m mm m m mm m m mm m m • • • • —i 1 1 — — — i 1 1 1 1 1 10 20 30 40 5 0 6 0 70 8 0 90 CHYMOSIN 175 165" 155-145 (0 a 135-1 o a 125 z Ul a. 115 105 95-1 85 a a • * m m t • • m 85 95 105 115 125 135 145 CHYMOSIN 155 175 Figure 8a. Diagonal p l o t of the primary amino acid sequences of p e n i c i l l o p e p s i n and chymosin for residues 1 to 85 and 85 to 170. 260 250-240-230- . i t 220-210-200-190-180-I 170-• • mm SITE , • mmm m m m m mmm 170 ~180~ 335 n 325 315 Si 305-1 o 2 295-j in a. 285-1 275 265 190 200 210 220 230 240 250 260 CHYMOSIN 255 • • • • • » 255 265 275 285 295 305 CHYMOSIN 315 325 T35 Figure 8b. Diagonal p l o t of the primary amino acid sequences of p e n i c i l l o p e p s i n and chymosin for residues 170 to 255 and 255 to 330. - 98 -T a b l e 5. Degree of s i m i l a r i t y between various p a i r s of a s p a r t y l pro-t e i n a s e s based on the diagonal p l o t method. S i m i l a r i t y Proteinase p a i r (?o) pepsin - chymosin 57.3 chymosin - Mucor miehei proteinase 24.7 pepsin - Mucor miehei proteinase 25.7 pepsin - p e n i c i l l o p e p s i n 27.1 chymosin - p e n i c i l l o p e p s i n 25.3 S i m i l a r i t y (%) was c a l c u l a t e d by d i v i d i n g the number of a s t e r i s k s that lay on the 45° diagonal by the t o t a l number of residues compared. Comparisons of M. miehei proteinase with chymosin and pepsin were based on the p a r t i a l sequence of M. miehei proteinase. - 99 -residues being i d e n t i c a l (Table 5). The fact that pepsin and chymosin showed a higher degree of homology than the comparison of pepsin or chymosin with Mucor miehei proteinase or p e n i c i l l o p e p s i n may be because pepsin and chymosin are g a s t r i c enzymes while Mucor miehei proteinase and p e n i c i l l o p e p s i n are m i c r o b i a l enzymes. Proteinases from s i m i l a r sources may have higher homologies. In comparison to chymosin-pepsin, the degree of s i m i l a r i t y between the other a s p a r t y l proteinases was r e l a t i v e l y low, despite the observation that as a c l a s s , the a s p a r t y l proteinases have- been shown to have very s i m i l a r three-dimensional s t r u c t u r e s . Schulz and Schirmer (1978) stated that i n the e v o l u t i o n of homologous p r o t e i n s , important p o s i t i o n s such as a c t i v e s i t e s are almost i n v a r i a n t , however, very s i m i l a r three-dimensional s t r u c t u r e s can r e s u l t from q u i t e d i f f e r e n t amino acid sequences. It was i n t e r e s t i n g to note that areas adjacent to the a c t i v e s i t e a s p a r t i c a c i d residues ( i . e . Asp32 and Asp2i5 using the pepsin numbering system) were h i g h l y homologous i n a l l proteinase p a i r s examined. F o l t -mann and Pedersen (1976) i n a comparison of the primary s t r u c t u r e s of a c i d i c proteinases and of t h e i r zymogens, reported that the two c a t a l y -t i c a l l y a c t i v e a s p a r t i c acid residues were located i n h i g h l y i d e n t i c a l surroundings. B. SECONDARY STRUCTURE PREDICTION I t i s now g e n e r a l l y accepted that the three-dimensional s t r u c t u r e of a p r o t e i n d i c t a t e s i t s a c t i o n (Barry et a l . , 1974). From renatura-t i o n experiments (Anfinsen et a l . , 1961) i t has been hyothesized that - 100 -the three-dimensional s t r u c t u r e of a p r o t e i n i s a unique f u n c t i o n of i t s amino ac i d sequence. An important f i r s t step i n the p r e d i c t i o n of the o v e r a l l three-dimensional s t r u c t u r e of a p r o t e i n may be the p r e d i c t i o n of secondary s t r u c t u r e ( P t i t s y n and F i n k e l s t e i n , 1983). In t h i s l i g h t , two methods were used to determine secondary s t r u c t u r e from the primary amino ac i d sequences, namely the Chou and Fasman (1978b) method as computerized by Pham (1981) and the hydrophobicity p r o f i l e method of Cid et a l . (1982). Secondary s t r u c t u r e p r e d i c t i o n s were l i m i t e d to those proteinases for which the complete or near complete sequences had been determined. The proteinases examined were chymosin, pepsin, p e n i c i l l o -pepsin and Mucor miehei proteinase ( p a r t i a l sequence). The Chou and Fasman (1978b) method i s a simple p r e d i c t i v e method i n which the secondary s t r u c t u r e forming p o t e n t i a l of the twenty amino acids are used to c a l c u l a t e the " - h e l i x , 8-sheet and B-turn p o t e n t i a l f o r any p r o t e i n segment i n the primary amino ac i d sequence. E m p i r i c a l r u l e s set f o r t h by the authors aid i n l o c a t i n g the s p e c i f i c secondary s t r u c t u r e s . Figures 9, 10 and 11 are the pr e d i c t e d secondary s t r u c t u r e s for chymosin, pepsin and p e n i c i l l o p e p s i n , r e s p e c t i v e l y . The p r e d i c t e d s t r u c t u r e of chymosin (Figure 9) revealed a r e l a t i v e l y high p r o p o r t i o n of B-sheet f r a c t i o n s , with only minor s e c t i o n s of the t o t a l secondary s t r u c t u r e p r e d i c t e d as " - h e l i x . The a c t i v e s i t e a s p a r t i c a c i d residues (Asp3i+ and Asp2i6) were p r e d i c t e d to be i n B-turn regions. The pre-d i c t e d secondary s t r u c t u r e of pepsin (Figure 10), l i k e chymosin, was dominated by 8-sheet f r a c t i o n s . Only one s e c t i o n of " - h e l i x (residues 65 to 70) was p r e d i c t e d . The a c t i v e s i t e a s p a r t i c a c i d residues ( A s p 3 2 - 101 -F i g u r e 9 . P r e d i c t e d secondary s t r u c t u r e of chymosin using the Chou and Fasman method. - 102 -Figure 10. P r e d i c t e d secondary s t r u c t u r e of pepsin using the Chou and Fasman method. - 103 -Fig u r e 11. P r e d i c t e d secondary s t r u c t u r e of p e n i c i l l o p e p s i n using the Chou and Fasman method. - 104 -and ASP215) were also p r e d i c t e d to be located i n B-turn regions. The B-sheet s t r u c t u r e was pre d i c t e d to be the main s t r u c t u r a l feature of the secondary s t r u c t u r e of p e n i c i l l o p e p s i n (Figure 11). Al p h a - h e l i x regions were a minor p o r t i o n of the t o t a l p r e d i c t e d secondary s t r u c t u r e although the amount of t h i s secondary s t r u c t u r e was s l i g h t l y higher i n p e n i c i l l o -pepsin than i n the other proteinases examined. A s p a r t i c acid33 and Asp2i3« the a c t i v e s i t e r e s i d u e s , were located i n B-turn regions. Hsu et a l . (1977) reported that the two a c t i v e s i t e a s p a r t i c acid residues of p e n i c i l l o p e p s i n were located at the ends of B-strands near B-turn regions. As was apparent from the above observations, s e v e r a l secondary s t r u c t u r e features were common among the three a s p a r t y l proteinases examined. The high p r o p o r t i o n of B-sheet observed i s c o n s i s t e n t with the r e s u l t s obtained from X-ray d i f f r a c t i o n a n a l y s i s for s e v e r a l aspar-t y l p r o t e i n a s e s . Andreeva et a l . (1976) examined the X-ray data obtained from pepsin and found that the major s t r u c t u r a l components of the molecule were 8-sheet s t r u c t u r e s . The c r y s t a l s t r u c t u r e s f or the m i c r o b i a l proteinases from Endothia p a r a s i t i c a (Jenkins et a l . , 1976) as w e l l as from Rhizopus c h i n e n s i s (Subramanian et a l . , 1976) have also been e l u c i d a t e d . In both these proteinases the B-sheet s t r u c t u r e was found to be a major secondary s t r u c t u r e f r a c t i o n . Recently, James and S i e l e c k i (1983) examined the c r y s t a l s t r u c t u r e of p e n i c i l l o p e p s i n at 1.8A r e s o l u t i o n and found that approximately two-thirds of the t o t a l amino acid residues of the m i c r o b i a l proteinase have ¥ conformational angles that describe those of p a r a l l e l or a n t i - p a r a l l e l B-sheets. - 105 -Although the Chou and Fasman (1978b) method p r e d i c t e d a r e l a t i v e l y high p r o p o r t ion of B-sheet i n p e n i c i l l o p e p s i n (approximately 0.2), the amount was s u b s t a n t i a l l y lower than that observed by James and S i e l e c k i (1983). Kabsch and Sander (1983) examined various methods used i n the p r e d i c t i o n of p r o t e i n secondary s t r u c t u r e from the amino a c i d sequence and found that the Chou and Fasman (1978b) method had only a 50 percent o v e r a l l p r e d i c t i o n accuracy. The d i f f e r e n c e between r e s u l t s obtained using t h i s p r e d i c t i o n method (Chou and Fasman) and those obtained c r y s -t a l l o g r a p h i c a l l y have been a t t r i b u t e d t o : (1) ambiguities of some of the r u l e s set f o r t h by Chou and Fasman, and (2) the v a r i a b i l i t y i n the d e f i n i t i o n s of secondary s t r u c t u r e used by c r y s t a l l o g r a p h e r s . Although the method of Chou and Fasman (1978b) may y i e l d erroneous r e s u l t s one could argue that a s i n g l e program was used, thus d i f f e r e n c e s i n the i n t e r p r e t a t i o n would be c o n s i s t e n t for a l l p r o t e i n s examined; t h e r e f o r e , t h i s method may allow us a means for the r e l a t i v e comparison of the a s p a r t y l p r o t e i n a s e s . Furthermore, the p r e d i c t i o n of the a c t i v e s i t e a s p a r t i c a c i d residues for the three proteinases o c c u r r i n g i n 8-turn regions i s reasonable, c o n s i d e r i n g the r o l e of these r e s i d u e s . In order to be i n v o l v e d i n the h y d r o l y s i s of a peptide substrate ( i . e . c a t a l y s i s ) , i t would be imperative that these residues be on the surface of the enzyme. Kuntz (1972) observed that a s u b s t a n t i a l number of 8-turn f r a c t i o n s were located on the surface of g l o b u l a r p r o t e i n s . The type and sequence of amino acids are important for determining the two- and three-dimensional s t r u c t u r e of a p r o t e i n ; other important f a c t o r s are the forces that a f f e c t the f o l d i n g of the polypeptide. - 106 -Kauzmann (1959) was one of the f i r s t researchers to recognize the importance of hydrophobic forces i n a f f e c t i n g p r o t e i n s t r u c t u r e . Cid et a l . (1982) proposed a simple method for the p r e d i c t i o n of secondary s t r u c t u r e by means of a hydrophobicity p r o f i l e . B a s i c a l l y i n t h i s method the bulk hydrophobicity for each amino acid residue i s p l o t t e d against residue number and the secondary s t r u c t u r e f r a c t i o n i s deter-mined based on the p r o f i l e e x h i b i t e d . The hydrophobicity p r o f i l e method of Cid et a l . (1982) was used to p r e d i c t the secondary s t r u c t u r e s f o r chymosin, pepsin, p e n i c i l l o p e p s i n and Mucor miehei proteinase and the r e s u l t s are presented i n Figures 12a-12b, 13a-13b, 14a-14b and 15a-15b. Using t h i s method i t was found that c h a r a c t e r i z a t i o n of secondary s t r u c t u r e s other than exposed 6-strands or 8-turns was d i f f i c u l t , which g r e a t l y r e s t r i c t e d the capa-b i l i t i e s of t h i s method. I t should be noted that for Mucor miehei proteinase the region of sequence 160 to 180 should be i n t e r p r e t e d with some caution s i n c e , although the amino a c i d composition i s known, the exact sequence has not been e s t a b l i s h e d . As with the Chou and Fasman method which had p r e d i c t e d 8-sheet as a major secondary s t r u c t u r e f r a c t i o n , the hydrophobicity p r o f i l e method also p r e d i c t e d 6-sheet as a major f r a c t i o n i n the four p r o t e i n a s e s . The 6-sheet f r a c t i o n i s repre-sented by the " z i g - z a g " p a t t e r n (Figures 12 to 15) r e s u l t i n g from a l t e r -nating h y d r o p h i l i c and hydrophobic r e s i d u e s . Kanehisa and Tsong (1980) had p r e v i o u s l y described 8-strands on the surface of p r o t e i n s as a l t e r -nating h y d r o p h i l i c and hydrophobic r e s i d u e s . Figure 12a. The bulk hydrophobicity p r o f i l e of chymosin for residues 1 to 100 and 100 to 200. Figure 12b. The bulk hydrophobicity p r o f i l e of chymosin for residues 200 to 323. Figure 13a. The bulk hydrophobicity p r o f i l e of pepsin for residues 1 to 100 and 100 to 200. Figure 13b, The bulk hydrophobicity p r o f i l e of pepsin for residues 200 to 327. F i g u r e 14a. The bulk hydrophobicity p r o f i l e of p e n i c i l l o p e p s i n for residues 1 to 100 and 100 to 200. Figure 14b. The bulk hydrophobicity p r o f i l e of p e n i c i l l o p e p s i n for residues 200 to 323. Figure 15a. The bulk hydrophobicity p r o f i l e of Mucor miehei proteinase for residues 1 to 100 and 100 to 200. Figure 15b. The bulk hydrophobicity p r o f i l e of Mucor miehei proteinase for residues 200 to 300 and 300 to 369. - 115 -The hydrophobicity p r o f i l e method revealed that the a c t i v e s i t e a s p a r t i c acid residues for the four proteinases were located i n areas of low hydrophobicity ( i . e . h y d r o p h i l i c ) . These areas were i d e n t i f i e d as 6-turn regions. Rose (1978) hypothesized that "turns occur at those s i t e s i n the polypeptide chain where the hydrophobicity i s at a l o c a l minimum". Kuntz (1972), i n a paper on p r o t e i n f o l d i n g , observed that the residues located at B-turns were r e l a t i v e l y polar i n nature. Areas adjacent to the a c t i v e s i t e were p r e d i c t e d as 8-sheet and i t was also noted that these areas were r e l a t i v e l y hydrophobic. These observations may be of importance i n d e f i n i n g the i n t e r a c t i o n between enzyme and s u b s t r a t e . Raap et a l . (1983) analyzed the c i r c u l a r dichroism spectra for peptides c o n t a i n i n g the l a b i l e Phe-Met bond of K - c a s e i n ( i n d i l u t e sodium dodecyl s u l f a t e s o l u t i o n s ) and found that these peptides had 8-structure forming p o t e n t i a l . S i m i l a r r e s u l t s were obtained by the authors when they analyzed the peptide sequence using various secondary s t r u c t u r e p r e d i c t i v e methods ( i . e . Lim (1974) and Chou and Fasman (1978b)). The r e s u l t s obtained i n the present study appear to support the hypothesis put f o r t h by Jenkins et a l . (1976) and by Raap et a l . (1983) that K - c a s e i n may i n t e r a c t with the a c t i v e s i t e region of the enzyme through B-sheet-B-sheet i n t e r a c t i o n s during the m i l k - c l o t t i n g process. The r e s u l t s obtained from the diagonal p l o t and the secondary s t r u c t u r e p r e d i c t i o n methods revealed that various s t r u c t u r a l features were common to chymosin, pepsin, p e n i c i l l o p e p s i n and Mucor miehei proteinase which belong to the a s p a r t y l proteinases. V i s s e r (1981), i n - 116 -a review on p r o t e o l y t i c enzymes, stated that the o v e r a l l conformation of one proteinase molecule may be very s i m i l a r to that of other proteinases from the same c l a s s . C. CIRCULAR DICHROISM SPECTRA 1. Far-UV s p e c t r a (190 to 240 nm) The CD spectra of various a s p a r t y l proteinases as a f u n c t i o n of pH (pH 5.0 to 8.0) were measured over the wavelength range 190 to 240 nm and are represented as f o l l o w s : chymosin (Figure 16); pepsin (Figure 17); Mucor miehei proteinase (Figure 18); Mucor p u s i l l u s proteinase (Figure 19); Endothia p a r a s i t i c a proteinase (Figure 20); A s p e r g i l l u s  s a i t o i proteinase (Figure 21) and p e n i c i l l o p e p s i n (Figure 22). Chymosin showed three d i s t i n c t i v e patterns i n the CD s p e c t r a over the pH range (Fig u r e 16). The magnitude of the peak at 193 nm of the p a t t e r n e x h i b i t e d at low pH values (pH 5.0 and 5.3) was lower i n compar-i s o n to those spectra at pH values above 5.3. At pH 5.8, 6.3 and 7.0 a shoulder at 215 nm also became evident. At pH 8.0 a blue s h i f t i n the maxima at 193 nm occurred although peak magnitude was s i m i l a r to those spectra recorded at pH values greater than 5.3. The change i n CD spectra seen as a f u n c t i o n of pH may be due to the a s s o c i a t i v e p r o p e r t i e s of chymosin. Baldwin and Wake (1959) using sedimentation s t u d i e s demonstrated that chymosin i n s o l u t i o n was suscep-t i b l e to p o l y m e r i z a t i o n ; the extent of p o l y m e r i z a t i o n was dependent on both i o n i c strength and the concentration of the enzyme i n s o l u t i o n . Dimers were found to form at high concentrations of enzyme and low i o n i c - 117 -0 o E o CD n CM E o CD CD D ) CD CO o X o 6 4 2 0 - 2 - 4 - 6 - 8 -10 c, d ,e a b c d e f pH 5.0 pH 5.3 pH 5.8 pH 6.3 pH 7.0 pH 8.0 180 190 200 210 220 230 240 250 WAVELENGTH, nm Figure 16. The e f f e c t of pH on the far-UV CD spectra of chymosin. - 118 -6 4 2 0 - 2 - 4 - 6 - 8 -10 a b c d e f pH 5.0 pH 5.3 pH 5.8 pH 6.3 pH 7.0 pH 8.0 180 190 200 210 220 230 240 250 WAVELENGTH, nm F i g u r e 17. The e f f e c t of pH on the far-UV CD spectra of pepsin. - 1 1 9 -i o E o <D TJ CM E o CD CO k_ O ) 0) T J CO I o X a pH 5.0 b pH 5.3 c pH 5.8 d pH6.3 e pH 7.0 f pH 8.0 180 190 200 210 220 230 240 250 W A V E L E N G T H , nm - 8 -10 Figure 18. The e f f e c t of pH on the far-UV CD spectra of Mucor miehei p r o t e i n a s e . - 120 -6 4 2 0 - 2 - 4 - 6 - 8 -10 a b c d e f pH 5.0 pH 5.3 pH 5.8 pH 6.3 pH 7.0 PH 8.0 180 190 200 210 220 230 240 250 WAVELENGTH , nm Figure 19. The e f f e c t of pH on the far-UV CD spectra of Mucor p u s i l l u s p r o t e i n a s e . - 121 -6 I-4 2 L 0 - 2 - 4 - 6 - 8 -10 a b c d e f pH 5.0 pH 5.3 pH 5.8 pH 6.3 pH 7.0 pH 8.0 180 190 200 210 220 230 240 250 WAVELENGTH , nm F i g u r e 20. The e f f e c t of pH on the far-UV CD spectra of Endothia  p a r a s i t i c a p roteinase. - 122 -6 4 2 0 - 2 - 4 - 6 - 8 -10 r a b c d e f pH 5.0 pH 5.3 pH 5.8 pH 6.3 pH 7.0 pH 8.0 180 190 200 210 220 230 240 250 WAVELENGTH, nm Figure 21. The e f f e c t of pH on the far-UV CD spectra of A s p e r g i l l u s  s a i t o i p r oteinase. - 123 -a pH 5.0 d pH 6.3 f pH 8.0 6 1-imole 4 deci 2 CM E o 0 A degree - 2 i (V / CO - 4 \ / ' o \ / X - 6 \ 'CD - 8 -10 i _ l 1 180 190 2 0 0 210 2 2 0 2 3 0 2 4 0 2 5 0 W A V E L E N G T H , nm Figure 22. The e f f e c t of pH on the far-UV CD spect r a of p e n i c i l l o -pepsin. - 124 -st r e n g t h , however, at low chymosin concentrations only the monomer was evident. D j u r t o f t et a l . (1963) i n a s i m i l a r study showed that the sed-imentation c o e f f i c i e n t for chymosin increased with i n c r e a s i n g concentra-t i o n ; increases i n sedimentation were h i g h l y n o t i c e a b l e at chymosin conc e n t r a t i o n s greater than 3 mg/mL. In the present study.chymosin concentrations of l e s s than 1.0 mg/mL were g e n e r a l l y used at an i o n i c s t r e n g t h of 0.01. The CD spectra seen at pH 5.0 and pH 5.3 might also be associated with a conformational change which may occur as chymosin approaches i t s i s o e l e c t r i c point at pH 4.6. The appearance of a p r e c i p i t a t e was noted at pH 5.0 and 5.3. The p r e c i p i t a t e s were removed from the p r o t e i n s o l u -t i o n s p r i o r to CD measurement. The CD spectrum of chymosin seen at pH 8.0 may be the r e s u l t of the p r o t e i n u n f o l d i n g at a l k a l i n e pH values. Pepsin showed v i r t u a l l y no change i n the spectra from pH 5.0 to pH 6.3 (Figure 17). However, at pH values greater than 6.3 a d r a s t i c change i n spectra occurred. Ahmad and McPhie (1978) working with the d i a z o a c e t y l g l y c i n e e t h y l e s t e r d e r i v a t i v e of swine pepsin (to prevent a u t o l y s i s ) observed that the CD spectrum of the proteinase underwent a s u b s t a n t i a l conformational change at pH 7.0. In the present study the change i n conformation as r e f l e c t e d i n CD spectra at pH values greater than 6.3 may be due to p r o t e i n u n f o l d i n g as a r e s u l t of breakage of hydrogen bonds i n v o l v i n g carboxyl groups (Edelhoch, 1958a and 1958b). Nakayama et a l . (1983) examined the e l e c t r o n s p i n resonance (ESR) spectr a of 4-(3-diazo-2-oxopropylidene)-2,2,6,6-tetramethylpiperidine-1-oxyl l a b e l l e d porcine pepsin over a pH range of 1.8 to 12.0. Between - 125 -pH 1.8 and 6.2 the ESR spectra remained unchanged and were representa-t i v e of native porcine pepsin. At pH values greater than 7.4 the shape of the ESR spectra changed to a broad t r i p l e t which was assumed to represent denatured porcine pepsin. ESR spectra at pH values between 6.2 and 7.1 were a composite of the nativ e and denatured s p e c t r a . The authors postulated that the change i n conformation seen at a l k a l i n e pH values may be associated with the i n a c t i v a t i o n of pepsin reported i n the l i t e r a t u r e . Mucor miehei proteinase (Figure 18) showed very l i t t l e change i n the CD spectra over the pH range, although a s l i g h t change i n the CD spectrum was noted at pH 8.0. Ottesen and R i c k e r t (1970a) and A l a i s and Lagrange (1972) reported the l o s s of enzymatic a c t i v i t y f or Mucor miehei proteinase at pH values near 8.O.. The CD spectra of Mucor p u s i l l u s proteinase (Figure 19) also remained v i r t u a l l y unchanged for pH values 5.0 to 7.0. A decrease i n the amplitude of the peak i n the 190 to 200 nm range became evident at pH 8.0. Arima et a l . (1970) reported that the m i l k - c l o t t i n g a c t i v i t y of the enzyme decreased markedly at pH values above 7.0. Endothia p a r a s i t i c a proteinase (Figure 20) showed very l i t t l e change i n the CD spectra over the pH range although a s l i g h t blue s h i f t i n the peak maximum i n the 190 to 195 nm range was evident at pH values of 7.0 and 8.0. This s l i g h t change i n the CD spectra at pH 7.0 and pH 8.0 may be associated with a l o s s of enzymatic a c t i v i t y reported i n the l i t e r a t u r e . Whitaker (1970), i n s t a b i l i t y t e s t s with the p r o t e i n a s e , reported that a c t i v i t y was r a p i d l y l o s t above pH 6.5 while l o s s of a c t i v i t y was almost instantaneous above pH 8.0. - 126 -The A s p e r g i l l u s s a i t o i proteinase CD spectra (Figure 21) remained r e l a t i v e l y unchanged from pH 5.0 to pH 6.3, however, at pH values greater than 6.3 a breakdown i n the 190 to 200 nm region was evident. Ichishima and Yoshida (1967) studied the o p t i c a l r o t a t o r y p r o p e r t i e s of A s p e r g i l l u s s a i t o i proteinase and found that the ORD spectra did not change at pH values between 2.7 and 5.7, but noted a breakdown i n the ORD spectra on exposure to a l k a l i c o n d i t i o n s ( i . e . pH values greater than 7.0). The CD spectra of p e n i c i l l o p e p s i n at pH 5.0 and pH 6.3 (Figure 22) were v i r t u a l l y i d e n t i c a l , however, the CD spectrum at pH 8.0 no longer showed a peak i n the 190 to 200 nm region. This change i n CD spectrum at pH 8.0 was very s i m i l a r to that seen for pepsin at pH 7.0 and pH 8.0. Sodek and Hofmann (1970b) examined the e f f e c t of pH on the s t a b i l -i t y of p e n i c i l l o p e p s i n and found that at pH values greater than 6.6 there was a ra p i d drop i n a c t i v i t y , s i m i l a r to that reported for pepsin by Ahmad and McPhie (1978). Based on the examination of the CD spectra f or the various aspar-t y l proteinases some general observations could be made. The CD sp e c t r a were c h a r a c t e r i z e d by a maximum i n the 190 to 200 nm range as w e l l as a minimum around 210 nm. With the exception of chymosin, the CD sp e c t r a fo r the proteinases remained r e l a t i v e l y constant i n the lower pH range; the CD spec t r a were found to change at or above the n e u t r a l pH range depending on the proteinase examined. 2. Secondary s t r u c t u r e determination (far-UV) In order to q u a n t i t a t e the CD spectra of the various proteinases - 127 -as a f u n c t i o n of pH, the method of Provencher and GlOckner (1981) was used to analyze the CD data for the determination of secondary s t r u c -t u r e . The r e s u l t s of the secondary s t r u c t u r e determination are pre-sented i n Table 6. As would be expected, changes observed i n the CD s p e c t r a tended to be r e f l e c t e d by changes i n the amounts of secondary s t r u c t u r e determined. The a s p a r t y l proteinases were g e n e r a l l y charac-t e r i z e d by large amounts of 8-sheet. The X-ray d i f f r a c t i o n a n a l y s i s of various a s p a r t y l proteinases conducted by s e v e r a l researchers (Andreeva et a l . , 1976; Jenkins et a l . , 1976; Subramanian et a l . , 1976; James and S i e l e c k i , 1983) has shown that a large p o r t i o n of these molecules c o n s i s t of p a r a l l e l and a n t i - p a r a l l e l 8-sheet s t r u c t u r e s . The X-ray r e s u l t s together with the CD r e s u l t s of the present study would tend to i n d i c a t e that as a c l a s s the a s p a r t y l proteinases have s i m i l a r secondary s t r u c t u r e s . Of the a s p a r t y l proteinases examined, chymosin contained a r e l a -t i v e l y high p r o p o r t i o n of " - h e l i x , a feature which r e a d i l y d i s t i n g u i s h e d i t from the other a s p a r t y l p r o t e i n a s e s . At the low pH values (pH 5.0 and pH 5.3) the amount of " - h e l i x was lower than that seen at pH values greater than 5.3 while the opposite was true for the B-sheet f r a c t i o n s . At pH values of 6.3 and lower, the secondary s t r u c t u r e f r a c t i o n s of pepsin compared favourably to those obtained by Ahmad and McPhie (1978) who c a l c u l a t e d 0.00, 0.58 and 0.42 for " - h e l i x , B-sheet and unordered f r a c t i o n s r e s p e c t i v e l y for pepsin at pH 4.5 using the method of White (1976). Rao and Dunn (1981) using the CD spectrum of porcine pepsin at pH 5.5 determined that the secondary s t r u c t u r e c o n s i s t e d of - 128 -Table 6. Secondary s t r u c t u r e determination from CD spectra for various p r o t e i n a s e s . Secondary s t r u c t u r e f r a c t i o n P r o t e i n " - h e l i x 8-sheet 8-turn random 5.0 0.09 0.44 0.19 0.28 5.3 0.15 0.43 0.17 0.25 5.8 0.24 0.37 0.14 0.25 6.3 0.23 0.35 0.15 0.27 7.0 0.22 0.37 0.16 0.25 8.0 0.22 0.36 0.16 0.26 5.0 0.12 0.59 0.14 0.15 5.3 0.13 0.60 0.13 0.14 5.8 0.13 0.58 0.14 0.15 6.3 0.12 0.58 0.15 0.15 7.0 0.12 0.48 0.16 0.24 8.0 0.12 0.44 0.15 0.29 5.0 0.01 0.61 0.22 0.16 5.3 0.01 0.62 0.21 0.16 5.8 0.01 0.60 0.22 0.17 6.3 0.02 0.60 0.21 0.17 7.0 0.03 0.60 0.20 0.17 8.0 0.03 0.58 0.22 0.17 5.0 0.01 0.62 0.20 0.17 5.3 0.00 0.61 0.22 0.17 5.8 0.00 0.61 0.21 0.18 6.3 0.01 0.61 0.20 0.18 7.0 0.03 0.60 0.19 0.18 8.0 0.03 0.58 0.20 0.19 5.0 0.00 0.62 0.23 0.15 5.3 0.00 0.63 0.22 0.15 5.8 0.00 0.62 0.21 0.17 6.3 0.03 0.58 0.22 0.17 7.0 0.02 0.59 0.22 0.17 8.0 0.03 0.57 0.19 0.21 Aspartyl proteinases chymosin pepsin M. miehei proteinase M. p u s i l l u s proteinase pH PH pH pH E. p a r a s i t i c a proteinase pH (continued ...) - 129 -Table 6. (continued) Secondary s t r u c t u r e f r a c t i o n P r o t e i n " - h e l i x 8-sheet B-turn random A. s a i t o i proteinase pH 5.0 0.00 0.62 0.23 0.15 5.3 0.00 0.62 0.23 0.15 5.8 0.00 0.63 0.22 0.15 6.3 0.00 0.63 0.21 0.16 7.0 0.00 0.55 0.24 0.21 8.0 0.03 0.51 0.20 0.26 p e n i c i l l o p e p s i n pH 5.0 0.00 0.70 0.23 0.07 6.3 0.00 0.69 0.22 0.09 8.0 0.01 0.43 0.22 0.34 Non-aspartyl proteinases papain pH 6.3 0.14 0.36 0.22 0.28 "-chymotrypsin PH 6.3 0.03 0.50 0.29 0.18 t r y p s i n pH 6.3 0.06 0.47 0.28 0.19 - 130 -0.11 " - h e l i x , 0.39 B-sheet and 0.50 random s t r u c t u r e . The content of B-sheet determined by these authors was low compared to the r e s u l t s of the present study and might have been due to the l e a s t squares method used for the a n a l y s i s . This method for the determination of secondary s t r u c t u r e f r a c t i o n s used reference p r o t e i n spectra which were generated from only f i v e p r o t e i n s . In the present study the pr o p o r t i o n of B-sheet decreased at pH 7.0 and pH 8.0 while the f r a c t i o n of unordered s t r u c t u r e increased. This l o s s of organized s t r u c t u r e would tend to suggest that the molecule was u n f o l d i n g . Edelhoch (1957) found that as pepsin was exposed to a l k a l i n e pH c o n d i t i o n s , a change i n v i s c o s i t y occurred which was a t t r i b u t e d to the unfo l d i n g of the p r o t e i n to a l i n e a r p o l y e l e c -t r o l y t e . Secondary s t r u c t u r e determination from the CD spectra of Endothia  p a r a s i t i c a proteinase over the pH range i n d i c a t e d a B-sheet content of approximately 0.6 with very l i t t l e " - h e l i x content. Jenkins et a l . (1976) using three d i f f e r e n t methods for the a n a l y s i s of the CD spectrum fo r _E. p a r a s i t i c a proteinase at pH 4.48, found that the amount of B-sheet ranged from 0.34 to 0.59 while the proportion of " - h e l i x ranged from 0.03 to 0.08 depending on the algorithm used. As the pH was increased from 5.0 to 8.0 s l i g h t decreases i n B-sheet with corresponding s l i g h t increases i n both " - h e l i x and unordered f r a c t i o n s were observed. S i m i l a r trends i n the secondary s t r u c t u r e f r a c t i o n s over the pH range were noted for both Mucor miehei proteinase and Mucor p u s i l l u s p r o t e i n -ase. Secondary s t r u c t u r e data revealed that these two proteinases were almost i d e n t i c a l i n s t r u c t u r a l nature. Etoh et a l . (1979) studied the - 131 -p h y s i c a l - c h e m i c a l and immunochemical p r o p e r t i e s of Mucor miehei p r o t e i n -ase and Mucor p u s i l l u s proteinase and found that a high s t r u c t u r a l s i m i l a r i t y e x i s t s between the two p r o t e i n a s e s . Secondary s t r u c t u r e determination of A s p e r g i l l u s s a i t o i p r oteinase i n d i c a t e d a B-sheet f r a c t i o n of approximately 0.6 and no " - h e l i x at pH values of 6.3 and lower. Ichishima and Yoshida (1966a) on the basis of ORD and i n f r a r e d data concluded that the proteinase from A s p e r g i l l u s  s a i t o i was void of h e l i c a l conformation and that the molecule e x i s t e d i n the a n t i - p a r a l l e l B - s t r u c t u r e . A s p e r g i l l u s s a i t o i p r o t e i n a s e , as with the other m i c r o b i a l p r o t e i n a s e s , also e x h i b i t e d a decrease i n the 8-sheet f r a c t i o n with a corresponding increase i n the unordered f r a c t i o n . Ichishima and Yoshida (1967) found that at pH values above 7.0 the r o t a t o r y d i s p e r s i o n curve resembled that of an unordered c o i l found i n denatured p r o t e i n s . P e n i c i l l o p e p s i n was determined to have a high p-sheet content of approximately 0.7 at pH 5.0 and pH 6.3, analogous to the other a s p a r t y l proteinases from m i c r o b i a l sources. This r e s u l t compares favourably to that obtained by James and S i e l e c k i (1983) who c a l c u l a t e d a B-sheet content of 0.66 based on r e s u l t s obtained from X-ray d i f f r a c t i o n analy-s i s of p e n i c i l l o p e p s i n . In the present study the f r a c t i o n of B-sheet decreased markedly from approximately 0.7 to 0.4 at pH 8.0; the propor-t i o n of unordered f r a c t i o n g r e a t l y increased from approximately 0.1 at pH 5.0 and pH 6.3 to 0.3 at pH 8.0. These changes would suggest that the molecule was unfolded as a r e s u l t of exposure to a l k a l i n e pH c o n d i -t i o n s . The s t r u c t u r a l behaviour e x h i b i t e d by p e n i c i l l o p e p s i n i s thus - 132 -very s i m i l a r to that displayed by pepsin. Mains et a l . (1971) found that many of the 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 of p e n i c i l l o p e p s i n and pepsin were very s i m i l a r . The secondary s t r u c t u r e data for the non-aspartyl p r o t e i n a s e s papain, "-chymotrypsin and t r y p s i n at pH 6.3 are a l s o presented i n Table 6. The fact that the three non-aspartyl proteinases had r e l a t i v e l y high contents of B-sheet may aid i n e x p l a i n i n g the reported m i l k - c l o t t i n g a b i l i t y of these enzymes (Ernstrom, 1974). On the b a s i s of the r e s u l t s obtained for the a s p a r t y l proteinases i t would appear that an increase i n pH to the n e u t r a l - a l k a l i n e range r e s u l t s i n a t r a n s i t i o n of the secondary s t r u c t u r e from B-sheet to unordered f r a c t i o n , which may be i n d i c a t i v e of denaturation. The extent of t r a n s i t i o n , however, was proteinase dependent. Chymosin, Mucor  miehei proteinase, Mucor p u s i l l u s proteinase and Endothia p a r a s i t i c a proteinase showed r e l a t i v e l y strong r e s i s t a n c e to s t r u c t u r a l change with an increase i n pH, whereas pepsin, A s p e r g i l l u s s a i t o i proteinase and p e n i c i l l o p e p s i n were markedly a f f e c t e d by the pH change. 3. Near-UV s p e c t r a (240 to 320 nm) The near-UV (240 to 320 nm) spectra for chymosin, pepsin, Mucor  miehei p r o t e i n a s e , Mucor p u s i l l u s p roteinase, Endothia p a r a s i t i c a pro-t e i n a s e and A s p e r g i l l u s s a i t o i proteinase are presented i n Figures 23, 24, 25, 26, 27 and 28, r e s p e c t i v e l y . In contrast to the far-UV CD spectra which i s i n d i c a t i v e of secondary s t r u c t u r e , the near-UV CD s p e c t r a r e f l e c t s changes i n the t e r t i a r y s t r u c t u r e ( S t r i c k l a n d , 1974). - 133 -E o 8.0 6.0 4.0 2.0 0.0 - 2.0 - 4.0 - 6.0 - 8.0 - 10.0 -12.0 a pH 5 0 b pH 5.3 c pH 5.8 d pH6.3 e pH 7.0 f PH8.0 240 250 260 270 280 290 300 310 320 WAVELENGTH,nm Figure 23. The e f f e c t of pH on the near-UV CD spectra of chymosin. - 134 -E o < -8.0 h 6.0 r-4.0 h 2.0 0.0 2.0 - 4.0 - 6.0 - 8 0 - 10.0 12.0 h 240 250 260 270 280 290 300 310 320 WAVELENGTH , nm F i g u r e 24. The e f f e c t of pH on the near-UV CD s p e c t r a of pepsin. - 135 -- 10.0 - 12.0 \-240 250 260 270 280 290 300 310 320 WAVELENGTH , nm F i g u r e 25. The e f f e c t of pH on the near-UV CD spe c t r a of Mucor miehei p r o t e i n a s e . - 136 -a pH 5.0 b pH 5.3 8.0 -c d pH 5 8 pH 6.3 6.0 -e f pH 7.0 pH 8.0 4.0 ' " \ / \ a , b,c , d , e 2.0 f ^ ^ - ^ 0.0 2.0 4.0 6.0 8.0 10.0 12.0 -, '- i i i i 240 250 260 270 280 290 300 310 320 WAVELENGTH , nm F i g u r e 26. The e f f e c t of pH on the near-UV CD spectra of Mucor p u s i l l u s p r o t e i n a s e . - 137 -E 8.0 - 10.0 U - 12.0 L 240 250 260 270 280 290 300 310 320 WAVELENGTH , nm F i g u r e 27. The e f f e c t of pH on the near-UV CD s p e c t r a of Endothia para- s i t i c a p r o t e i n a s e . - 138 8.0 6.0 4.0 2.0 0.0 2.0 4.0 6.0 8.0 10.0 -12.0 r-a pH 5.0 b pH 5.3 pH 5.8 pH 6.3 pH 7.0 f PH 8.0 240 250 260 270 280 290 300 310 320 WAVELENGTH , nm F i g u r e 28. The e f f e c t of pH on the near-UV CD s p e c t r a of A s p e r g i l l u s  s a i t o i p r o t e i n a s e . - 139 -In the near-UV region, the aromatic r i n g s of t y r o s i n e , tryptophan and phenylalanine give r i s e to CD bands through i n t e r a c t i o n s with the amino a c i d moiety or with nearby groups i n the p r o t e i n . In a d d i t i o n , the d i s u l f i d e chromophore of c y s t i n y l residues may c o n t r i b u t e to the near-UV sp e c t r a ( S t r i c k l a n d , 1974; Heindl et a l . , 1980). In general, the changes observed i n the CD spectra from the 190 to 240 nm range were r e f l e c t e d i n the CD spectra from the near-UV range. The r e l a t i v e i n t e n s i t y of the near-UV spectra f or chymosin (Figure 23) was low ( i n the range of 0 to 2 M""1crTr1) which may r e s u l t from the r e l a t i v e l y l a rge number of aromatic residues found i n the molecule. Chymosin contains approximately 12 percent aromatic amino a c i d s . P r o t e i n s c o n t a i n i n g large numbers of aromatic side chains may not have very large CD bands as a r e s u l t of c a n c e l l a t i o n s by p o s i t i v e and nega-t i v e c o n t r i b u t i o n s ( S t r i c k l a n d , 1974). Since intense CD bands occur when aromatic groups are i n clos e proximity to one another i t i s also p o s s i b l e that the aromatic amino acids found i n the t e r t i a r y s t r u c t u r e of chymosin are not clos e to one another. Due to the low i n t e n s i t y of the CD spectra e s p e c i a l l y at low pH values (pH 5.0 and pH 5.3), the i d e n t i f i c a t i o n of the f i n e s t r u c t u r e f or the various aromatic groups was d i f f i c u l t . Chymosin also contains three d i s u l f i d e l i n k a g e s (Foltmann et a l . , 1977), however the shape and i n t e n s i t i e s of d i s u l f i d e CD bands have not been w e l l c h a r a c t e r i z e d ( S t r i c k l a n d , 1974); t h e r e f o r e , no attempts were made to i d e n t i f y these s t r u c t u r e s . I d e n t i f i c a t i o n of the d i s u l f i d e f i n e s t r u c t u r e was not attempted for any of the other a s p a r t y l proteinases examined. - 140 -Tentative i d e n t i f i c a t i o n of phenylalanine f i n e s t r u c t u r e f or chymosin was made at 261 nm and 269 nm while the 0+850 cm - 1 1L( 3 and the 0-0 cm - 1 bands of tryptophan were assigned to peaks centered at 285 nm and 292 nm, r e s p e c t i v e l y . The 0+800 cm"1 band of t y r o s i n e was i d e n t i f i e d at 276 nm, however, the 0-0 cm-1 band was not detected and may have been obscured by the tryptophan f i n e s t r u c t u r e . No change i n the pat t e r n of f i n e s t r u c t u r e was noted u n t i l the pH was increased to 8.0 where the l o s s of tryptophan f i n e s t r u c t u r e (bands at 285 nm and 292 nm) became apparent. This l o s s of f i n e s t r u c t u r e may i n d i c a t e a s l i g h t p e r t u r b a t i o n of the t e r t i a r y s t r u c t u r e r e s u l t i n g from the m o t i l i t y of the tryptophan side chains. In s t u d i e s with model compounds i t has been shown that an increase i n the m o t i l i t y of the aromatic side chains tends to decrease the CD i n t e n s i t y ( S t r i c k l a n d , 1974). Although no change i n CD f i n e s t r u c t u r e was apparent at pH 5.0 and pH 5.3, the decreased i n t e n s i t y of the spectra may i n d i c a t e a change i n t e r t i a r y s t r u c t u r e . The i n t e n s i t y of the near-UV spectra i s a f f e c t e d by the r i g i d i t y of the p r o t e i n , i n t e r a c t i o n s of aromatic r i n g s with i t s surroundings and the number of aromatic residues ( S t r i c k l a n d , 1974). The near-UV CD spectra of pepsin i s presented i n Figure 24. Un-l i k e the chymosin CD spectra which was mainly negative, the CD spectra of pepsin was p o s i t i v e . Tentative i d e n t i f i c a t i o n of the phenylalanine f i n e s t r u c t u r e was made at 266 nm and 271 nm, the 0+800 cm - 1 band of t y r o s i n e at 281 nm and the 0+850 cm - 1 1 L b and the 0-0 cm - 1 1 L b bands of tryptophan at 287 nm and 292 nm, r e s p e c t i v e l y . Again, as with chymo-s i n the 0-0 cm - 1 band of t y r o s i n e was absent. The appearance of a - 141 -shoulder i n the 295 to 305 nm region may i n d i c a t e the presence of the 1 L a band of tryptophan, however, the CD band due to d i s u l f i d e s may al s o be c o n t r i b u t i n g to the region. Pepsin contains three d i s u l f i d e bonds (Moravek and Kostka, 1974). No change i n the spectra with an increase i n pH was observed u n t i l pH 7.0 when a general decrease i n CD i n t e n s i t y was noted. As with chymosin, a decrease i n i n t e n s i t y was accompanied by a l o s s of tryptophan f i n e s t r u c t u r e which may i n d i c a t e a change i n t e r t i a r y s t r u c t u r e . The near-UV spectra of Mucor miehei proteinase (Figure 25) and Mucor p u s i l l u s proteinase (Figure 26) were r e l a t i v e l y s i m i l a r . This s i m i l a r i t y i n t e r t i a r y s t r u c t u r e supports the work of Etoh et a l . (1979), who concluded that the two proteinases shared a "morphological i d e n t i t y " . In comparison to chymosin and pepsin, the r e s o l u t i o n of some f i n e s t r u c t u r e s for M. miehei and M. p u s i l l u s proteinases was improved, e s p e c i a l l y for phenylalanine. With Mucor miehei proteinase the phenyl-alanine f i n e s t r u c t u r e was i d e n t i f i e d at 257 nm and 263 nm, with the 0+800 cm - 1 band of t y r o s i n e at 276 nm and the 0+850 cm - 1 1 L j 3 band and 0-0 cm - 1 1L| 3 band of tryptophan at 285 nm and 292 nm, r e s p e c t i v e l y . As the pH was increased, very l i t t l e change occurred i n the near-UV spectra other than the disappearance of the phenylalanine peak at 263 nm. Only minor changes accompanied an increase i n pH which would sug-gest that the t e r t i a r y s t r u c t u r e of Mucor miehei proteinase i s r e l a t i v e -l y s t a b l e to pH change. A s i m i l a r s t a b i l i t y to pH was also seen i n the far-UV sp e c t r a f o r t h i s p r o t e i n a s e . The near-UV spectra of Mucor p u s i l l u s proteinase (Figure 26) i n d i c a t e d that the phenylalanine f i n e s t r u c t u r e was c h a r a c t e r i z e d by - 142 -bands at 259 nm and 265 nm, the 0+800 cm - band of t y r o s i n e at 274 nm and the 0+850 cm - 1 1 L b band and the 0-0 cm - 1 1L|-) band of tryptophan at 287 nm and 293 nm, r e s p e c t i v e l y . Some evidence of the 0-0 cm - 1 band of t y r o s i n e was seen at 277 nm, although the band was very weak. As with the near-UV spectra of \A. miehei pr o t e i n a s e , both CD i n t e n s i t y and band shape were v i r t u a l l y unaffected u n t i l pH 8.0. At pH 8.0 a n o t i c e -able decrease i n CD i n t e n s i t y , e s p e c i a l l y with regards to t y r o s i n e and tryptophan f i n e s t r u c t u r e , became apparent with the disappearance of the 0-0 cm - 1 band of t y r o s i n e . The disappearance of t h i s band may be the r e s u l t of the i o n i z a t i o n of the t y r o s i n e side c h a i n . Like M. miehei pro t e i n a s e , the t e r t i a r y s t r u c t u r e of M. p u s i l l u s proteinase appeared to be r e l a t i v e l y r e s i s t a n t to pH change. The near-UV CD spectra of Endothia p a r a s i t i c a proteinase (Figure 27) was hi g h l y r e s o l v e d . S i m i l a r h i g h l y resolved CD spectra were al s o obtained by Jenkins et a l . (1976). In the present study tryptophan f i n e s t r u c t u r e predominated the CD spectra with the 0+850 cm - 1 1L|-) band lo c a t e d at 288 nm and the 0-0 cm - 1 ^L^ band lo c a t e d at 297 nm. In a d d i t i o n to the 1L| 3 bands, the ^ L a band was t e n t a t i v e l y i d e n t i f i e d at 262 nm. The presence of t h i s 1 L a band may have obscured by the presence of the c h a r a c t e r i s t i c double band of phenylalanine which i s found i n t h i s wavelength r e g i o n . No change i n CD i n t e n s i t y or band p o s i t i o n was noted u n t i l pH values greater than 6.3 were achieved. At pH 7.0 a general decrease i n the i n t e n s i t y of the spectra occurred with no change i n band p o s i t i o n . At pH 8.0 a completely d i f f e r e n t s p e c t r a from those at the other pH values was observed with the appearance of - 143 -two new bands at 280 nm and 285 nm. The r e s u l t s obtained would suggest that u n l i k e the other m i c r o b i a l proteinases (M. miehei proteinase and j ^ . p u s i l l u s p r o t e i n a s e ) , the t e r t i a r y s t r u c t u r e of Endothia p a r a s i t i c a proteinase was more s u s c e p t i b l e to conformational change as pH was incre a s e d . The near-UV CD spectra of A s p e r g i l l u s s a i t o i proteinase ( F i g u r e 28) showed some s i m i l a r i t y to the spectra e x h i b i t e d by Endothia p a r a s i - t i c a proteinase with respect to tryptophan f i n e s t r u c t u r e . Tryptophan f i n e s t r u c t u r e was i d e n t i f i e d as negative peaks at 287 nm (0+850 cm - 1 band) and at 294 nm (0-0 cm - 1 band) and a p o s i t i v e peak at 257 nm ( 1 L a band). The presence of the l L a band of tryptophan made i t d i f f i c u l t to d i s t i n g u i s h the phenylalanine f i n e s t r u c t u r e , although t e n t a t i v e i d e n t i f i c a t i o n was made at 267 nm and 272 nm. The 0+800 cm - 1 band of t y r o s i n e was located at 277 nm. Increasing the pH to above 6.3 r e s u l t e d i n a general l o s s of CD i n t e n s i t y with the broadening of some bands (e.g. 0+850 cm - 1 1L|-) band of tryptophan) with a consequent l o s s of r e s o l u t i o n . The l o s s of both i n t e n s i t y and r e s o l u t i o n may i n d i c a t e a change i n the t e r t i a r y s t r u c t u r e . From the r e s u l t s of the near-UV CD spectra study i t was concluded that as the pH was increased, changes i n the t e r t i a r y s t r u c t u r e occurred, the extent and degree of which was proteinase dependent. A s i m i l a r type of conc l u s i o n was made when the far-UV region was examined. The fa c t that changes i n the near-UV CD s p e c t r a l range were observed for a l l a s p a r t y l proteinases examined may i n d i c a t e the impor-tance of the aromatic groups i n maintaining the s t r u c t u r a l s t a b i l i t y of - 144 -the nat i v e p r o t e i n . S t r u c t u r a l s t a b i l i t y i n the a s p a r t y l proteinases may be a fu n c t i o n of hydrophobicity due to the inherent r e l a t i o n s h i p between aromatic amino acids and hydrophobicity (Bigelow, 1967). The importance of hydrophobic forces to the s t r u c t u r a l s t a b i l i t y of the a s p a r t y l proteinases may be r e i n f o r c e d from the r e s u l t s of X-ray c r y s t a l l o g r a p h y f or some of these enzymes. These X-ray r e s u l t s have i n d i c a t e d that a s i g n i f i c a n t p o r t i o n of the a s p a r t y l proteinases c o n s i s t of hydrophobic, p a r a l l e l and a n t i - p a r a l l e l B-sheet s t r u c t u r e s (Tang, 1979; V i s s e r , 1981). D. BIGELOW AVERAGE HYDROPHOBICITY Hydrophobic forces may play an important r o l e i n the s t r u c t u r a l s t a b i l i t y of a p r o t e i n and also i n the i n t e r a c t i o n between enzyme and su b s t r a t e . On the ba s i s of h y d r o l y t i c s t u d i e s using s y n t h e t i c peptide s u b s t r a t e s , Tang (1963) postulated that there was a s i t e on the pepsin molecule that binds the hydrocarbon side-chains of the amino acids of the substrate through hydrophobic bonding which was e s s e n t i a l for enzy-matic a c t i o n . Hydrophobic forces may be involved i n the enzymatic (or primary) phase of the m i l k - c l o t t i n g process. Green and Marshall (1977) working with chemically modified casein m i c e l l e s found that the neu-t r a l i z a t i o n of the negative charge on the m i c e l l e s by a p o s i t i v e l y charged a d d i t i v e r e s u l t e d i n an increased a f f i n i t y of chymosin f or the m i c e l l e s . The average h y d r o p h o b i c i t i e s ( B $ A V G ) for the various proteinases using the method of Bigelow (1967) are presented i n Table 7. A compari-son of the average h y d r o p h o b i c i t i e s of the non-aspartyl proteinases - 145 -Table 7. Bigelow average hydrophobicity values (H$AyG-) obtained for va r i o u s p r o t e i n a s e s . P r o t e i n H<3?.un c a l r e s -Aspartyl proteinases chymosin 1120 pepsin 1063 _M. miehei proteinase 1109 M. p u s i l l u s proteinase 1041 E_. p a r a s i t i c a proteinase 955 A_. s a i t o i proteinase 973 p e n i c i l l o p e p s i n 933 Non-aspartyl proteinases papain 1159 "-chymotrypsin 1030 t r y p s i n 1034 - 146 -(papain, "-chymotrypsin and t r y p s i n ) to those of the a s p a r t y l p r o t e i n -ases showed no d i s t i n c t d i f f e r e n c e as evidenced by the overlap of some values between the two groups. Within the group of a s p a r t y l proteinases examined, the H3?AVG values ranged from 933 c a l r e s - 1 f o r p e n i c i l l o p e p -s i n to the highest value of 1120 c a l r e s - 1 for chymosin. This r e l a -t i v e l y large range i s contrary to the f i n d i n g s of Bigelow (1967) who reported that p r o t e i n s r e l a t e d to each other g e n e r a l l y have s i m i l a r hydrophobicity values; (e.g. f or various hemoglobins and myoglobins, the !~®AVG values ranged from 1060 to 1180 c a l r e s - 1 ) . A narrower range of hydrophobicity values would have been expected for a s p a r t y l proteinases due to the sequence homology displayed by various a s p a r t y l proteinases examined. The use of average hydrophobicity values as a means for c l a s s i f i -c a t i o n may have some l i m i t a t i o n i f one assumes that f u n c t i o n i s deter-mined by t e r t i a r y s t r u c t u r e . Average hydrophobicity i n v o l v e s the summa-t i o n of the c o n t r i b u t i o n of a l l amino acids i n the p r o t e i n (Bigelow, 1967) and the r e f o r e would not be expected to give an i n d i c a t i o n of the hydrophobicity on the surface of the native p r o t e i n s t r u c t u r e . E. HYDROPHOBICITY USING FLUORESCENT PROBES In a d d i t i o n to an e m p i r i c a l c a l c u l a t i o n of Bigelow hydrophobicity, the hydrophobicity of the various proteinases was measured using f l u o r e s c e n t probes. Various f l u o r e s c e n t probes have been used to study aspects of the s t r u c t u r e and i n t e r a c t i o n s of p r o t e i n i n r e l a t i o n to p r o t e i n hydrophobicity. In the present study the fl u o r e s c e n t probes - 147 -c i s - p a r i n a r i c a c i d (CPA) and 1-anilino-8-naphthalene s u l f o n a t e (ANS) were used. CPA i s a conjugated polyene f a t t y acid and would bind a l i p h a t i c regions on the p r o t e i n surface ( S k l a r et a l . , 1977), whereas ANS being aromatic i n nature would bind aromatic regions on the p r o t e i n surface (Figure 29). The support for the use of c i s - p a r i n a r i c a c i d as a hydrophobic probe was provided by Townsend (1982) who found a s i g n i f i -cant c o r r e l a t i o n (P<0.01) between the p o l a r i t y of a s e r i e s of s o l v e n t s (as described by Snyder (1978)) and the quantum y i e l d of c i s - p a r i n a r i c a c i d i n these s o l v e n t s (as described by Sklar et a l . (1977)). The use of ANS as a hydrophobic probe has been w e l l documented i n the l i t e r a t u r e ( S t r y e r , 1965; Weber and D a n i e l , 1966; S t r y e r , 1968) where i t has been demonstrated that the quantum y i e l d of fluorescence and the wavelength of maximum emission of ANS bound to p r o t e i n i s dependent on the p o l a r i t y of the binding r e g i o n . The r e s u l t s using the two f l u o r e s c e n t probes (CPA and ANS) for the hydrophobicity determination of the various proteinases are presented i n Table 8. No d i s t i n c t d i f f e r e n c e was observed between a s p a r t y l p r o t e i n -ases and non-aspartyl proteinases as i n d i c a t e d by the overlap of the hydrophobicity values between the two groups using both probes. These r e s u l t s were ther e f o r e s i m i l a r to the r e s u l t s obtained using the Bigelow average hydrophobicity c a l c u l a t i o n . Of the a s p a r t y l p r o t e i n a s e s , chymo-s i n showed high values for both CPA and ANS i n comparison to the other p r o t e i n a s e s , which would i n d i c a t e that chymosin had a r e l a t i v e l y a l i p h a -t i c and aromatic surface area. Endothia p a r a s i t i c a p r o t e i n a s e , on the other hand, had a high CPA value but a low ANS value, i n d i c a t i n g a - 148 -(CH2) COO-c i s t rans trans c i s c i s - p a r i n a r i c a c i d (CPA) N • H SO. 1-anilino-8-naphthalene s u l f o n a t e (ANS) Figure 29. St r u c t u r e s of c i s - p a r i n a r i c acid and 1-anilino-8-naphtha-lene s u l f o n a t e . - 149 -Table 8. Hydrophobicity values obtained for various proteinases using f l u o r e s c e n t probes. 1 - a n i l i n o - 8 -c i s - p a r i n a r i c a c i d naphthalene s u l f o n a t e P r o t e i n (CPA) (ANS) Aspartyl proteinases chymosin 96.0 1 48.0 1 pepsin 6.0 1.0 _M. miehei proteinase 21.0 2.0 M. p u s i l l u s proteinase 3.0 7.0 E_. p a r a s i t i c a proteinase 113.0 7.0 A_. s a i t o i proteinase 73.0 6.0 p e n i c i l l o p e p s i n 23.0 3.0 Non-aspartyl proteinases papain 19.0 12.0 "-chymotrypsin 9.0 5.0 t r y p s i n 6.0 12.0 Values represent the mean of d u p l i c a t e determinations. - 150 -surface area which was r e l a t i v e l y a l i p h a t i c i n nature with l i t t l e aroma-t i c c h a r a c t e r i s t i c . Pepsin had low values for both CPA and ANS i n d i -c a t i n g that the surface area of pepsin was neither very a l i p h a t i c nor aromatic i n nature. These low values may be r e l a t e d to the f a c t that pepsin contains a large amount of charged groups, e s p e c i a l l y a s p a r t i c a c i d , which would r e s t r i c t the binding of hydrophobic probes. The non-aspartyl proteinases (papain, <*-chymotrypsin and t r y p s i n ) g e n e r a l l y had low CPA and ANS values which may also i n d i c a t e a h i g h l y charged surface. The surface hydrophobicity c h a r a c t e r i s t i c s of the a s p a r t y l proteinases were qu i t e v a r i e d and were l i k e l y a f u n c t i o n of the f o l d i n g of the p r o t e i n molecule. The f o l d i n g of the p r o t e i n i n t o the t e r t i a r y s t r u c t u r e w i l l u l t i m a t e l y decide the surface c h a r a c t e r i s t i c s of the p r o t e i n by determining which residues i n the primary amino ac i d sequence w i l l be exposed and which residues w i l l be bu r i e d . Further e l u c i d a t i o n of the exact nature of the surface c h a r a c t e r i s t i c s of the proteinases may requ i r e high r e s o l u t i o n X-ray d i f f r a c t i o n a n a l y s i s . The s i m i l a r i -t i e s i n secondary s t r u c t u r e s as p r e v i o u s l y demonstrated for the a s p a r t y l proteinases were not shown to be r e f l e c t e d i n surface hydrophobicity c h a r a c t e r i s t i c s . F. CHARGE RATIOS In f u r t h e r attempts to d i s t i n g u i s h the a s p a r t y l proteinases from the non-aspartyl proteinases, various charge r a t i o s were c a l c u l a t e d on the basis of amino ac i d composition data (Table 9). No d i s t i n c t d i f f e r -ences between a s p a r t y l and non-aspartyl proteinases were seen when the - 151 -Table 9 . Charge r a t i o s of various p r o t e i n a s e s . P r o t e i n T o t a l charge 1 A c i d i c 3 B a s i c 4 A c i d i c T o t a l AA 2 Total AA Total AA Basic Aspartyl proteinases chymosin 0.156 0.096 0.062 1.55 pepsin 0.141 0.128 0.012 10.67 M. miehei proteinase 0.157 0.106 0.052 2.04 M. p u s i l l u s proteinase 0.292 0.228 0.064 3.56 E. p a r a s i t i c a proteinase 0.167 0.118 0.049 2.41 A. s a i t o i proteinase 0.249 0.197 0.052 3.80 p e n i c i l l o p e p s i n 0.108 0.084 0.025 3.39 Non-aspartyl proteinases papain 0.184 0.071 0.113 0.63 "-chymotrypsin 0.139 0.057 0.082 0.70 t r y p s i n 0.265 0.161 0.085 1.89 Total charge = A c i d i c + Basic r e s i d u e s . T o t a l AA = To t a l number of amino a c i d s . A c i d i c = Asp, Glu re s i d u e s . Basic = Lys, His, Arg re s i d u e s . - 152 -r a t i o s i n v o l v i n g t o t a l charged groups and a c i d i c groups were examined, however, some d i f f e r e n c e s were noted when the r a t i o i n v o l v i n g b a s i c amino acids and the r a t i o of a c i d i c to basic amino acids were examined. The proportion of basic amino acids was r e l a t i v e l y low for the a s p a r t y l proteinases i n comparison to the non-aspartyl p r o t e i n a s e s . On the other hand, the pro p o r t i o n of a c i d i c amino acids i n the a s p a r t y l proteinases was g e n e r a l l y much higher than the proportion of the bas i c amino a c i d s , r e s u l t i n g i n r a t i o s greater than one. Examination of the a c i d i c to bas i c r a t i o i n d i c a t e d that pepsin had a s u b s t a n t i a l l y higher r a t i o than the other a s p a r t y l p r o t e i n a s e s . This excess of a c i d i c to bas i c groups may p a r t i a l l y e x p l a i n the conforma-t i o n a l change seen i n the CD spectra for pepsin at pH values greater than 6.3. Lowenstein (1974), working with chemically modified pepsin, postulated that at pH values greater than 6.0 an e l e c t r o s t a t i c expansion of the ne g a t i v e l y charged polypeptide may occur which could lead to denaturation. Chymosin, Mucor miehei proteinase, Mucor p u s i l l u s pro-t e i n a s e and Endothia p a r a s i t i c a proteinase which had low a c i d i c to basic r a t i o s were r e l a t i v e l y s t a b l e to conformational change with i n c r e a s i n g pH as detected by CD s p e c t r a l a n a l y s i s (both far-UV and near-UV). However, the r e l a t i o n s h i p between a low a c i d i c to basic amino a c i d r a t i o and conformation s t a b i l i t y could not be extended to p e n i c i l l o p e p s i n and A s p e r g i l l u s s a i t o i p r o t e i n a s e . The a b i l i t y to l i n k charge r a t i o s to a f u n c t i o n a l property such as conformational s t a b i l i t y may s u f f e r from the same type of l i m i t a t i o n that was experienced with the use of average hydrophobicity as a means for c l a s s i f i c a t i o n . Both parameters (average - 153 -hydrophobicity and charge r a t i o ) describe the t o t a l p r o t e i n r e g ardless of the l o c a t i o n of s p e c i f i c residues i n the topography of the n a t i v e p r o t e i n , which i s undoubtedly important i n determining the f u n c t i o n of the p r o t e i n . In a d d i t i o n , the charge r a t i o of a proteinase may not r e f l e c t the charge density at a s p e c i f i c pH since charge density i s a f f e c t e d by the various pK values of the a c i d i c and basic side chains. G. ZETA POTENTIAL An i n d i c a t i o n of a p r o t e i n ' s net charge may be obtained from the zeta p o t e n t i a l or the e l e c t r i c a l p o t e n t i a l at the surface of a p a r t i c l e coated with p r o t e i n . The zeta p o t e n t i a l of the various proteinases was measured over the pH range of 5.0 to 8.0 and the r e s u l t s are presented i n Figure 30. The net charge of a p r o t e i n at a s p e c i f i c pH i s dependent on the degree of i o n i z a t i o n of the amino acid side chains. Negative charges r e s u l t from carboxyl groups whereas E-amino groups, im i d a z o l e , i n d o l e and guanidyl groups c o n t r i b u t e to the p o s i t i v e charges. S u l f h y d r y l and phenolic groups may also c o n t r i b u t e to negative charges at high pH values (Townsend, 1982). The a s p a r t y l proteinases studied showed a net negative charge over the pH range examined, with the degree of n e g a t i v i t y i n c r e a s i n g with i n c r e a s i n g pH. This increase i n n e g a t i v i t y can be a t t r i b u t e d to the d i s s o c i a t i o n of the c a r b o x y l i c side chains coupled with the deprotona-t i o n of the amino groups. The net negative charge observed for the proteinases would be expected since the a s p a r t y l proteinases have - 154 -40 -Figure 30. The e f f e c t of pH on the z e t a p o t e n t i a l of various p r o t e i n -ases. Chymosin A , pepsin • , M. miehei proteinase • , _M. p u s i l l u s proteinase O , E_. p a r a s i t i c a proteinase # , A_. s a i - t o i proteinase • , p e n i c i l l o p e p s i n • , papain m , "-chymo-t r y p s i n A and t r y p s i n © . - 155 -i s o e l e c t r i c p o i n t s below pH 5.0. Attempts were made to e x t r a p o l a t e the zeta p o t e n t i a l curves of the a s p a r t y l proteinases using l i n e a r regres-s i o n a n a l y s i s i n order to determine the i s o e l e c t r i c p o i n t s . Results were g e n e r a l l y unsuccessful when compared to l i t e r a t u r e values. Abram-son et a l . (1964) demonstrated that e x t r a p o l a t i o n of the zeta p o t e n t i a l versus pH curve to zero zeta p o t e n t i a l may y i e l d erroneous i s o e l e c t r i c p o i n t s because of a c u r v i l i n e a r r e l a t i o n s h i p between zeta p o t e n t i a l and pH. With the exception of A s p e r g i l l u s s a i t o i p r o t e i n a s e , the other a s p a r t y l proteinases had lower ( i . e . more negative) zeta p o t e n t i a l s than d i d chymosin over the pH range. The large negative zeta p o t e n t i a l values obtained for pepsin are undoubtedly r e l a t e d to the low i s o e l e c -t r i c point of t h i s p r o t e i n a s e . Fruton (1970) reported that the i s o e l e c -t r i c point of porcine pepsin i s below 1.0 since even at t h i s pH the molecule i s s t i l l m igrating as an anion. The high negative charge of pepsin i s also c o n s i s t e n t with the c a l c u l a t e d charge r a t i o of a c i d i c to b a s i c groups (Table 9) since pepsin has a d e f i n i t e predominance of a c i d i c groups over basic groups. Curves of higher zeta p o t e n t i a l values ( i . e . l e s s negative) might be expected for the other a s p a r t y l p r o t e i n -ases which have lower a c i d i c to b a s i c amino a c i d r a t i o s , and would l i k e l y have higher i s o e l e c t r i c p o i n t s as compared to pepsin. Higher i s o e l e c t r i c p o i n t s have been reported i n the l i t e r a t u r e f o r the other a s p a r t y l proteinases (e.g. Mucor miehei proteinase with an i s o e l e c t r i c point of p i = 4.2 (Ottesen and R i c k e r t , 1970b)). Chymosin which had a nearly equal proportion of a c i d i c to basic groups (Table 9) also had high ( i . e . l e s s negative) zeta p o t e n t i a l - 156 -values. The zeta p o t e n t i a l curves of Mucor miehei pr o t e i n a s e , Mucor  p u s i l l u s proteinase and Endothia p a r a s i t i c a proteinase were intermediate to chymosin and pepsin as might be expected i f one assumes that charge r a t i o provides an i n d i c a t i o n of zeta p o t e n t i a l . A s p e r g i l l u s s a i t o i proteinase which had a greater p r o p o r t i o n of a c i d i c to basic groups (Table 9), had higher zeta p o t e n t i a l values ( i . e . l e s s negative) than those of chymosin. The zeta p o t e n t i a l values of p e n i c i l l o p e p s i n on the other hand were lower than expected based on charge r a t i o s . The a v a i l a b i l i t y of c e r t a i n a c i d i c or ba s i c groups may be a f f e c t e d by unfo l d i n g of the p r o t e i n molecule during zeta p o t e n t i a l measurement. P r o t e i n s that are adsorbed onto a c a r r i e r surface (e.g. 3,3'-dimethylbiphenyl) may not have the zeta p o t e n t i a l values expected fo r the same p r o t e i n s d i s s o l v e d i n the absence of a c a r r i e r . Abramson et a l . (1964) sta t e d that changes i n p r o t e i n conformation may occur upon adsorption to a c a r r i e r thus a f f e c t i n g the exposure of charged groups. H. ACCESSIBLE SURFACE AREA Lee and Richards (1971) introduced the term " a c c e s s i b l e surface area" to describe the proportion of the p r o t e i n surface which could form contacts with water. Since the nativ e s t r u c t u r e of a p r o t e i n e x i s t s only i n the presence of water (Bernal et a l . , 1938), the determination of a c c e s s i b l e surface area may y i e l d information p e r t i n e n t to the t h e o r i e s of p r o t e i n s t r u c t u r e and p r o t e i n - p r o t e i n i n t e r a c t i o n (Kauzmann, 1959). Janin (1976), on the basis of the work by Chothia (1975), formu-l a t e d an equation (Eq. 23) which r e l a t e s the molecular weight of a - 157 -p r o t e i n to i t s a c c e s s i b l e surface area ( A s ) . The a c c e s s i b l e surface areas obtained for the various proteinases are presented i n Table 10. The a s p a r t y l proteinases were found to have s i m i l a r a c c e s s i b l e surface areas i n the range of 11000 to 13000 A 2, whereas the non-aspartyl proteinases had A s values i n the range of 9000 to 10000 A 2. These r e s u l t s would be expected due to the nature of the r e l a t i o n s h i p between a c c e s s i b l e surface area and molecular weight, and the f a c t that the a s p a r t y l proteinases are c h a r a c t e r i z e d by having r e l a t i v e l y s i m i l a r molecular weights (Table 10). As a c l a s s , the a s p a r t y l proteinases a l s o have s i m i l a r t e r t i a r y s t r u c t u r e s (Jenkins et a l . , 1976), thus i t f o l l o w s t h a t molecules with s i m i l a r s i z e and shape would have s i m i l a r A s values. A c c e s s i b l e surface area may help to e x p l a i n p r o t e i n f u n c t i o n . However, the non-aspartyl proteinases which are also known to e x h i b i t m i l k - c l o t t i n g a b i l i t y (Ernstrom, 1974), were found to have lower A s values than the a s p a r t y l proteinases ( m i l k - c l o t t i n g enzymes). I t i s t h e r e f o r e apparent that other parameters i n a d d i t i o n to A s must be c o n t r i b u t i n g to enzymatic a c t i v i t y (e.g. m i l k - c l o t t i n g ) . I . MILK-CLOTTING TO PROTEOLYTIC ACTIVITY RATIO The m i l k - c l o t t i n g to p r o t e o l y t i c a c t i v i t y r a t i o has been used i n attempts to i d e n t i f y proteinases which are s u i t a b l e as m i l k - c l o t t i n g agents i n the cheese-making process (Puhan and I r v i n e , 1973; de Koning et a l . , 1978). Proteinases which are s u c c e s s f u l l y used i n cheese-making have a high m i l k - c l o t t i n g to p r o t e o l y t i c a c t i v i t y r a t i o ( V i s s e r , 1981). - 158 -Table 10. A c c e s s i b l e surface areas (A s) of various p r o t e i n a s e s . P r o t e i n Aspartyl proteinases chymosin pepsin _M. miehei proteinase M. p u s i l l u s proteinase E_. p a r a s i t i c a proteinase A_. s a i t o i proteinase p e n i c i l l o p e p s i n 30700 35000 38000 30600 37500 34500 32000 11300 12300 13000 11200 12900 12200 11600 Non-aspartyl proteinases papain "-chymotrypsin t r y p s i n 20900 21600 24000 8700 8900 9600 Molecular weight. - 159 -Both m i l k - c l o t t i n g and p r o t e o l y t i c a c t i v i t i e s are c a l c u l a t e d on the basis of proteinase weight, but due to i m p u r i t i e s i n some of the proteinase p r e p a r a t i o n s , i t was f e l t that the use of the m i l k - c l o t t i n g to p r o t e o l y t i c a c t i v i t y r a t i o would reduce the e f f e c t s of non-proteoly-t i c components w i t h i n the pre p a r a t i o n s . The m i l k - c l o t t i n g to proteo-l y t i c a c t i v i t y r a t i o s obtained for the various proteinases are presented i n Table 11. Bf the enzymes examined, chymosin e x h i b i t e d the highest m i l k - c l o t t i n g to p r o t e o l y t i c a c t i v i t y r a t i o s over the pH range. Ernstrom (1974) a l s o reported that chymosin had the highest m i l k - c l o t -t i n g to p r o t e o l y t i c a c t i v i t y r a t i o among p r o t e o l y t i c enzymes i n d i c a t i n g that chymosin had the highest s p e c i f i c m i l k - c l o t t i n g a c t i v i t y . The m i c r o b i a l proteinases from Mucor miehei, Mucor p u s i l l u s and Endothia  p a r a s i t i c a g e n e r a l l y showed r a t i o s that were intermediate to those of chymosin and porcine pepsin. A s p e r g i l l u s s a i t o i p r o t e i n a s e , p e n i c i l -l o p e p s i n and the non-aspartyl proteinases (papain, «-chymotrypsin and t r y p s i n ) a l l showed low r a t i o s . According to Huang and Dooley (1976), the majo r i t y of cheese manu-f a c t u r i n g operations i n the United States u t i l i z e the proteinases from Mucor miehei, Mucor p u s i l l u s and Endothia p a r a s i t i c a ; a s i m i l a r s i t u a t i o n may e x i s t i n Canada. Arima et a l . (1970), i n a comparison of the m i l k - c l o t t i n g to p r o t e o l y t i c a c t i v i t y r a t i o s of various p r o t e i n a s e s , found that the r a t i o obtained for Endothia p a r a s i t i c a proteinase was lower than that of Mucor p u s i l l u s var. Lindt proteinase, which i n turn was lower than that obtained f or chymosin. The authors observed that low r a t i o s were obtained f or proteinases such as t r y p s i n , papain and - 160 -Table 11. M i l k - c l o t t i n g to p r o t e o l y t i c a c t i v i t y r a t i o s for various p r o t e i n a s e s . M i l k - c l o t t i n g to p r o t e o l y t i c a c t i v i t y r a t i o 1 (x10- 2) pH  P r o t e i n 5.0 5.3 5.8 6.3 7.0 8.0 Aspartyl proteinases chymosin 159 .61 132.81 97.06 95.75 13.64 N.D.2 pepsin 74 .43 57.89 53.12 47.43 N.D. N.D. M. miehei proteinase 114 .31 105.96 97.58 71.05 6.93 N.D. M. p u s i l l u s proteinase 127 .30 90.01 86.04 63.37 8.32 N.D. E. p a r a s i t i c a proteinase 82 .11 62.52 62.49 60.36 N.D. N.D. A. s a i t o i proteinase 1 .45 0.93 0.58 N.D. N.D. N.D. p e n i c i l l o p e p s i n 22 .95 _3 - 0.75 - N.D. INlon-aspartyl proteinases papain - 2.17 "-chymotrypsin - - - 3.07 t r y p s i n - 0.02 R a t i o s were c a l c u l a t e d using the mean values of d u p l i c a t e determina-t i o n s f or both m i l k - c l o t t i n g and p r o t e o l y t i c a c t i v i t y . 2N.D. = Not detected. Not determined. - 161 -A s p e r g i l l u s s a i t o i proteinase and were g e n e r a l l y the r e s u l t of low m i l k - c l o t t i n g a b i l i t y to high general p r o t e o l y t i c a c t i v i t y . Examination of the a s p a r t y l proteinases showed that as pH was increased from 5.0 to 8.0, the m i l k - c l o t t i n g to p r o t e o l y t i c a c t i v i t y r a t i o decreased i n d i c a t i n g l o s s of enzymatic a c t i v i t y . The decrease i n enzymatic a c t i v i t y for the a s p a r t y l proteinases could be p a r t i a l l y e xplained by a change i n the far-UV CD spectra as r e f l e c t e d by changes i n the p r o t e i n secondary s t r u c t u r e f r a c t i o n s . At pH 8.0 no enzymatic a c t i v i t y was detected for any of the a s p a r t y l p r o t e i n a s e s , which c o r r e s -ponded to changes i n the far-UV CD spectra noted at t h i s pH. However, not a l l of the decrease i n enzymatic a c t i v i t y could be a t t r i b u t e d to changes i n secondary s t r u c t u r e since v i r t u a l l y no change i n secondary s t r u c t u r e was seen at pH values of 6.3 or lower for most of the a s p a r t y l proteinases (except chymosin). I t was i n t e r e s t i n g to note that the proteinases (pepsin, A s p e r g i l l u s s a i t o i proteinase and p e n i c i l l o p e p s i n ) which showed r e l a t i v e l y l a r ge changes i n secondary s t r u c t u r e at pH values greater than 6.3 (Table 6 ) , showed greater l o s s e s of enzymatic a c t i v i t y (Table 11) than those a s p a r t y l proteinases which showed only small changes i n secondary s t r u c t u r e . The r e l a t i o n s h i p between the secondary s t r u c t u r e of a p r o t e i n and i t s f u n c t i o n has been documented i n the l i t e r a t u r e ( P t i t s y n and F i n k e l s t e i n , 1983). I t i s undoubtedly the f o l d i n g of the secondary s t r u c t u r e f r a c t i o n s i n t o the t e r t i a r y s t r u c t u r e that governs the intimate contact between enzyme and substrate which e v e n t u a l l y r e s u l t s i n c a t a l y s i s . From the present study i t i s apparent that changes i n - 162 -secondary s t r u c t u r e may a f f e c t enzymatic a c t i v i t y . A l o s s of enzymatic a c t i v i t y was not always associated with a change i n secondary s t r u c t u r e i n the present study, however, su b t l e changes i n the secondary s t r u c t u r e may have occurred, but were not detected by the method of a n a l y s i s . The r e l a t i o n s h i p seen between the far-UV CD spectra and the milk-c l o t t i n g to p r o t e o l y t i c a c t i v i t y r a t i o was s i m i l a r to that observed between the near-UV CD spectra and the m i l k - c l o t t i n g to p r o t e o l y t i c a c t i v i t y r a t i o . The l a r g e s t reductions i n enzymatic a c t i v i t y for the various a s p a r t y l proteinases g e n e r a l l y occurred at pH values greater than 6.3. I t was also at these pH values that changes i n the near-UV CD f i n e s t r u c t u r e were noted, the extent of these changes being proteinase dependent. Losses i n enzymatic a c t i v i t y i n the pH range of 5.0 to 6.3 were not detected when the near-UV CD spectra of the a s p a r t y l p r o t e i n -ases were examined. Minor changes i n the t e r t i a r y s t r u c t u r e may have occurred i n the pH range of 5.0 to 6.3, but were not detected. Cheeseman (1969) examined the l o s s of m i l k - c l o t t i n g a c t i v i t y by measuring changes i n the UV absorption spectra obtained for chymosin tr e a t e d with urea and subjected to d i f f e r e n t pH values. Results i n d i -cated that l o s s of a c t i v i t y occurred with changes i n absorption s p e c t r a and that the rate of change increased as both urea concentration and pH value increased. However, i t was also found that 50 percent i n a c t i v a -t i o n was obtained with changes varying from 27 to 94 percent of the t o t a l s p e c t r a l change, which may suggest that there i s only a p a r t i a l c o r r e l a t i o n between the degree of u n f o l d i n g and the l o s s of a c t i v i t y . Factors other than the a v a i l a b i l i t y of the chromophore groups as a - 163 -r e s u l t of the u n f o l d i n g of the molecule must i n f l u e n c e the l o s s of enzyme a c t i v i t y . Kay and V a l l e r (1981) noted that the a s p a r t y l proteinases were s u s c e p t i b l e to a l k a l i n e denaturation due to high contents of a c i d i c amino ac i d r e s i d u e s . Ma (1979) stu d i e d the chemical m o d i f i c a t i o n of carboxyl groups i n porcine pepsin and stated that decreases i n enzymatic a c t i v i t y were due e i t h e r to conformational changes i n the p r o t e i n mole-cule or to changes i n the charge d i s t r i b u t i o n ; both changes could e f f e c t the binding between enzyme and s u b s t r a t e . In the present study, pepsin which had a high predominance of a c i d i c to basic residues (Table 9) showed no detectable enzymatic a c t i v i t y at pH values greater than 6.3. Chymosin, on the other hand, which had a lower r a t i o of a c i d i c to b a s i c residues (Table 9) showed s u b s t a n t i a l l y higher enzymatic a c t i v i t y at pH values greater than 6.3 i n d i c a t i n g greater enzymatic s t a b i l i t y . Mucor  miehei p r o t e i n a s e , Mucor p u s i l l u s proteinase and Endothia p a r a s i t i c a proteinase which had charge r a t i o s intermediate to chymosin and pepsin showed m i l k - c l o t t i n g to p r o t e o l y t i c a c t i v i t y r a t i o s which were interme-d i a t e to chymosin and pepsin. However, p e n i c i l l o p e p s i n and A s p e r g i l l u s  s a i t o i proteinase which also had intermediate a c i d i c to basic amino ac i d r a t i o s showed no detectable enzymatic a c t i v i t y at pH values greater than 6.3, t h e r e f o r e , charge r a t i o s cannot t o t a l l y e x p l a i n enzymatic s t a b i l i t y to a l k a l i n e c o n d i t i o n s . In a d d i t i o n to the charge r a t i o , the s p e c i f i c arrangement of the charged groups i n the p r o t e i n ' s t e r t i a r y s t r u c t u r e must be important for determining enzymatic s t a b i l i t y . Futhermore, i t i s undoubtedly the charge and conformation of the residues i n the a c t i v e - 164 -s i t e which have a major i n f l u e n c e on the enzyme-substrate i n t e r a c t i o n and subsequent c a t a l y s i s . Most work concerning the a c t i v e s i t e region of the a s p a r t y l pro-t e i n a s e s has been c a r r i e d out on pepsin. I f i t i s assumed that a l l a s p a r t y l proteinases react i n a s i m i l a r manner, then some of the hypo-theses proposed for the pH dependency of pepsin c a t a l y z e d r e a c t i o n s may be g e n e r a l i z e d to the a s p a r t y l proteinases as a group. Clement (1973) reported that the pK a values of the c a t a l y t i c a l l y e s s e n t i a l a s p a r t i c a c i d residues of pepsin, Asp3 2 and ASP215, were 1.5 and 4.5, r e s p e c t i v e -l y . The d i f f e r e n c e i n pK a values was a t t r i b u t e d to the existence of a hydrogen bond between the two c a r b o x y l i c a c i d groups. Antonov (1976), also working with pepsin, proposed that the decrease i n c a t a l y t i c a c t i v -i t y with an increase i n pH may be due to the l o s s of a proton from the hydrogen bonded e s s e n t i a l a s p a r t i c a c i d groups. Once t h i s proton has been e l i m i n a t e d due to the d i s s o c i a t i o n of the c a r b o x y l i c a c i d group of ASP215, the o r i e n t a t i o n of the carboxyl groups may change. Hsu et a l . (1977) discussed the p o s s i b i l i t y of t h i s type of mechanism for the a c t i v e s i t e a s p a r t i c a c i d residues i n p e n i c i l l o p e p s i n . A s i m i l a r s i t u a -t i o n could occur with other a s p a r t y l proteinases leading to the l o s s of c a t a l y t i c a c t i v i t y with i n c r e a s i n g pH. Raymond and B r i c a s (1979) were able to determine the pK values of the groups e s s e n t i a l for the r e a c t i o n between chymosin and s y n t h e t i c peptide s u b s t r a t e s ; pK values of 3.3 and 5.7 were obtained on the b a s i s of a p l o t of log k c a t / K m vs pH. The authors postulated that the c a t a l y t i c a c t i v i t y of chymosin was dependent on the i o n i z a t i o n of two - 165 -c a r b o x y l groups, s i m i l a r to the r e a c t i o n mechanism proposed for pepsin (Fruton, 1970). V i s s e r et a l . (1980) studied the h y d r o l y s i s of t r y p t i c fragments of bovine K-casein by chymosin and found that the apparent pK^ and pl<2 values of c a t a l y t i c a l l y important groups on the enzyme-substrate complexes were i n the region of 4.0 to 4.2 and 6.5 to 6.7, respec-t i v e l y . I t was assumed that these pK values represent the pK values of the a c t i v e s i t e a s p a r t i c a c i d residues although the values for the free enzyme may be d i f f e r e n t due to substrate b i n d i n g . The d i f f e r e n c e s i n l o s s of enzymatic a c t i v i t y as a f u n c t i o n of pH e x h i b i t e d by the various a s p a r t y l proteinases examined i n the present study may be p a r t i a l l y due to the d i f f e r e n c e s i n the pK a values of the a c t i v e s i t e a s p a r t i c a c i d r e s i d u e s . The pK a values of the a c t i v e s i t e a s p a r t i c acid residues may be a f f e c t e d by the residues adjacent to them i n the a c t i v e s i t e , and by the c h a r a c t e r i s t i c s of the s u b s t r a t e . K i t s o n and Knowles (1971) observed that by chemically modifying a r g i n i n e r e s i -dues (by phenylglyoxal) i n pepsin and thereby changing the microenviron-ment, i t was p o s s i b l e to change the pK a value of one of the a c t i v e s i t e a s p a r t i c a c i d residues (Asp 32). Raap et a l . (1983) examined various peptide substrates for chymo-s i n and stated that i n order to b e t t e r understand the s p e c i f i c i t y and the k i n e t i c mechanism of enzyme a c t i o n both the conformation of the a c t i v e s i t e of the enzyme and the conformation of the substrate molecule are important. I t i s p o s s i b l e that the decrease i n enzymatic a c t i v i t y with i n c r e a s i n g pH observed i n the present study for the various aspar-t y l proteinases may have also been due to changes i n the s u b s t r a t e ( s ) . No attempts were made to measure any substrate conformation. - 166 -J . PRINCIPAL COMPONENT ANALYSIS Data c o l l e c t e d for the various proteinases examined i n the present study was subjected to p r i n c i p a l component a n a l y s i s (PCA) i n attempts to i d e n t i f y parameters important for the c l a s s i f i c a t i o n of these p r o t e i n -ases. The s i x t e e n v a r i a b l e s used i n the PCA were p r e v i o u s l y described (see M a t e r i a l s and Methods). M i l k - c l o t t i n g to p r o t e o l y t i c a c t i v i t y r a t i o was excluded i n t h i s a n a l y s i s . When the s i x t e e n o r i g i n a l v a r i a b l e s for each of the proteinase samples were entered i n the p r i n c i p a l component a n a l y s i s program, four f a c t o r s were returned which met the c r i t e r i a of t h e i r eigenvalues exceeding 1.0 (Table 12). Aishima (1979a; 1979b; 1979c) used the same c r i t e r i a for s e l e c t i n g p r i n c i p a l components. The four f a c t o r s or p r i n -c i p a l components obtained accounted for more than 85 percent of the t o t a l variance (Table 12), where the t o t a l variance i s the sum of the i n d i v i d u a l variances for each of the o r i g i n a l v a r i a b l e s . Each f a c t o r represents a combination of the o r i g i n a l v a r i a b l e s . The f a c t o r loadings for each f a c t o r give an i n d i c a t i o n of the importance of the o r i g i n a l v a r i a b l e s to that f a c t o r . The f a c t o r loadings for the four f a c t o r s are presented i n Table 13. Examination of the f a c t o r loadings i n d i c a t e d that f a c t o r 1 was p r i m a r i l y concerned with c i r c u l a r dichroism s p e c t r a l data. V a r i a b l e s c o n t r i b u t i n g to f a c t o r 2 included molar e l l i p t i c i t y values at wave-lengths 193, 198, 200 and 202 nm. I t i s i n t e r e s t i n g to note that these wavelengths corresponded to a c h a r a c t e r i s t i c wavelength for each of the major secondary s t r u c t u r e f r a c t i o n s . The CD band at 193 nm i s one of the bands c h a r a c t e r i s t i c of " - h e l i x , the 198 nm band c h a r a c t e r i s t i c of - 167 -Table 12. The variance explained and the cumulative p r o p o r t i o n of t o t a l variance accounted for by each f a c t o r derived from the p r i n -c i p a l component a n a l y s i s . Variance explained Cumulative p r o p o r t i o n of Factor (eigenvalue) t o t a l variance 1 8.113 0.5071 2 2.793 0.6816 3 1.867 0.7984 4 1.280 0.8783 5 0.749 0.9251 6 0.562 0.9603 7 0.380 0.9840 8 0.145 0.9931 9 0.061 0.9969 10 0.028 0.9987 11 0.011 0.9994 12 0.006 0.9997 13 0.002 0.9998 14 0.001 0.9999 15 0.001 0.9999 16 0.000 1.0000 - 168 -Table 13. Factor loadings f or the f a c t o r s whose eigenvalues exceed 1.0. Factor loadings O r i g i n a l v a r i a b l e Factor 1 Factor 2 Factor 3 Factor 4 213 nm 0.978 0.000 0.000 0.000 202 nm 0.974 0.000 0.000 0.000 210 nm 0.965 0.000 0.000 0.000 224 nm 0.954 0.000 0.000 0.000 225 nm 0.945 0.000 0.000 0.000 190 nm -0.905 0.000 0.000 0.000 193 nm -0.879 0.359 0.000 0.000 ^AVG -0.719 0.000 0.000 0.394 ANS -0.626 0.000 -0.495 -0.259 198 nm 0.000 0.971 0.000 0.000 200 nm 0.000 0.967 0.000 0.000 202 nm 0.537 0.811 0.000 0.000 A c i d i c / B a s i c 0.000 0.000 0.870 0.000 Zeta p o t e n t i a l 0.000 0.000 -0.692 0.000 A s 0.000 0.000 0.000 0.794 CPA 0.000 0.000 -0.470 -0.780 - 169 -8-sheet, the 200 nm band c h a r a c t e r i s t i c of the unordered f r a c t i o n and the 202 nm band i s c h a r a c t e r i s t i c of the 6-turn f r a c t i o n . Factor 3 was c h a r a c t e r i z e d by ANS hydrophobicity, a c i d i c to basic amino a c i d r a t i o , z e ta p o t e n t i a l and CPA hydrophobicity. V a r i a b l e s c o n t r i b u t i n g to f a c t o r 4 included Bigelow average hydrophobicity, ANS hydrophobicity, access-i b l e surface area, as w e l l as CPA hydrophobicity. Two-dimensional p l o t s i n v o l v i n g the f i r s t three f a c t o r s are presented i n Figures 31 and 32. Alphabetic codes used to i d e n t i f y the proteinases are given i n the f i g u r e legends. The p l o t of f a c t o r 2 versus f a c t o r 1 (Figure 31) i n d i c a t e d that the proteinases from micro-b i a l sources ( i . e . M. miehei, M. p u s i l l u s , E_. p a r a s i t i c a and A_. s a i t o i p roteinases and p e n i c i l l o p e p s i n ) were c l o s e l y associated with one another, while pepsin at pH values 5.0 to 6.3 formed one group, and chymosin at pH values 5.8, 6.3 and 7.0 formed another group. The non-a s p a r t y l proteinases (papain, "-chymotrypsin and t r y p s i n ) were not associated with any group. Chymosin at pH 5.0, 5.3 and 8.0, as w e l l as pepsin at pH 7.0 and 8.0, were not associated with t h e i r r e s p e c t i v e groups which could be explained by noting that these samples showed d i f f e r e n t CD s p e c t r a l patterns than those from the p r e v i o u s l y mentioned pH values. Since f a c t o r 1 and f a c t o r 2 were almost e x c l u s i v e l y con-cerned with CD s p e c t r a l data, any change i n s p e c t r a l c h a r a c t e r i s t i c s of a sample w i t h i n a group may r e s u l t i n the e x c l u s i o n of that sample from that group. The far-UV spectra of the a s p a r t y l proteinases examined g e n e r a l l y changed at pH values greater than 6.3. This change i n CD s p e c t r a was - 170 -CM O (0 Li-es C4 C5 Di 02 D3 D4 C6 Ci K4 K3 A1A2 KiJCfc F5 F6 C 2 D5 D6 G6 F a c t o r 1 F i g u r e 31. The p l o t of f a c t o r 2 vs f a c t o r 1 obtained from the p r i n c i p a l component a n a l y s i s of the various s t r u c t u r a l and i n t r i n s i c p r o p e r t i e s of the pr o t e i n a s e s . A - Mucor miehei p r o t e i n a s e , K - Endothia p a r a s i t i c a p r o t e i n a s e , C - chymosin, D -pepsin, E - Mucor p u s i l l u s p r o t e i n a s e , F - A s p e r g i l l u s  s a i t o i p r o t e i n a s e , G - p e n i c i l l o p e p s i n , H - t r y p s i n , I -oc-chymotrypsin, and J - papain. The numbers 1,2,3,4,5,6 represent pH 5.0, 5.3, 5.8, 6.3, 7.0, 8.0 r e s p e c t i v e l y . - 171 -D6 05 D4 Di Gt, CO 6 CO L L F6 F5 G4 A6 Gi 45 • E6 E 5 A 2 K4 Ki C5 C4 C 3 C2 C1 F a c t o r 2 F i g u r e 32. The p l o t of f a c t o r 3 vs f a c t o r 2 obtained from the p r i n c i p a l component a n a l y s i s of the various s t r u c t u r a l and i n t r i n s i c p r o p e r t i e s of the p r o t e i n a s e s . A - Mucor miehei p r o t e i n a s e , K - Endothia p a r a s i t i c a p r o t e i n a s e , C - chymosin, D -pepsin, E - Mucor p u s i l l u s p r o t e i n a s e , F - A s p e r g i l l u s  s a i t o i p r o t e i n a s e , G - p e n i c i l l o p e p s i n , H - t r y p s i n , I -a-chymotrypsin, and 3 - papain. The numbers 1,2,3,4,5,6 represent pH 5.0, 5.3, 5.8, 6.3, 7.0, 8.0 r e s p e c t i v e l y . - 172 -r e f l e c t e d i n a downward s h i f t of the points representing these samples i n the p l o t . The degree of downward s h i f t was r e l a t e d to the degree of change i n the CD spectra ( i . e . the greater the change i n the CD s p e c t r a , the l a r g e r the downward s h i f t ) . Changes i n the v e r t i c a l d i r e c t i o n (Y-axis) of the p l o t would be c o n t r o l l e d by f a c t o r 2 while changes i n the h o r i z o n t a l d i r e c t i o n (X-axis) would be c o n t r o l l e d by f a c t o r 1. The s h i f t s or changes observed were i n the v e r t i c a l d i r e c t i o n which would tend to imply that changes o c c u r r i n g i n the short wavelength far-UV CD range were more c r i t i c a l than those seen at the long wavelength far-UV CD range. I f one examines the reference spectra for the four secondary s t r u c t u r e f r a c t i o n s ( " - h e l i x , B-sheet, B-turn and unordered) as proposed by Chang et a l . (1978) (Figure 33) i t i s apparent that the wavelengths comprising f a c t o r 2 show the l a r g e s t d i f f e r e n c e s i n molar e l l i p t i c i t y values among the secondary s t r u c t u r e s . These wavelengths may t h e r e f o r e have some a b i l i t y to d i s c r i m i n a t e among the four secondary s t r u c t u r e s . Unlike the p l o t of f a c t o r 2 versus f a c t o r 1, i n which both f a c t o r s were concerned with CD s p e c t r a l data, the p l o t of f a c t o r 3 versus f a c t o r 2 (Figure 32) was a p l o t of p r o p e r t i e s associated with s t r u c t u r e ( z e t a p o t e n t i a l , a c i d i c to ba s i c amino a c i d r a t i o , ANS and CPA hydrophobicity) versus CD s p e c t r a l p r o p e r t i e s (molar e l l i p t i c i t y values at wavelengths 193, 198, 200 and 202 nm). The p l o t of f a c t o r 3 versus f a c t o r 2 (Figure 32) again showed that the m i c r o b i a l proteinases were c l o s e l y associated with one another. Chymosin as a group, however, was much more c l o s e l y associated with the m i c r o b i a l proteinases than i n the p l o t of f a c t o r 2 versus f a c t o r 1 (Figure 31). P e n i c i l l o p e p s i n at pH 5.0 and pH 6.3 formed a group intermediate to the m i c r o b i a l enzymes and a group formed - 173 -Figure 33. Reference spectra for " - h e l i x , B-sheet, B-turn and random c o i l f r a c t i o n s . (Adapted from Chang et a l . , 1978.) - 174 -from pepsin at pH 5.0, pH 5.3, pH 5.8 and pH 6.3. Again papain, "-chymotrypsin and t r y p s i n were not associated with any group. In Figure 32 i t was i n t e r e s t i n g to note that as the pH of the a s p a r t y l proteinases samples was increased, the p o i n t s representing those samples tended to s h i f t h o r i z o n t a l l y to the l e f t - h a n d side of the p l o t . Since f a c t o r 2 represents CD s p e c t r a l data and i s p l o t t e d on the a b s c i s s a , any change i n CD s p e c t r a l data would be expected to cause changes along that a x i s . The samples corresponding to the p o i n t s which had been s h i f t e d h o r i z o n t a l l y also had lower m i l k - c l o t t i n g to p r o t e o l y -t i c a c t i v i t y r a t i o s than those p o i n t s on the right-hand side of the p l o t . In attempts to i d e n t i f y the v a r i a b l e s that c o n t r i b u t e to the m i l k - c l o t t i n g to p r o t e o l y t i c a c t i v i t y r a t i o , the m i l k - c l o t t i n g to p r o t e o l y t i c a c t i v i t y r a t i o which acted as the dependent v a r i a b l e was regressed on the p r i n c i p a l components that were derived from the s i x t e e n o r i g i n a l v a r i a b l e s . Regression of the dependent v a r i a b l e on the s i x t e e n o r i g i n a l v a r i a b l e s using standard m u l t i p l e l i n e a r r e g r e s s i o n a n a l y s i s (both forward and backward stepwise) was unsuccessful due l a r g e l y to the i n t e r c o r r e l a t i o n found among the independent v a r i a b l e s . L u kovits (1983), i n a study on the q u a n t i t a t i v e s t r u c t u r e - a c t i v i t y r e l a t i o n s h i p s (QSAR) employing independent quantum chemical i n d i c e s , also pointed out the problem of i n t e r c o r r e l a t i o n s of various measured i n d i c e s used i n a QSAR a n a l y s i s . Due to these i n t e r c o r r e l a t i o n s i n t e r p r e t a t i o n of the r e g r e s s i o n equations between a c t i v i t y and the chemical i n d i c e s of molecules was reported to be d i f f i c u l t . The author used p r i n c i p a l - 175 -component a n a l y s i s to reduce the number of i n d i c e s , without l o s s of in f o r m a t i o n , and also to decompose the i n d i c e s i n t o mutually independent components which could then be regressed against the dependent v a r i a b l e . In the present study p r i n c i p a l components were entered i n t o the r e g r e s s i o n model on the b a s i s of t h e i r c o r r e l a t i o n with the dependent v a r i a b l e (Table 14); the component having the l a r g e s t c o e f f i c i e n t was entered f i r s t . M u l t i v a r i a t e l i n e a r r e g r e s s i o n i n d i c a t e d that three components were necessary to describe the dependent v a r i a b l e (Table 14). The r e g r e s s i o n model c o n t a i n i n g the three components was h i g h l y s i g n i f i c a n t (P<0.01) and included components four, seven and two, the components being entered i n order of c o r r e l a t i o n to the dependent v a r i -able. Component four represented zeta p o t e n t i a l , Bigelow average hydrophobicity, a c i d i c to b a s i c amino ac i d r a t i o , a c c e s s i b l e surface area and CPA hydrophobicity; component seven represented the molar e l l i p t i c i t y value at 190 nm, zeta p o t e n t i a l , Bigelow average hydrophobi-c i t y , a c i d i c to b a s i c amino ac i d r a t i o and ANS hydrophobicity; and component two represented molar e l l i p t i c i t y values at wavelengths 193, 198, 200 and 202 nm. A n a l y s i s of the components i n d i c a t e d that the p r o p e r t i e s and the combination of the p r o p e r t i e s measured for the pro-t e i n a s e s were important i n p a r t i a l l y d e s c r i b i n g the m i l k - c l o t t i n g to p r o t e o l y t i c a c t i v i t y r a t i o . Since a s i g n i f i c a n t r e l a t i o n s h i p (P<0.01) was e s t a b l i s h e d between the dependent v a r i a b l e and various combinations of p r o p e r t i e s determined for the p r o t e i n a s e s , t h i s may imply that proteinases with s i m i l a r com-b i n a t i o n s have s i m i l a r enzymatic a c t i v i t i e s . Although the r e l a t i o n s h i p was s i g n i f i c a n t , some of the v a r i a b i l i t y observed f o r the dependent - 176 -Table 14. Regression a n a l y s i s f or the m i l k - c l o t t i n g to p r o t e o l y t i c a c t i v i t y r a t i o on the p r i n c i p a l components computed from various s t r u c t u r a l and i n t r i n s i c p r o p e r t i e s of the p r o t e i n -ases. Number of components (p) Number of cases (N) Index of components en t e r i n g F-value Regression model R 2 1 42 4 a 5.58** .1224 2 42 7 b 6.06** .2370 3 42 2 C 6.83** .3503 ** S i g n i f i c a n t at P<0.01. R e p r e s e n t s zeta p o t e n t i a l , Bigelow average hydrophobicity, a c i d i c / b a s i c amino a c i d r a t i o , a c c e s s i b l e surface area and CPA hydrophobicity. ^Represents the molar e l l i p t i c i t y value at 190 nm, zeta p o t e n t i a l , Bigelow average hydrophobicity, a c i d i c / b a s i c amino ac i d r a t i o and ANS hydrophobicity. Represents molar e l l i p t i c i t y values at wavelengths 193, 198, 200 and 202 nm. - 177 -v a r i a b l e was not accounted for by the independent v a r i a b l e s ( p r i n c i p a l components) which would i n d i c a t e that other p r o p e r t i e s not examined i n the present study were c r i t i c a l for d e s c r i b i n g enzymatic a c t i v i t y . For example, the proteinase from A s p e r g i l l u s s a i t o i had measured c h a r a c t e r -i s t i c s g e n e r a l l y s i m i l a r to those of the m i c r o b i a l proteinases examined and was grouped c l o s e to these proteinases, but had a very much lower m i l k - c l o t t i n g to p r o t e o l y t i c a c t i v i t y r a t i o . In attempts to improve the reg r e s s i o n model i t was decided to exclude the data from A s p e r g i l l u s s a i t o i proteinase since t h i s p r o t e i n -ase was the l e a s t pure of the enzymes examined. The r e s u l t i n g regres-s i o n model was also s i g n i f i c a n t (P<0.01) and was described by the same three components ( i . e . components, 2, 4 and 7) as p r e v i o u s l y d i s c u s s e d , however, the order i n which the components were entered i n t o the equa-t i o n was d i f f e r e n t . The c o e f f i c i e n t of determination was increased from .35 to .43 (Table 15). It may be p o s s i b l e to examine the two-dimensional p r i n c i p a l component p l o t s and on the ba s i s of f a c t o r loadings and standardized scores, hypothesize how a manipulation of the o r i g i n a l v a r i a b l e s may r e s u l t i n a p r o t e i n with s i m i l a r s t r u c t u r a l combination c h a r a c t e r i s t i c s having s i m i l a r f u n c t i o n a l p r o p e r t i e s . For example, i n the p l o t of f a c t o r 3 versus f a c t o r 2 (Figure 32) no change i n f a c t o r 2 (CD s p e c t r a l c h a r a c t e r i s t i c s ) would be necessary to move pepsin at pH values 5.0 to 6.3 c l o s e r to chymosin since the r e s p e c t i v e groups are s i t u a t e d above one another i n the p l o t . However, a change i n f a c t o r 3 (r e p r e s e n t i n g a c i d i c to bas i c amino a c i d r a t i o , zeta p o t e n t i a l , ANS and CPA hydropho-b i c i t y ) would be necessary to move pepsin c l o s e r to chymosin i n - 178 -Table 15. Regression a n a l y s i s f o r the m i l k - c l o t t i n g to p r o t e o l y t i c a c t i v i t y r a t i o on the p r i n c i p a l components computed for various s t r u c t u r a l and i n t r i n s i c p r o p e r t i e s of the p r o t e i n -ases i n the absence of A s p e r g i l l u s s a i t o i p r o t e i n a s e . Index of Number of Number of components F-value components (p) cases (N) en t e r i n g Regression model R 2 1 36 7 a 7.30** 0.1768 2 36 2 b 7.71** 0.3186 3 36 4° 8.14** 0.4329 ** S i g n i f i c a n t at P<0.01. g Represents molar e l l i p t i c i t y value at 190 nm, zeta p o t e n t i a l , Bigelow average hydrophobicity, a c i d i c / b a s i c amino acid r a t i o and ANS hydropho-b i c i t y . ^Represents molar e l l i p t i c i t y values at wavelengths 193, 198, 200 and 202 nm. Represents zeta p o t e n t i a l , Bigelow average hydrophobicity, a c i d i c / b a s i c amino a c i d r a t i o , a c c e s s i b l e surface area and CPA hydrophobicity. - 179 -Figure 32. Since chymosin had a low a c i d i c to basic amino a c i d r a t i o , high zeta p o t e n t i a l values and high ANS and CPA hydrophobicity values as compared to pepsin, h y p o t h e t i c a l l y a l t e r i n g those values f o r pepsin to more c l o s e l y resemble those of chymosin would undoubtedly move pepsin c l o s e r to chymosin. A s i m i l a r approach could be used for the other proteinases i n the p l o t . Although a r e l a t i o n s h i p between m i l k - c l o t t i n g to p r o t e o l y t i c a c t i v i t y r a t i o and s t r u c t u r a l and i n t r i n s i c parameter combinations has been e s t a b l i s h e d , whether or not such changes could r e s u l t i n enzymatic p r o p e r t i e s s i m i l a r to those of a d e s i r e d enzyme (e.g. chymosin) i s s p e c u l a t i v e and must await f u r t h e r experimental evidence (e.g. chemical m o d i f i c a t i o n s t u d i e s ) . - 180 -CONCLUSIONS The major o b j e c t i v e s of the present study were: to examine v a r i -ous s t r u c t u r a l and i n t r i n s i c p r o p e r t i e s of some a s p a r t y l proteinases as w e l l as some non-aspartyl proteinases; to c l a s s i f y these proteinases using p r i n c i p a l component a n a l y s i s ; and to i d e n t i f y parameters important for the m i l k - c l o t t i n g to p r o t e o l y t i c a c t i v i t y r a t i o . Using the diagonal p l o t method, i t was found that pepsin and chymosin had the highest degree of primary sequence homology when compared to the other p a i r s of a s p a r t y l p r o t e i n a s e s . I t was also found that the a c t i v e s i t e regions were h i g h l y homologous between the a s p a r t y l proteinases examined. The secondary s t r u c t u r e p r e d i c t i o n methods of Chou and Fasman (1978b) and Cid et a l . (1982) i n d i c a t e d that chymosin, pepsin, p e n i c i l l o p e p s i n and Mucor miehei proteinase had a high proportion of B-sheet and that the a c t i v e s i t e a s p a r t i c a c i d residues were located i n B-turn r e g i o n s . Examination of the far-UV CD spectra of the a s p a r t y l proteinases i n d i -cated that changes i n the spectra occurred i n the n e u t r a l to a l k a l i n e pH range, the extent of change being proteinase dependent. Secondary s t r u c t u r e determination from far-UV CD s p e c t r a l data demonstrated that the a s p a r t y l proteinases had a high proportion of B-sheet. The propor-t i o n of B-sheet g e n e r a l l y decreased at pH values greater than 6.3. Results obtained from the near-UV CD spectra of the a s p a r t y l proteinases i n d i c a t e d a change i n spectra i n the n e u t r a l to a l k a l i n e pH range and may i m p l i c a t e the importance of aromatic groups to t e r t i a r y s t r u c t u r e s t a b i l i t y . - 181 -Bigelow average hydrophobicity showed no c l e a r d i s t i n c t i o n between a s p a r t y l and non-aspartyl p r o t e i n a s e s . Of the a s p a r t y l p r o t e i n a s e s , chymosin had the highest hydrophobicity value. The determination of hydrophobicity using f l u o r e s c e n t probes (CPA and ANS) again i n d i c a t e d no c l e a r d i s t i n c t i o n between a s p a r t y l and non-aspartyl p r o t e i n a s e s ; chymo-s i n showed r e l a t i v e l y high values for both probes. Charge r a t i o s i n d i -cated that the a s p a r t y l proteinases g e n e r a l l y had low proportions of basic amino acids and correspondingly high a c i d i c to ba s i c amino a c i d r a t i o s as compared to the non-aspartyl p r o t e i n a s e s . For a l l a s p a r t y l proteinases examined, the zeta p o t e n t i a l became more negative with i n c r e a s i n g pH. C a l c u l a t i o n of the a c c e s s i b l e surface area i n d i c a t e d that the a s p a r t y l proteinases had s i m i l a r a c c e s s i b l e surface areas that were higher than those of the non-aspartyl proteinases examined. The m i l k - c l o t t i n g to p r o t e o l y t i c a c t i v i t y r a t i o of the a s p a r t y l proteinases decreased with i n c r e a s i n g pH. Changes i n the f a r - and near-UV CD spectra were p a r t i a l l y c o r r e l a t e d to changes i n the m i l k -c l o t t i n g to p r o t e o l y t i c a c t i v i t y r a t i o . Of the a s p a r t y l p r o t e i n a s e s , chymosin showed the highest m i l k - c l o t t i n g to p r o t e o l y t i c a c t i v i t y r a t i o , whereas p e n i c i l l o p e p s i n , A s p e r g i l l u s s a i t o i proteinase and the non-a s p a r t y l proteinases had low r a t i o s . P r i n c i p a l component a n a l y s i s of various s t r u c t u r a l and i n t r i n s i c p r o p e r t i e s of a s p a r t y l and non-aspartyl proteinases i n d i c a t e d that a s p a r t y l proteinases formed d i s t i n c t groups; the non-aspartyl p r o t e i n -ases were not associated with any of these groups. 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P r e n t i c e - H a l l , Inc., Engle-wood C l i f f s , NJ. Zimmerman, S. S. and Scheraga, H. A. 1977. Local i n t e r a c t i o n s i n bends of p r o t e i n s . Proc. N a t l . Acad. S c i . U.S.A. 74: 4126. - 202 -APPENDIX Appendix 1. L i s t i n g of a For t r a n IV ve r s i o n of the computer program used by S i e g e l et a l . (1978) to determine secondary s t r u c -ture f r a c t i o n s from CD s p e c t r a l data. - 203 -2 C * * 3 C * T H I S PROGRAM C A L C U L A T E S PERCENTAGE H E L I X ACCORDING TO THE METHOD * 4 C * OF S I EGEL , STE INMETZ AND LONG. 1 9 8 0 . A N A L . B I O C H E M . 104 : 1 6 0 - 1 6 7 * 5 C * * g Q **************************************************** 7 C 8 C V A R I A B L E S : 9 C CORCTN : REFERENCE E L L I P T I C I T Y CORRESPONDING TO THE C TERM 10 C FRACTN : F R A C T I O N OF H E L I X AT S P E C I F I C WAVELENGTH 11 C H E L I X : F R A C T I O N OF H E L I X DETERMINED 12 C N : NUMBER OF WAVELENGTHS EXAMINED 13 C PROTN : P R O T E I N EXAMINED 14 C REFTHT : REFERENCE E L L I P T I C I T Y CORRESPONDING TO THE H E L I X 15 C SAMPLE : MEASURED E L L I P T I C I T Y OF SAMPLE P R O T E I N 16 C SHEET : F R A C T I O N OF SHEET DETERMINED 17 C SSODC : VAR IANCE CORRESPONDING TO THE C TERM 18 C SSOTHC : VAR IANCE CORRESPONDING TO THE H E L I X AND C TERM 19 C SSQTHT : STANDARD D E V I A T I O N OF RES IDUAL E L L I P T I C I T Y 2 0 C STHETA : V A R I A N C E CORRESPONDING TO THE H E L I X REFERENCE 21 C 22 C V A L U E S FOR CORCTN, R E F T H T , SSODC, SSOTHC, SSOTHT, 23 C AND STHETA ARE OBTA INED FROM THE PAPER BY 24 C S I E G E L ET A L . ( 1 9 8 0 ) 25 C 26 C * * ' DATA ENTRY * * 27 C - NAME OF P R O T E I N I S ENTERED F I R S T FORMAT 80A1 28 C - NUMBER OF WAVELENGTHS FORMAT 6 X . I 6 29 C - MEASUED E L L I P T I C I T I E S ARE ENTERED AT WAVE LENGTHS : 3 0 C 2 1 0 , 2 1 3 , 2 1 6 , 2 1 8 . 2 2 0 , 2 2 2 , 2 2 4 . 2 2 7 , 2 2 9 , 2 3 1 . 31 C 2 3 4 , 2 3 7 , AND 2 4 0 . 32 C - MEASURED E L L I P T I C I T Y ON ONE L I N E FORMAT 6 X . E 1 0 . 4 33 C - THE NEXT L I N E CONTA INS VALUES FOR CORCTN, R E F T H T , 34 C SSODC, SSOTHC, SSOTHT, AND STHETA FORMAT AS PER L I N E 54 35 C 36 INTEGER N, I 37 INTEGER P R 0 T N ( 8 O ) 38 REAL S A M P L E , C O R C T N , R E F T H T , F R A C T N , S S O D C , S T H E T A , S S O T H T , S S O T H C , H E L I X , 39 1 SUM0NE , SUMTWO,FACTOR,C0RFAC 4 0 D IMENS ION S A M P L E ( 5 0 ) , C 0 R C T N ( 5 O ) , R E F T H T ( 5 0 ) , F R A C T N ( 5 0 ) . S S O D C ( 5 0 ) , 41 1 S S O T H T ( 5 0 ) . S T H E T A ( 5 0 ) . F A C T O R ( 5 0 ) , C O R F A C ( 5 0 ) . S S O T H C ( 5 0 ) 42 SUMONE=0 4 3 SUMTWO=0 44 1 = 1 45 R E A D ( 5 , 1 )PROTN 46 1 FORMAT (80A1 ) 47 R E A D ( 5 , 1 0 ) N 4 8 10 F O R M A T ( 6 X , 1 6 ) 4 9 2 0 l F ( I . G T . N ) GO TO 5 0 5 0 R E A D ( 5 , 3 0 ) S A M P L E ( I ) 51 3 0 F O R M A T ( 6 X , E 1 0 . 4 ) 52 R E A D ( 5 , 4 0 ) C O R C T N ( I ) . R E F T H T ( I ) , S S O D C ( I ) , S S O T H T ( I ) . S T H E T A ( I ) . S S Q T H C 53 1 ( 1 ) 54 4 0 F 0 R M A T ( 6 X , E 1 0 . 4 , 2 X , E 1 0 . 4 , 2 X , E 1 O . 4 . 2 X , E 1 O . 4 , 2 X , E 1 O . 4 , 2 X , E 1 0 . 4 ) 5 5 F R A C T N ( I ) = ( S A M P L E ( I ) - C O R C T N ( I ) ) / R E F T H T ( I ) 56 F A C T O R ( I ) = ( S S O D C ( I ) + S S Q T H T ( I ) * * 2 ) / ( R E F T H T ( I ) * * 2 ) + ( ( S A M P L E ( I ) -57 1 C 0 R C T N ( I ) ) * * 2 ) * S T H E T A ( I ) / ( R E F T H T ( I ) * * 4 ) 5 8 C O R F A C ( I ) = F A C T O R ( I ) + 2 * ( S A M P L E ( I ) - C O R C T N ( I ) ) * S S Q T H C ( I ) / ( R E F T H T ( I - 204 -5 9 1 ) * * 3 ) 6 0 S U M 0 N E = S U M 0 N E + ( F R A C T N ( I ) / C 0 R F A C ( I ) ) 61 SUMTW0=SUMTW0+ (1 . 0/CORFAC ( I ) ) 6 2 1 = 1 + 1 6 3 GO TO 2 0 6 4 5 0 CONTINUE 6 5 H E L I X = ( S U M O N E / S U M T W O ) * 1 0 0 66 S H E E T = ( - 0 . 7 2 9 * H E L I X ) + 0 . 5 8 3 6 7 W R I T E ( 6 , 5 5 ) PROTN 68 55 F O R M A T ( ' 1 ' , 8 0 A 1 ) 6 9 W R I T E ( 6 , 6 0 ) H E L I X 7 0 6 0 F O R M A T ( ' 0 ' . ' T H E PERCENTAGE OF H E L I X 71 W R I T E ( 6 , 7 0 ) SHEET 72 7 0 F O R M A T ( ' 2 ' . ' T H E PERCENTAGE OF SHEET 73 STOP 74 END - 205 -Appendix 2. L i s t i n g of a Fortran IV computer program s i m i l a r to that used by Chang et a l . (1978) to determine the 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 l data. - 206 -•j c ***************************************** 2 C * * 3 C * T H I S PROGRAM C A L C U L A T E S THE SECONDARY STRUCTURE OF PROTE INS * 4 C * U S I N G THE METHOD OF CHANG ET A L . ( 1 9 7 8 ) A N A L . B I O C H E M 9 1 : 1 3 * 5 C * * g Q ******************************************************************** 7 C 8 C T H I S PROGRAM U T I L I Z E S THE SUBROUTINE DBEST WHICH I S AN I T E R A T I V E 9 C ALGOR ITHM WHICH I S S U B J E C T TO A NUMBER OF L INEAR EQUAL ITY 10 C C O N S T R A I N T S . T H I S PROGRAM I S DOCUMENTED IN THE UBC MATRIX MANUAL. 11 C C O N S T R A I N T S ARE ENTERED AS THE F I R S T SET OF DATA. TO HAVE THE 12 C SECONDARY STRUCTURE FRACT IONS TOTAL ONE, 1 . 0 ' S SHOULD CONST ITUTE 13 C THE F I R S T ROW OF THE REFERENCE E L L I P T I C I T Y MATR I X . A VALUE OF 14 C ONE SHOULD ALSO BE THE F I R S T VALUE OF THE OBSERVED E L L I P T I C I T Y 15 C DATA VECTOR. 16 C 17 C 18 C V A R I A B L E S : 19 C DA : I S A TWO D IMENS IONAL ARRAY CONTA IN ING THE REFERENCE 2 0 C E L L I P T I C I T I E S FOR THE FOUR SECONDARY STRUCTURE F R A C T I O N S . 21 C DB : I S A VECTOR C O N T A I N I N G THE OBSERVED E L L I P T I C I T I E S 22 C DETA : I S A CONVERGENCE C R I T E R I O N 23 C DRES : I S A VECTOR C O N T A I N I N G THE RES IDUES 24 C DX : I S A VECTOR CONTA ING THE SOLUT ION 25 C NHRS : I S THE NUMBER OF VECTORS CONTA IN ING THE OBSERVED 26 C E L L I P T I C I T I E S 27 C 28 R E A L * 8 D A . D B , D E T A , D T O L , D X , D R E S 29 D I M E N S I O N D A ( 1 0 0 , 2 0 ) , D B ( 1 0 0 , 1 ) . D X ( 1 0 0 . 1 ) , D R E S ( 1 0 0 , 1 ) 3 0 C 31 C READ IN AND WRITE OUT THE REFERENCE E L L I P T I C I T I E S 32 C THE REFERENCE E L L I P T I C I T I E S ARE OBTA INED FROM THE OUTPUT 33 C OF PROGRAM YANG WHICH GENERATES THE REFERENCE E L L I P T I C I T I E S 34 C 35 R E A D ( 5 , 1 0 ) ( ( D A ( I , J ) , J = 1 . 4 ) , I = 1 ,52 ) 36 10 F O R M A T ( 4 F 1 0 . 1 ) 37 W R I T E ( 6 , 2 0 ) 38 2 0 F O R M A T ( ' 1 ' , ' REFERENCE E L L I P T I C I T I E S ' / ) 39 W R I T E ( 6 , 3 0 ) 4 0 3 0 F O R M A T ( ' 0 ' , ' H E L I X B - S H E E T B -TURN R A N D O M ' / / ) 41 W R I T E ( 6 , 4 0 ) ( ( D A ( I , 0 ) , J = 1 , 4 ) , 1 = 1 , 5 2 ) 4 2 4 0 F O R M A T ( 1 X . 4 F 1 0 . 1 ) 4 3 NRHS=1 44 C 4 5 C READ IN OBSERVED E L L I P T I C I T I E S 2 4 0 - 1 9 0 N M , AT 1NM I N T E R V A L S 46 C 47 R E A D ( 5 , 5 0 ) ( ( D B ( I , J ) , I = 1 , 5 2 ) , d = 1 , N R H S ) 4 8 5 0 F O R M A T ( 1 0 F 1 0 . 1 ) 4 9 W R I T E ( 6 , 6 0 ) 5 0 6 0 F O R M A T ( / / / ' OBSERVED E L L I P T I C I T I E S 2 4 0 - 1 9 0 N M , 1NM I N T E R V A L S ' / ) 51 W R I T E ( 6 , 7 0 ) ( ( D B ( I , d ) , I = 1 . 5 2 ) , J = 1 , N R H S ) 5 2 7 0 F O R M A T ( 1 X , 1 0 F 1 0 . 1 ) 5 3 C 54 C C A L L SUBROUT INE DBEST TO F IND THE BEST LEAST SQUARES SOLUT ION 5 5 C 56 D T 0 L = 1 . D - 8 5 7 D E T A = 5 . D - 7 5 8 C A L L D B E S T ( D A , D B , N R H S , 1 , 5 2 , 4 , 1 0 0 , D E T A , D T O L , D X , D R E S ) - 207 -5 9 C 6 0 C WRITE OUT THE SOLUT ION 61 C 6 2 W R I T E ( 6 , 8 0 ) 6 3 8 0 F O R M A T ( / / / ' SECONDARY STRUCTURE F R A C T I O N S ' / ' H E L I X B - SHEET 6 4 1 B -TURN RANDOM ' / ) 6 5 W R I T E ( 6 , 9 0 ) ( ( D X ( I , d ) , 1 = 1 , 4 ) , J = 1.NRHS) 6 6 9 0 F O R M A T ( 1 X , 4 G 1 2 . 4 ) 6 7 W R I T E ( 6 , 1 0 0 ) ( ( D R E S ( I , d ) , I = 1 , 5 2 ) , J = 1 , N R H S ) 6 8 100 F O R M A T ( / / / ' R E S I D U A L S ' / ( 1 X , 1 0 G 1 2 . 4 ) ) 6 9 STOP 7 0 END - 208 -Appendix 3. L i s t i n g of a Fortran IV computer program to generate the reference e l l i p t i c i t y values for the various secondary s t r u c t u r e f r a c t i o n s ( " - h e l i x , B-sheet, B-turn and random f r a c t i o n s ) . - 2 0 9 -1 Q** ****************** it******,****,*********************************** 2 C * * 3 C * T H I S PROGRAM CORRECTS THE H E L I X REFERENCE E L L I P I T I C I T Y US ING * 4 C * THE WAVELENGTH DEPENDENT FACTOR AND NUMBER OF RE S IDUES PER H E L I X , * 5 C * FORMATS THE DATA FOR THE PROGRAM THAT C A L C U L A T E S THE SECONDARY * 6 C * STRUCTURE U S I N G THE METHOD OF CHANG ET A L . ( 1 9 7 8 ) A N A L . B I O C H E M * 7 C * 9 1 : 1 3 OR THE S I M P L E X - L E A S T SQUARES METHOD * 8 C * * 9 Q * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * 10 C 11 C V A R I A B L E S 12 C RES : NUMBER OF AMINO A C I D RE S IDUES PER H E L I X 13 C WDF : WAVELENGTH DEPENDENT FACTOR 14 C H E L I X : REFERENCE E L L I P I T I C I T Y - H E L I X 15 C BSHT : REFERENCE E L L I P I T I C I T Y - BETA SHEET 16 C BTURN : REFERENCE E L L I P I T I C I T Y - BETA TURN 17 C RAND : REFERENCE E L L I P I T I C I T Y - RANDOM C O I L 18 C E L L P : OBSERVED E L L I P I T I C I T I E S AT WAVELENGTHS 2 4 0 - 1 9 0 NM 19 C 2 0 C R E F E R E N C E V A L U E S FOR H E L I X , WAVELENGTH DEPENDENT FACTOR, BETA SHEET, 21 C BETA TURN AND RANDOM C O I L WERE OBTA INED FROM J . T . Y A N G ( U N P U B L I S H E D 22 C DATA ) AND ARE CONTA INED IN F I L E R E F E R E N C E . 23 C 24 C * * DATA ENTRY * * 25 C NUMBER OF RE S IDUES PER H E L I C A L REG ION I S ENTERED F I R S T , I F 26 C UNKNOWN 1 0 . 4 SHOULD BE ENTERED. T H I S I S AN AVERAGE VALUE 27 C BASESD ON X - R A Y DATA . 28 C REFERENCE DATA I S THEN ENTERED IN THE FOLLOWING ORDER: H E L I X , 29 C WAVELENGTH DEPENDENT FACTOR, BETA SHEET , BETA TURN AND 3 0 C RANDOM C O I L . 31 C - OBSERVED E L L I P T I C I T I E S ARE THE ENTERED AT 1NM I N T E R V A L S 2 4 0 32 C TO 190 NM ( 51 DATA P O I N T S ) . 33 C 34 C * P O S S I B L E CHANGES TO THE PROGRAM * 35 C - I F THE METHOD OF CHANG ET A L . ( 1 9 7 8 ) I S TO BE USED THEN THE 36 C DO LOOP C O N T A I N I N G STATEMENT 6 0 SHOULD BE CHANGED TO 1 = 1 , 5 2 . 37 C THE I N I T I A L ENTRY OF 1 .0 INTO THE VAR IOUS REFERENCE 38 C E L L I P T I C I T I E S ALLOWS FOR THE CONSTRA INT OF THE SUM OF THE 39 C SECONDARY FRACT IONS TO TOTAL ONE TO BE MET. 4 0 C - FOR THE S I M P L E X METHOD 1=2 , 52 SHOULD BE USED 41 C 4 2 C * * DATA OUTPUT * * 4 3 C - OUTPUT FROM T H I S PROGRAM COULD BE USED I N E I THER THE METHOD OF 44 C CHANG ET A L . ( 1 9 7 8 ) OR THE S I M P L E X METHOD DEPEND ING ON 1= S T A T E -4 5 C MENT. 46 C 47 D I M E N S I O N B S H T ( 1 0 0 ) , B T U R N ( 1 0 0 ) , H E L I X ( 1 0 0 ) , W D F ( 1 0 0 ) , R A N D ( 1 0 0 ) , E L L P ( 48 1 1 0 0 ) 4 9 R E A D ( 5 , 1 0 ) RES 5 0 10 F O R M A T ( F 4 . 1 ) 51 H E L I X ( 1 ) = 1 . 0 52 R E A D ( 5 , 2 0 ) ( H E L I X ( I ) , 1 = 2 , 5 2 ) 53 2 0 F O R M A T ( 1 0 F 1 0 . 0 / 1 0 F 1 0 . 0 / 1 0 F 1 0 . 0 / 1 0 F 1 0 . 0 / 1 0 F 1 0 . 0 / F 1 0 . 0 ) 54 W D F ( 1 ) = 1 . 0 55 R E A D ( 5 , 3 0 ) ( W D F ( I ) , I = 2 , 5 2 ) 5 6 3 0 F O R M A T ( 1 0 F 6 . 1 / 1 0 F 6 . 1 / 1 0 F 6 . 1 / 1 0 F 6 . 1 / 1 0 F 6 . 1 / F 6 . 1 ) 57 DO 4 0 1 = 2 , 5 2 5 8 H E L I X ( I ) = H E L I X ( I ) * ( 1 . 0 - ( W D F ( I ) / R E S ) ) - 210 -59 4 0 CONT INUE 6 0 B S H T ( 1 ) = 1 . 0 61 R E A D ( 5 , 2 0 ) ( B S H T ( I ) , 1 = 2 , 5 2 ) 6 2 B T U R N ( 1 ) = 1 . 0 6 3 R E A D ( 5 , 2 0 ) ( B T U R N ( I ) , 1 = 2 , 5 2 ) 6 4 RAND(1 ) = 1.0 6 5 R E A D ( 5 , 2 0 ) ( R A N D ( I ) , 1 = 2 , 5 2 ) 6 6 E L L P ( 1 ) = 1 . 0 6 7 R E A D ( 5 , 2 0 ) ( E L L P ( I ) , 1 = 2 , 5 2 ) 6 8 DO 6 0 1=1,52 6 9 W R I T E ( 6 , 5 0 ) H E L I X ( I ) , B S H T ( I ) , B T U R N ( I ) , R A N D ( I ) 7 0 5 0 F O R M A T ( 4 F 1 0 . 1 ) 71 6 0 CONT INUE 72 W R I T E ( 6 , 7 0 ) ( E L L P ( I ) , 1 = 1 , 5 2 ) 73 7 0 F O R M A T ( 1 0 F 1 0 . 1 ) 74 STOP 7 5 END - 211 -1 2 - 3 5 1 0 . . - 4 6 3 0 . . - 6 0 0 0 . , - 7 6 4 0 . , - 9 5 6 0 . , - 1 1 8 0 0 . . - 1 4 2 0 0 . , - 1 6 2 0 0 . . - 1 8 9 0 0 . , - 2 1 5 0 0 . : 3 - 2 4 0 0 0 . , - 2 6 7 0 0 . . - 2 9 0 0 0 . .. - 3 1 2 0 0 . , - 3 3 2 0 0 . , - 3 5 5 0 0 . , - 3 7 0 0 0 . . - 3 7 5 0 0 . . - 3 7 4 0 0 . , - 3 6 9 0 0 . ; 4 - 3 6 3 0 0 . , - 3 5 7 0 0 . . - 3 5 3 0 0 . , - 3 5 0 0 0 . . - 3 4 8 0 0 . , - 3 5 0 0 0 . , - 3 5 3 0 0 . , - 3 6 0 0 0 . , - 3 6 8 0 0 . , - 3 7 3 0 0 . ; 5 - 3 8 0 0 0 . , - 3 7 5 0 0 . . - 3 6 0 0 0 . . - 3 3 2 0 0 . , - 2 8 8 0 0 . , - 2 2 6 0 0 . . - 1 4 5 0 0 . . - 4 5 3 0 . , 7 0 6 0 . . 2 0 0 0 0 . ; 6 3 6 7 0 0 . . 4 7 6 0 0 . , 6 1 0 0 0 . , 7 3 2 0 0 . . 8 3 4 0 0 . , 9 1 0 0 0 . , 9 5 6 0 0 . , 9 7 0 0 0 . , 9 5 3 0 0 . , 9 0 7 0 0 . ; 7 8 3 8 0 0 . : 8 2 . 5 , 2 . 5 , 2 . 5 . 2 . 5 , 2 . 5 , 2 . 5 . 2 . 5 , 2 . 5 , 2 . 5 , 2 . 5 : 9 2 . 5 . 2 . 5 , 2 . 5 , 2 . 5 . 2 . 5 , 2 . 5 . 2 . 5 . 2 . 5 , 2 . 5 , 2 . 5 ; 10 2 . 6 , 2 . 6 , 2 . 7 , 2 . 7 , 2 . 8 . 2 . 9 , 3 . 0 . 3 . 1 , 3 . 1 , 3 . 2 ; 11 3 . 3 , 3 . 4 , 3 . 5 . 3 . 6 . 3 . 7 , 4 . 1 , 4 . 8 , 9 . 4 . 1 . 5 , 1 . 2 ; 12 1 . 8 , 2 . 1 , 2 . 2 . 2 . 3 , 2 . 3 , 2 . 4 , 2 . 4 , 2 . 4 . 2 . 4 , 2 . 4 ; 13 2 . 4 ; 14 9 7 0 . . 1 2 3 2 . , 1436 . . 154 1 . , 1564 . , 1578 . . 1 7 0 8 . , 1 8 7 6 . , 2 1 37 . , 1 3 1 3 . : 15 2 2 2 7 . , 2167- . , 2 0 2 4 . , 155 1 . , 1077 . , 4 5 4 .', - 5 9 6 . , - 9 8 1 . , T 1763 . , - 2 4 8 8 . , 16 - 3 2 0 8 . , - 3 8 5 8 . , - 4 6 5 7 . , - 5 2 5 7 . . - 5 8 1 4 . , - 6 2 4 0 . , - 6 4 8 6 . . - 6 4 9 2 . . - 6 3 2 4 . . - 5 7 0 7 . ; 17 - 4 9 2 1 . . - 4 5 0 4 . , - 3 9 8 1 . , - 3 2 0 1 . , - 2 2 7 8 . , - 1 196 . , 6 9 . , 9 5 7 . , 2 1 6 4 . , 4 8 8 2 . ; 18 8 1 0 7 . . 9 5 8 4 . , 1 0 2 8 7 . , 1 0 0 1 5 . , 8 7 4 8 . , 7 4 9 1 . , 4 9 5 5 . , 1 5 8 4 . , - 161 1 . . - 3 4 7 6 . : 19 - 5 8 0 4 . ; 2 0 1 8 4 0 . . 2 174 . . 2 6 8 4 . . 3 3 4 2 . . 3 8 8 1 . . 4 3 4 4 . , 5 1 3 3 . , 5 4 2 4 . , 6 9 5 7 . . 8 1 2 0 . ; 21 1 0 8 3 7 . , 1 3 3 1 9 . , 1 5 4 0 7 . . 1 7 2 5 7 . , 186 14 . . 2 0 4 8 8 . . 2 1 9 2 8 . . 2 1 3 5 0 . , 2 0 1 3 9 . . 1 8 7 2 8 . ; 22 1 7 0 5 7 . , 1 5 2 7 9 . . 1 3 6 3 4 . . 1204 1 . , 1 0 2 9 1 . . 8 9 5 1 . , 7 7 1 8 . , 6 9 7 3 . . 6 0 3 3 . . 5 1 5 6 . : 2 3 6 2 9 9 . , 7 3 0 1 . . 8 0 7 9 . . 1 0 6 4 1 . , 134 76 . '. 1 6 8 3 4 . , 19434 . . 2 1 2 9 0 . , 2 4 258 . . 2 2 6 0 9 . ; 24 1 6 8 8 1 . . 14 134 . . 2 4 9 0 . . - 1 1 0 7 7 . . - 2 5 6 3 4 . . - 3 8 6 8 1 . . - 5 0 7 8 1 . , - 6 1 3 2 4 . . - 7 0 2 8 4 . . - 7 5 5 5 6 . : 25 - 7 7 4 3 5 26 - 1 8 7 9 . , - 2 2 3 5 . , - 2 5 1 5 . . - 2 8 1 6 . . - 2 9 8 6 . , - 3 1 3 3 . , - 3 6 3 9 . , - 4 5 0 4 . , - 5 6 9 1 . , - 6 3 4 4 . ; 27 - 8 6 6 1 . , - 1 0 2 4 9 . , - 1 1 6 3 6 . , - 1 2 4 8 5 . . - 1 3 0 2 3 . , - 1 3 2 2 2 . , - 1 2 8 7 0 . ', - 1 2 8 9 0 . . - 1 2 28 1 . , - 1 1 7 8 2 . ; 28 - 1 1053 . , - 1 0 2 4 5 . . - 9 1 5 9 . , - 8 130 . . - 7 0 3 4 . . - 6 1 1 2 . . - 5 5 1 9 . . - 5 2 5 3 . . - 5 0 1 6 . , -540 .1 . : 2 9 - 6 6 6 6 . . - 7 9 9 0 . . - 8 9 7 4 . , - 1 1 0 1 8 . , - 1 3 4 4 0 . , - 1 6 4 7 2 . , - 2 0 1 3 3 . , - 2 3 4 4 7 . , - 2 5 8 4 0 . , - 3 2 0 5 4 . ; 3 0 - 3 7 2 0 6 . , - 3 7 2 0 8 . . - 3 5 0 6 4 . , - 3 1 5 2 8 . , - 2 6 3 8 3 . , - 2 1 2 1 6 . , - 1 5 2 4 3 . , - 8 0 5 9 . . - 2 8 5 . , 5 9 9 9 . ; 3 1 1 1985 . ; 32 33 34 35 36 37 This f i l e contains the reference e l l i p t i c i t i e s f o r «-helix ( l i n e s 2-7), 6-sheet ( l i n e s 14-19), 8-turn ( l i n e s 20-25) and random ( l i n e s 26-31). Lines 8 to 13 contain the wavelength dependent f a c t o r s . The reference e l l i p t i c i t i e s as w e l l as wavelength dependent f a c t o r s were obtained from Yang (unpublished d a t a ) . The average number of residues per h e l i x (n) i s entered i n l i n e one, while the observed e l l i p t i c i t i e s (240-190 nm) for the p r o t e i n i n question are entered i n t o l i n e s 32 to 37. - 212 -A p p e n d i x 4. L i s t i n g of a Fo r t r a n IV computer program which u t i l i z e s the simplex o p t i m i z a t i o n algorithm of Morgan and Deming (1974) to determine the 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 l data. - 213 -1 c ****************************************************************** 2 C * * 3 C * T H I S PROGRAM U T I L I Z E S THE MORGAN-DEMING S I M P L E X ALGORITHM * 4 C * TO C A L C U L A T E THE SECONDARY STRUCTURE OF PROTE INS US ING THE * 5 C * E L L I P T I C I T Y DATA FROM C D . DATA * 6 C * * 8 C 9 C 10 C V A R I A B L E S : 11 C A : RESPONSE VALUES FOR S I M P L E X USED IN THE TERM INAT ION 12 C PROCEDURE 13 C BLOC : BEST LOCAT ION IN THE S I M P L E X M A T R I X . ROW NO. 14 C CENT : CENTROID V A L U E S 15 C CP : P V A L U E S OF S T A R T I N G S I M P L E X MATR IX 16 C CO : 0 V A L U E S OF S T A R T I N G S I M P L E X MATR IX 17 C CR : CONTRACT ION OF R RESPONSES 18 C CRP : MASS IVE CONTRACT ION OF R RESPONSES 19 C CW : CONTRACT ION OF W RESPONSES 2 0 C CWP : MASS IVE CONTRACT ION OF W RESPONSES 21 C EOBS : VECTOR OF OBSERVED E L L I P T I C I T I E S 22 C E X P A N : E X P A N S I O N RESPONSES 23 C I S I M N : S I M P L E X NUMBER 24 C I V E R T : VERTEX NUMBER 25 C L I M I T : VECTOR OF L I M I T S FOR EACH FACTOR, LOWER THEN UPPER 26 C N : NUMBER OF SECONDARY STRUCTURE FRACT IONS 27 C NW : NUMBER OF WAVELENGTHS EXAMINED 28 C RC : MATR IX OF REFERENCE E L L I P T I C I T I E S . ROW REPRESENTS A 29 C WAVELENGTH 3 0 C R E F L E C : R E F E L E C T I O N RESPONSES 31 C SD : REPONSES FOR EACH S I M P L E X COND IT ION 32 C SS : S I M P L E X MATR IX 33 C WLOC : WORST LOCAT ION IN S I M P L E X M A T R I X , ROW NO. 34 C 35 C * * DATA ENTRY * * 36 C 37 C - F I R S T SET DATA ENTERED I S N AND NW, FORMAT 2 1 3 38 C - L I M I T S FOR EACH FACTOR ARE THEN ENTERED LOWER F I R S T , 39 C THEN U P P E R , FORMAT 8 F 6 . 0 4 0 C - OBSERVED E L L I P T I C I T I E S ARE ENTERED 7 PER L I N E FOR 7 L I N E S , 41 C 2 DATA FOR THE 8TH L I N E , TOTAL OF 51 DATA PO INTS FROM 2 4 0 42 C TO 190 NM, 1 NM I N T E R V A L S 4 3 C - REFERENCE E L L I P T I C I T I E S ARE ENTERED 4 PER L I N E (REPRESENT THE 44 C 4 SECONDARY STRUCTURES ) FOR 51 WAVELENGTHS 2 4 0 TO 190 NM 4 5 C FORMAT 4 E 1 2 . 4 . THESE ARE V A L U E S OBTA INED FROM THE REFERENCE 46 C PROGRAM. REFERENCE PROGRAM CORRECTS FOR H E L I X REFERENCE FOR 4 7 C C H A I N LENGTH DEPENDENCY. REFERENCE E L L I P T I C I T Y PROGRAM I S 4 8 C RUN PR IOR TO T H I S PROGRAM. 4 9 C 5 0 REAL L I M I T 51 INTEGER WLOC,BLOC 52 D I M E N S I O N S S ( 1 0 0 , 1 0 0 ) , C O ( 1 0 0 ) . C P ( 1 0 0 ) , L I M I T ( 1 0 0 ) , R C ( 1 0 0 , 1 0 0 ) 5 3 D I M E N S I O N E O B S ( 1 0 0 ) , S D ( 1 0 ) , C E N T ( 1 0 ) , A ( 1 0 ) 5 4 D I M E N S I O N R E F L E C ( 1 0 ) , C W ( 1 0 ) , E X P A N ( 1 0 ) , C R ( 1 0 ) , C R P ( 1 0 ) . C W P ( 1 0 ) 5 5 56 R E A D ( 5 , 1 0 ) N.NW 5 7 10 F 0 R M A T ( 2 I 3 ) 5 8 L=N-M - 214 -5 9 M=N*2 6 0 I S IMN=1 61 I VERT=L 6 2 R E A D ( 5 . 2 0 ) ( L I M I T ( I ) , 1 = 1 , M ) 6 3 2 0 F 0 R M A T ( 8 F 6 . 0 ) 6 4 R E A D ( 5 , 2 5 ) ( E 0 B S ( I ) , I = 1 , N W ) 6 5 25 F 0 R M A T ( 7 ( F 7 . O . 6 F 8 . O , / ) , F 7 . O , F 8 . O ) 6 6 W R I T E ( 6 , 2 5 ) ( E O B S ( I ) , 1 = 1 , N W ) 67 R E A D ( 5 , 2 7 ) ( ( R C ( I , d ) , d = 1 , N ) , I = 1 , N W ) 6 8 27 F 0 R M A T ( 4 E 1 2 . 4 ) 6 9 W R I T E ( 6 , 2 7 ) ( ( R C ( I , d ) , 0 = 1 , N ) , 1 = 1 , N W ) 7 0 W R I T E ( 6 , 3 0 ) N 71 3 0 F O R M A T C NUMBER OF V A R I A B L E S ' ' , 1 3 / / / ) 72 P = ( 1 / ( N * S Q R T ( 2 . 0 ) ) ) * ( ( N - 1 ) + S Q R T ( N + 1 . 0 ) ) 73 Q = ( 1 / ( N * S Q R T ( 2 . 0 ) ) ) * ( S Q R T ( N + 1 . 0 ) - 1 . 0 ) 74 d=1 75 DO 6 0 1=1.N 76 W R I T E ( 6 , 4 0 ) I 77 4 0 F O R M A T ( ' V A R I A B L E ' , 1 3 / ) 78 W R I T E ( 6 . 5 0 ) L I M I T ( d ) . L I M I T ( d + 1 ) 7 9 5 0 F O R M A T C LOWER L I M I T = ' . F 6 . 2 , ' UPPER L IM IT= 8 0 C P ( I ) = ( L I M I T ( d + 1 ) - L I M I T ( d ) ) * P + L I M I T ( d ) 81 C Q ( I ) = ( L I M I T ( d + 1 ) - L I M I T ( d ) ) * 0 + L I M I T ( d ) 82 d = d+2 8 3 6 0 CONT INUE 84 d=1 8 5 DO 7 0 1=1,M,2 86 S S ( 1 , d ) = L I M I T ( I ) 87 d = d+1 8 8 7 0 CONT INUE 8 9 K= 1 9 0 DO 9 0 1=1,N 91 DO 8 0 d = 2 . L 9 2 S S ( d . I ) = C 0 ( K ) 9 3 8 0 CONT INUE 94 K=K+ 1 9 5 9 0 CONT INUE 96 d=1 9 7 1 = 1 9 8 DO 100 K = 2 , L 9 9 S S ( K , d ) = C P ( I ) 100 1 = 1+1 101 d = d+1 102 100 CONT INUE 103 C 104 C C O N S T R A I N T I N G V A L U E S IN THE S I M P L E X SUCH THAT 105 C THE SUM OF THE FRACT IONS EQUALS ONE 106 C 107 DO 107 1=1,L 108 SUM=0 .0 109 DO 103 d = 1 , N 1 10 SUM=SUM+SS ( I , d ) 1 1 1 103 CONT INUE 112 I F ( S U M . E Q . O . O ) GOTO 107 1 13 DO 105 d = 1 , N 114 S S ( I , d ) = S S ( I . d ) / S U M 115 105 CONT INUE 1 16 107 CONT INUE - 215 -1 17 120 W R I T E ( 6 , 1 2 1 ) 1 18 121 F O R M A T ( 1 2 5 ( 1 H * ) ) 1 19 W R I T E ( 6 , 1 2 5 ) I S I M N 120 125 F O R M A T ( / ' S I M P L E X NUMBER ' , 1 3 / 1 X , 1 7 ( 121 W R I T E ( 6 , 1 2 7 ) ( ( S S ( I , J ) , J = 1 , N ) ,1 = 1 , L ) 122 127 F O R M A T ( 4 G 1 2 . 4 ) 123 124 C 125 - C C A L C U L A T I O N OF S I M P L E X RESPONSES 126 C 127 128 I F ( I S I M N . N E . 1 ) GOTO 153 129 DO 150 K = 1 , L 1 30 A L S D = 0 . 0 131 DO 140 1=1,NW 132 C A L C = 0 . 0 133 DO 130 J = 1 , N 134 R E S U L T = R C ( I , J ) * S S ( K , J ) 135 CALC=CALC+RESULT 136 130 CONT INUE 137 A L S D = A L S D + ( E O B S ( I ) - C A L C ) * * 2 138 140 CONT INUE 139 S D ( K ) = A L S D 140 150 CONT INUE 141 153 W R I T E ( 6 , 1 5 5 ) 142 155 F O R M A T ( / ' S I M P L E X R E S P O N S E S ' / ) 143 W R I T E ( 6 , 1 5 7 ) ( S D ( K ) , K = 1 , L ) 144 157 F 0 R M A T ( 6 G 1 2 . 4 ) 145 146 C 147 C F IND THE WORST AND THE BEST RESPONSE 148 C 149 150 WORST=SD(1) 151 WLOC= 1 152 DO 160 K = 2 , L 153 I F ( W O R S T . G T . S D ( K ) ) GOTO 160 154 WORST=SD(K) 155 WLOC=K 156 160 CONT INUE 157 BEST = SD( 1 ) 158 BL0C=1 159 DO 170 K = 2 . L 160 I F ( B E S T . L T . S D ( K ) ) GOTO 170 161 B E S T = S D ( K ) 162 BLOC=K 163 170 CONT INUE 164 165 C 166 C T E R M I N A T I O N PROCEDURE 167 C 168 169 DO 180 I = 1 , N 170 A ( I ) = S D ( I ) 171 180 CONT INUE 172 LAST=N 173 NLAST =N-1 174 DO 2 0 0 J = 1 , N L A S T - 2 1 6 -175 ML IM=LAST - 1 176 DO 190 I = 1 , M L I M 177 I F ( A ( I ) . L T . A ( 1 + 1 ) ) GOTO 190 178 T E M P = A ( I ) 179 A ( I ) = A ( I + 1 ) 180 A ( 1 + 1 ) = T E M P 181 190 CONT INUE 182 L A S T = L A S T - 1 183 2 0 0 CONT INUE 184 C R I T = . 0 5 * A ( 1 ) 185 D I F F = A B S ( A ( N ) - A ( 1 ) ) 186 I F ( D I F F . L E . C R I T ) GOTO 5 7 0 187 188 C 189 C C A L C U L A T E CENTRO ID 190 C 191 192 2 2 0 DO 2 4 0 J = 1 , N 193 C E N T ( d ) = 0 . 0 194 DO 2 3 0 1=1 . L 195 I F ( I . E Q . W L O C ) GOTO 2 3 0 196 C E N T ( J ) = C E N T ( d ) + S S ( I , d ) 197 2 3 0 CONT INUE 198 C E N T ( J ) = C E N T ( J ) / ( L - 1 ) 199 2 4 0 CONT INUE 2 0 0 W R I T E ( 6 , 2 4 5 ) 201 2 4 5 F O R M A T ( / ' CENTRO ID V A L U E S ' / ) 2 0 2 W R I T E ( 6 , 2 4 7 ) ( C E N T ( d ) , d = 1 , N ) 2 0 3 247 F 0 R M A T ( 6 G 1 2 . 4 ) 2 0 4 2 0 5 C 2 0 6 C C A L C U L A T E AVERAGE 2 0 7 C 2 0 8 2 0 9 A V G = 0 . 0 2 1 0 DO 2 5 0 I = 1 , L 21 1 I F ( I . E Q . W L O C ) GOTO 2 5 0 212 I F ( I . E Q . B L O C ) GOTO 2 5 0 2 1 3 AVG = AVG+SD( I ) 2 1 4 2 5 0 CONT INUE 2 1 5 A V G = A V G / ( L - 2 ) 2 1 6 W R I T E ( 6 , 2 5 5 ) AVG 2 1 7 2 5 5 F O R M A T ( / ' AVERAGE R E S P O N S E ' , G 1 2 . 4 / ) 2 1 8 2 1 9 C 2 2 0 C C A L C U L A T I O N OF THE R E F L E C T I O N POINT 221 C 2 2 2 2 2 3 I VERT= IVERT+1 2 2 4 W R I T E ( 6 , 2 7 0 ) I VERT 2 2 5 2 7 0 F O R M A T ( / ' VERTEX N U M B E R ' . 1 3 / ) 2 2 6 W R I T E ( 6 , 2 7 5 ) 2 2 7 2 7 5 F O R M A T C R E F L E C T I O N ' / ) 2 2 8 DO 2 8 0 1=1,N 2 2 9 R E F L E C ( I ) = C E N T ( I ) + 1 . 0 * ( C E N T ( I ) - S S ( W L O C . 2 3 0 2 8 0 CONT INUE 231 C A L L C H E C K ( R E F L E C . L I M I T , M ) 2 3 2 C A L L C O N S T ( R E F L E C , N ) - 21 7 -2 3 3 C A L L L E A S T ( E O B S , R C , R E F L E C , N W , N . T O T A L ) 2 3 4 SD (L+1 )=TOTAL 2 3 5 W R I T E ( 6 , 2 9 0 ) ( R E F L E C ( I ) , I = 1 , N ) , S D ( L + 1 ) 2 3 6 2 9 0 F O R M A T ( 4 G 1 2 . 4 , 6 X , ' R E S P O N S E * ' , G 1 2 . 4 ) 2 3 7 I F ( S D ( L + 1 ) . L T . S D ( B L O C ) ) GOTO 3 4 0 2 3 8 I F ( S D ( L + 1 ) . L T . A V G ) GOTO 3 7 0 2 3 9 I F ( S D ( L + 1 ) . L T . S D ( W L 0 C ) ) GOTO 4 3 0 2 4 0 241 C 2 4 2 C CONTRACT ION OF W 2 4 3 C 2 4 4 2 4 5 I VERT= IVERT+1 2 4 6 W R I T E ( 6 , 2 7 0 ) I VERT 2 4 7 W R I T E ( 6 , 3 0 0 ) 2 4 8 3 0 0 F O R M A T ( / ' CONTRACT ION OF W ' / ) 2 4 9 DO 3 1 0 1=1,N 2 5 0 C W ( I ) = C E N T ( I ) - 0 . 5 * ( C E N T ( I ) - S S ( W L O C , I ) ) 251 3 1 0 CONT INUE 2 5 2 C A L L C H E C K ( C W , L I M I T , M ) 2 5 3 C A L L C O N S T ( C W . N ) 2 5 4 C A L L L E A S T ( E O B S , R C , C W , N W , N , T O T A L ) 2 5 5 S D ( L + 2 ) = T 0 T A L 2 5 6 W R I T E ( 6 , 2 9 0 ) ( C W ( I ) , I = 1 , N ) , S D ( L + 2 ) 2 5 7 I F ( S D ( L + 2 ) . G T . S D ( W L O C ) ) GOTO 5 3 0 2 5 8 DO 3 2 0 1=1,N 2 5 9 S S ( W L O C , I ) = C W ( I ) 2 6 0 3 2 0 CONT INUE 261 S D ( W L 0 C ) = S D ( L + 2 ) 2 6 2 W R I T E ( 6 , 3 3 0 ) 2 6 3 3 3 0 F O R M A T ( / ' CONTRACTION-W R E P L A C E S W O R S T ' / / ) 2 6 4 I S IMN= I S IMN+1 2 6 5 GOTO 120 2 6 6 2 6 7 C 2 6 8 C E X P A N S I O N C A L C U L A T I O N 2 6 9 C 2 7 0 271 3 4 0 I VERT= IVERT+1 2 7 2 W R I T E ( 6 , 2 7 0 ) I VERT 2 7 3 W R I T E ( 6 . 3 5 0 ) 2 7 4 3 5 0 F O R M A T ( ' E X P A N S I O N ' / ) 2 7 5 DO 3 6 0 I = 1 ,N 2 7 6 E X P A N ( I ) = C E N T ( I ) + 2 . 0 * ( C E N T ( I ) - S S ( W L O C , I ) ) 2 7 7 3 6 0 CONT INUE 2 7 8 C A L L C H E C K ( E X P A N , L I M I T , M ) 2 7 9 C A L L C O N S T ( E X P A N . N ) 2 8 0 C A L L L E A S T ( E O B S , R C , E X P A N , N W , N , T O T A L ) 281 S D ( L + 2 ) = T 0 T A L 2 8 2 W R I T E ( 6 , 2 9 0 ) ( E X P A N ( I ) , 1 = 1 , N ) , S D ( L + 2 ) 2 8 3 I F ( S D ( L + 2 ) . L T . S D ( L + 1 ) ) GOTO 4 0 0 2 8 4 2 8 5 C 2 8 6 C R E F L E C T I O N R E P L A C E S WORST 287 C 2 8 8 2 8 9 3 7 0 DO 3 8 0 1=1,N 2 9 0 S S ( W L O C , I ) = R E F L E C ( I ) - 21 8 -291 3 8 0 CONT INUE 2 9 2 S D ( W L 0 C ) = S D ( L + 1 ) 2 9 3 W R I T E ( 6 , 3 9 0 ) 2 9 4 3 9 0 F O R M A T ( / ' R E F L E C T I O N R E P L A C E S W O R S T ' / / ) 2 9 5 I S IMN= I S IMN+1 2 9 5 GOTO 120 2 9 7 2 9 8 C 2 9 9 C E X P A N S I O N R E P L A C E S WORST 3 0 0 C 301 3 0 2 4 0 0 DO 4 1 0 1=1.N 3 0 3 S S ( W L O C , I ) = E X P A N ( I ) 3 0 4 4 1 0 CONT INUE 3 0 5 S D ( W L 0 C ) = S D ( L + 2 ) 3 0 6 W R I T E ( 6 , 4 2 0 ) 3 0 7 4 2 0 F O R M A T ( / ' E X P A N S I O N R E P L A C E S W O R S T ' / / ) 3 0 8 I S IMN= I S IMN+1 3 0 9 GOTO 120 3 1 0 31 1 C 3 1 2 C CONTRACT ION OF R 3 1 3 C 3 1 4 3 1 5 4 3 0 I VERT= IVERT+1 3 1 6 W R I T E ( 6 , 2 7 0 ) I VERT 3 1 7 W R I T E ( 6 , 4 4 0 ) 3 1 8 4 4 0 F O R M A T ( / ' CONTRACT ION OF R ' / ) 3 1 9 DO 4 5 0 1=1,N 3 2 0 C R ( I ) = C E N T ( I ) + 0 . 5 * ( C E N T ( I ) - S S ( W L O C , I ) ) 321 4 5 0 CONT INUE 3 2 2 C A L L C H E C K ( C R , L IMIT., M) 3 2 3 C A L L C O N S T ( C R . N ) 3 2 4 C A L L L E A S T ( E O B S , R C , C R , N W , N , T O T A L ) 3 2 5 S D ( L + 2 ) = T 0 T A L 3 2 6 W R I T E ( 6 , 2 9 0 ) ( C R ( I ) , 1 = 1 , N ) , S D ( L + 2 ) 3 2 7 I F ( S D ( L + 2 ) . G T . S D ( L + 1 ) ) GOTO 4 8 0 3 2 8 DO 4 6 0 1=1,N 3 2 9 S S ( W L O C , I ) = C R ( I ) 3 3 0 4 6 0 CONT INUE 331 S D ( W L 0 C ) = S D ( L + 2 ) 3 3 2 W R I T E ( 6 , 4 7 0 ) 3 3 3 4 7 0 F O R M A T ( / ' CONTRACT ION OF R R E P L A C E S WORST 3 3 4 I S IMN= IS IMN+1 3 3 5 GOTO 120 3 3 6 3 3 7 C 3 3 8 C C A L C U L A T I O N - MASS IVE CONTRACT ION OF R 3 3 9 C 3 4 0 34 1 4 8 0 I VERT= IVERT+1 3 4 2 W R I T E ( 6 , 2 7 0 ) I VERT 3 4 3 W R I T E ( 6 , 4 9 0 ) 3 4 4 4 9 0 F O R M A T ( / ' MAS S I VE CONTRACT ION OF R ' / ) 3 4 5 DO 5 0 0 I = 1 ,N 3 4 6 CRP ( I )=CENT( I ) +0 . 2 5 * ( C E N T ( I ) - SS(WLOC , I ) ) 3 4 7 5 0 0 CONT INUE 3 4 8 C A L L C H E C K ( C R P , L I M I T , M ) - 2 1 9 -3 4 9 C A L L C O N S T ( C R P . N ) 3 5 0 C A L L L E A S T ( E O B S , R C , C R P , N W . N , T O T A L ) 351 S D ( L + 3 ) = T 0 T A L 3 5 2 W R I T E ( 6 . 2 9 0 ) ( C R P ( I ) , 1 = 1 , N ) , S D ( L + 3 ) 3 5 3 I F ( S D ( L + 3 ) . G T . S D ( L + 1 ) ) GOTO 3 7 0 3 5 4 DO 5 1 0 1=1,N 3 5 5 S S ( W L O C . I ) = C R P ( I ) 3 5 6 5 1 0 CONT INUE 3 5 7 S D ( W L 0 C ) = S D ( L + 3 ) 3 5 8 W R I T E ( 6 , 5 2 0 ) 3 5 9 5 2 0 F O R M A T ( / ' MASS IVE CONTRACT ION OF R R E P L A C E S W O R S T ' / / ) 3 6 0 I S IMN= I S IMN+1 361 GOTO 120 3 6 2 3 6 3 C 3 6 4 C M A S S I V E CONTRACT ION OF W 3 6 5 C 3 6 6 3 6 7 5 3 0 I VERT= IVERT+1 3 6 8 W R I T E ( 6 , 2 7 0 ) I VERT 3 6 9 W R I T E ( 6 , 5 3 5 ) 3 7 0 5 3 5 F O R M A T ( / ' MAS S I VE CONTRACT ION OF W ' / ) 371 DO 5 5 0 1=1,N 3 7 2 C W P ( I ) = C E N T ( I ) - 0 . 2 5 * ( C E N T ( I ) - S S ( W L O C , I ) ) 3 7 3 5 5 0 CONT INUE 3 7 4 C A L L C H E C K ( C W P , L I M I T , M ) 3 7 5 C A L L C O N S T ( C W . N ) 3 7 6 C A L L L E A S T ( E O B S , R C , C W P , N W . N , T O T A L ) 3 7 7 SD (WLOC)=TOTAL 3 7 8 DO 5 5 5 1=1,N 3 7 9 S S ( W L O C , I ) = C W P ( I ) 3 8 0 5 5 5 CONT INUE 381 W R I T E ( 6 , 5 6 0 ) 3 8 2 5 6 0 F O R M A T ( ' MAS S I VE CONTRACT ION OF W R E P L A C E S W O R S T ' / / ) 3 8 3 W R I T E ( 6 , 2 9 0 ) ( C W P ( I ) , 1 = 1 , N ) , S D ( W L O C ) 3 8 4 I S IMN= I S IMN+1 3 8 5 GO TO 120 3 8 6 5 7 0 W R I T E ( 6 . 5 8 0 ) 3 8 7 5 8 0 F O R M A T ( ' 1 ' , ' THE BEST S O L U T I O N ' / / ) 3 8 8 W R I T E ( 6 , 5 9 0 ) 3 8 9 5 9 0 F O R M A T ( ' H E L I X B - S H E E T B -TURN R A N D O M ' / / ) 3 9 0 WR ITE( 6 , 6 0 0 ) ( S S ( B L O C , J ) , <J= 1 ,N ) 391 6 0 0 F 0 R M A T ( 6 G 1 2 . 4 ) 3 9 2 STOP 3 9 3 END 3 9 4 3 9 5 C 3 9 6 C T H I S SUBROUT INE C A L C U L A T E S THE LEAST SQUARES D I F F E R E N C E 3 9 7 C 3 9 8 3 9 9 C V A R I A B L E S : 4 0 0 C S IMCO : S I M P L E X C O E F F I C I E N T S 401 C TOTAL : SUM OF SQUARES OF THE D I F F E R E N C E BETWEEN CALCULATED 4 0 2 c AND OBSERVED V A L U E S 4 0 3 c 4 0 4 SUBROUT INE L E A S T ( E O B S , R C , S I M C O , N W , N , T O T A L ) 4 0 5 D I M E N S I O N E O B S ( 1 0 0 ) , S I M C O ( 1 0 0 ) , R C ( 1 0 0 , 1 0 0 ) 4 0 6 T O T A L = 0 . 0 - 220 -4 0 7 DO 7 5 0 1=1,NW 4 0 8 R E S U L T = 0 . 0 4 0 9 DO 7 0 0 K=1 ,N 4 1 0 R E S U L T = R E S U L T + ( R C ( I , K ) * S I M C O ( K ) ) 4 11 7 0 0 CONT INUE 4 1 2 T O T A L = T O T A L + ( R E S U L T - E O B S ( I ) ) * * 2 4 1 3 7 5 0 CONT INUE 4 1 4 RETURN 4 1 5 END 4 1 6 4 1 7 C 4 1 8 C T H I S SUBROUT INE CHECKS FOR BOUNDARY V I O L A T I O N S - I F A FACTOR 4 1 9 C I S L E S S THAN THE LOWER L I M I T IT I S SET TO THE LOWER L I M I T . I F 4 2 0 C IT EXCEEDS THE UPPER L I M I T IT I S SET TO THE UPPER L I M I T 421 C 4 2 2 C V A R I A B L E S : 4 2 3 C FAC : S I M P L E X C O E F F I C I E N T S 4 2 4 C L I M I T : L I M I T S FOR THE FACTORS 4 2 5 C NUM : COUNTER 4 2 6 C I N D I C : IND ICATOR OF BOUNDARY V I O L A T I O N 4 2 7 C I F L A G : VECTOR C O N T A I N I N G FACTORS EXCEED ING BOUNDAR IE S . 4 2 8 C 4 2 9 4 3 0 SUBROUT INE C H E C K ( F A C , L I M I T , M ) 431 REAL L I M I T 4 3 2 D IMENS ION F A C ( 1 0 ) , L I M I T ( 2 0 ) , I F L A G ( 1 0 ) 4 3 3 I N D I C = 0 4 3 4 K= 1 4 3 5 J=1 4 3 6 DO 8 5 0 1 = 1 ,M ,2 4 3 7 I F ( F A C ( K ) . L T . L I M I T ( I ) ) GOTO 8 1 0 4 3 8 I F ( F A C ( K ) . G T . L I M I T ( 1 + 1 ) ) GOTO 8 2 0 4 3 9 K = K+1 4 4 0 GOTO 8 5 0 441 8 1 0 F A C ( K ) = L I M I T ( I ) 4 4 2 I F L A G ( J ) = K 4 4 3 IND IC=1 4 4 4 K = K+1 4 4 5 J = d+1 4 4 6 NUM=J-1 4 4 7 GOTO 8 5 0 4 4 8 8 2 0 F A C ( K ) = L I M I T ( I + 1 ) 4 4 9 I F L A G ( J ) =K 4 5 0 IND IC=1 451 K = K+ 1 4 5 2 J = J+1 4 5 3 NUM=J - 1 4 5 4 8 5 0 CONT INUE 4 5 5 I F ( I N D I C . E O . O ) GOTO 9 0 0 4 5 6 W R I T E ( 6 , 8 6 0 ) 4 5 7 8 6 0 F O R M A T C BOUNDARY V I O L A T I O N - THE FOLLOWING FACTORS EXCEEDED 4 5 8 1 L I M I T ' / ) 4 5 9 DO 8 7 0 J=1 ,NUM 4 6 0 W R I T E ( 6 , 8 6 5 ) I F L A G ( J ) 461 8 6 5 F O R M A T ( 1 X , 1 3 / ) 4 6 2 8 7 0 CONT INUE 4 6 3 9 0 0 RETURN 4 6 4 END - 221 -4 6 5 4 6 6 C 4 6 7 C T H I S SUBROUT INE CONSTRA INTS THE FRACT IONS SUCH THAT 4 6 8 C THE SUM OF THE FRACT IONS EQUALS ONE 4 6 9 C 4 7 0 C V A R I A B L E S : 471 C FRAC : S I M P L E X C O E F F I C I E N T S 4 7 2 C SUM : TOTAL OF S I M P L E X C O E F F I C I E N T S 4 7 3 C 4 7 4 4 7 5 SUBROUT INE C O N S T ( F R A C , N ) 4 7 6 D IMENS ION F R A C ( 1 0 ) 4 7 7 SUM=0 .0 4 7 8 DO 9 0 5 1=1,N 4 7 9 SUM=SUM+FRAC( I ) 4 8 0 9 0 5 CONT INUE 481 I F ( S U M . E Q . O . O ) GOTO 9 2 0 4 8 2 DO 9 1 0 1=1,N 4 8 3 F R A C ( I ) = F R A C ( I ) / S U M 4 8 4 9 1 0 CONT INUE 4 8 5 9 2 0 RETURN 4 8 6 END - 222 -A p p e n d i x 5. L i s t i n g of a Fortran IV computer program for the diagonal p l o t method. - 223 -1 c *********************************** 2 C * * 3 C * D IAGONAL PLOT PROGRAM U S I N G THE METHOD OF B E Y N 0 N ( 1 9 8 2 ) * 4 C * B IOCHEM I STRY MICROCOMPUTER GROUP 7 : 11 * 5 C * * g Q * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * 7 C 8 C V A R I A B L E S : 9 C B : TWO D IMENS IONAL ARRAY CONTA IN ING THE PR IMARY SEQUENCE 10 C OF P R O T E I N 1 11 C C : TWO D IMENS IONAL ARRAY CONTA IN ING THE PR IMARY SEQUENCE 12 C OF P R O T E I N 2 13 C D : VECTOR C O N T A I N I N G THE PR IMARY SEQUENCE DF P R O T E I N 1 14 C E : VECTOR CONTA IN ING THE PR IMARY SEQUENCE OF P R O T E I N 2 15 C I M I N : INTEGER VALUE FOR XMIN 1G C IMAX : INTEGER VALUE FOR XMAX 17 C N : NUMBER OF AMINO AC IDS TO BE COMPARED IN THE SEQUENCE 18 C P : NUMBER OF L I N E S OF DATA 19 C XM IN : START OF SEQUENCE TO BE COMPARED 2 0 C XMAX : END OF SEQUENCE TO BE COMPARED 21 C 2 2 C * * DATA ENTRY * * 23 C - AMINO A C I D SEQUENCE WAS D I G I T I Z E D US ING THE FOLLOWING CODE: 24 C 1 ( A L A ) , 2 ( A R G ) , 3 ( A S N ) , 4 ( A S P ) , 5 ( C Y S ) , 6 ( G L N ) , 7 ( G L U ) . 8 ( G L Y ) , 25 C 9 ( H I S ) , 1 0 ( I L E ) , 1 1 ( L E U ) , 1 2 ( L Y S ) . 1 3 ( M E T ) , 1 4 ( P H E ) , 1 5 ( P R 0 ) , 26 C 1 6 ( S E R ) , 1 7 ( T H R ) , 1 8 ( T R P ) , 1 9 ( T Y R ) , AND 2 0 ( V A L ) . 27 C - 2 0 DATA P O I N T S ARE ENTERED PER L I N E , FORMAT 2 0 1 2 28 C - SUBROUT INES A X I S AND SYMBOL ARE DOCUMENTED IN THE UBC PLOT 29 C MANUAL 3 0 C 31 C * P O S S I B L E CHANGES TO THE PROGRAM * 32 C XM IN ,XMAX W ILL HAVE TO BE CHANGED DEPEND ING ON THE REGION 33 C TO BE EXAM INED. 34 C - NAMES IN THE 2 A X I S SUBROUT INES WILL HAVE TO BE CHANGED 35 C DEPEND ING ON THE PROTE INS TO BE COMPARED ( I . E . P R O T E I N 1 36 C AND P R O T E I N 2 ) . 37 C - THE NUMBERS D I R E C T L Y FOLLOWING THE NAMES WILL HAVE TO BE 38 C CHANGED S I N C E THESE NUMBERS CORRESPOND TO THE NUMBER OF 39 C L E T T E R S IN THE P R O T E I N NAME. N . B . BLANKS MUST BE INCLUDED 4 0 C I N THE NUMBER COUNT. THE N E G A T I V E S IGN SHOULD BE KEPT FOR 41 C THE F I R S T A X I S SUBROUT INE . 42 C - I N I T I A L L Y FOR THE THE F I R S T COMPARISONS THE XMIN IN THE A X I S 43 C SUBROUT INES SHOULD BE R E P L A C E D BY 0 . 0 ( E . G . FOR COMPARISONS 44 C OF R E S I D U E S 1 TO 8 5 ) . FOR SUBSEQUENT COMPARISONS 0 . 0 SHOULD 4 5 C BE R E P L A C E D IN THE A X I S SUBROUT INES ( E . G . RE S I DUES 85 TO 1 7 0 ) . 4 6 C T H I S I S DONE TO F A C I L I T A T E THE SYNCHRON IZ ING OF RES IDUE NUMBER 47 C WITH PLOT NUMBER. 4 8 C - I N I T I A L L Y JA AND KA SHOULD BE SET TO 1 AND 0 , R E S P E C T I V E L Y . 4 9 C HOWEVER, FOR COMPARISONS OF RE S IDUES LATER ON IN THE SEQUENCE 5 0 C J A AND KA SHOULD BE RESET TO O AND - 1 , R E S P E C T I V E L Y ( E . G . R E S -51 C DUES 8 5 TO 170 J A = 0 , KA=-1 HOWEVER FOR RES IDUES 85 TO 170 52 C J A = 0 , K A = - 1 ) . A G A I N T H I S I S DONE TO SYNCHRONIZE RES IDUE 53 C NUMBER TO PLOT NUMBER. 54 C 55 C 56 INTEGER B ( 5 0 0 , 5 0 0 ) , C ( 5 0 0 . 5 0 0 ) , D ( 5 0 0 ) , E ( 5 0 0 ) , P 57 R E A D ( 5 , 1 0 ) N .P 58 10 F 0 R M A T ( 2 I 3 ) - 224 -5 9 R E A D ( 5 , 1 5 ) XM IN .XMAX 6 0 15 F 0 R M A T ( 2 F 6 . O ) 61 R E A D ( 5 , 2 0 ) ( ( B ( I , J ) , d = 1 . 4 0 ) , I = 1 .P ) 6 2 R E A D ( 5 , 2 0 ) ( ( C ( I , J ) , J = 1 , 4 0 ) , I = 1 , P ) 6 3 2 0 F 0 R M A T ( 4 O I 2 ) 6 4 1 = 1 6 5 DO 3 0 K = 1 , P 6 6 DO 3 0 M=1 , 40 6 7 D ( I ) = B ( K , M ) 6 8 E ( I ) = C ( K , M ) 6 9 1=1+1 7 0 3 0 CONT INUE 71 C A L L A X I S ( 0 . 0 , 0 . 0 . ' P R O T E I N 1 ' , - 9 , 9 . 0 , 0 . 0 , X M I N , 1 0 . 0 ) 72 C A L L A X I S ( 0 . 0 , 0 . 0 , ' P R O T E I N 2 ' , 9 , 9 . 0 , 9 0 . 0 , X M I N , 1 0 . 0 ) 73 I M I N = I F I T ( X M I N ) 74 I M A X = I F I T ( X M A X ) 75 J A = 0 76 DO 5 0 J = I M I N , I M A X 77 KA=-1 78 DO 4 0 K = I M I N , I M A X 79 KA=KA+1 8 0 I F ( • ( K ) . N E . E ( J ) ) GOTO 4 0 81 X = ( F L O A T ( K A ) - 0 . 0 ) / 1 0 . 0 82 Y = ( F L O A T ( J A ) - 0 . 0 ) / 1 0 . 0 83 C A L L S Y M B O L ( X . Y , 0 . 0 7 , 1 1 , 0 . 0 , - 1 ) 8 4 4 0 CONT INUE 8 5 JA=JA+1 86 5 0 CONT INUE 87 C A L L PLOTND 8 8 STOP 8 9 END - 225 -Appendix 6. L i s t i n g of a Fo r t r a n IV computer program for the p r e d i c t i o n of secondary s t r u c t u r e using the hydrophobicity p r o f i l e method of Cid et a l . (1982). - 226 -^ Q **************************************************** 2 C * * 3 C * SECONDARY STRUCTURE U S I N G HYDROPHOB IC ITY SCALE * 4 C * OF C I D ET A L . ( 1 9 8 2 ) FEBS L E T T E R S 1 5 0 : 2 4 7 * 5 C * * g Q **************************************************** 7 C 8 C V A R I A B L E S : 9 C A : BUFFER VECTOR 10 C C : TWO D IMENS IONAL ARRAY CONTA IN ING THE PR IMARY AMINO 11 C AC ID SEQUENCE OF THE P R O T E I N 12 C F : VECTOR C O N T A I N I N G THE BULK HYDROPHOB IC ITY VALUES FOR 13 C THE PR IMARY SEQUENCE. 14 C IMAX : INTEGER VALUE OF THE LAST RES IDUE OF THE SEQUENCE 15 C I M I N : INTEGER VALUE OF THE F I R S T RES IDUE OF THE SEQUENCE 16 C XMAX : LAST RES IDUE OF THE SEQUENCE TO BE EXAMINED 17 C XM IN : F I R S T RES IDUE OF THE SEQUENCE TO BE EXAMINED 18 C 19 C * * DATA ENTRY * * 2 0 C - SUBROUT INES A X I S AND SYMBOL ARE DOCUMENTED IN THE UBC PLOT 21 C MANUAL. 22 C - S I N C E A MAXIMUM X A X I S OF 10 INCHES WAS USED ONLY A C E R T A I N 23 C PORT ION OF ANY SEQUENCE CAN BE PLOTTED USUALLY C A . 100 R E S I D U E S . 24 C TO D E L I M I T THE RANGE OF RE S IDUES TO BE EXAMINED XM IN AND XMAX 25 C HAVE TO BE CHANGED ( E . G . I F RES IDUES 1 TO 100 ARE TO BE PLOTTED 26 C XMIN=0 AND X M A X = 1 0 0 ) . FOR SUBSEQUENT COMPARISONS XMIN SHOULD BE 27 C ST TO 1 0 0 , 2 0 0 , ETC . P L O T T I N G WILL START AT RES IDUE XM IN+1 . 28 C - PR IMARY AMINO A C I D SEQUENCE I S ENTERED 16 DATA PER L I N E 1615 29 C - AMINO AC ID SEQUENCE I S D I G I T I Z E D US ING THE FOLLOWING CODE: 3 0 C 1 ( A L A ) , 2 ( A R G ) , 3 ( A S N ) , 4 ( A S P ) . 5 ( C Y S ) , 6 ( G L N ) , 7 ( G L U ) , 8 ( G L Y ) , 31 C 9 ( H I S ) , 1 0 ( I L E ) , 1 1 ( L E U ) . 1 2 ( L Y S ) , 1 3 ( M E T ) , 1 4 ( P H E ) , 1 5 ( P R 0 ) , 32 C 1 6 ( S E R ) , 1 7 ( T H R ) , 1 8 ( T R P ) , 1 9 ( T Y R ) AND 2 0 ( V A L ) . 33 C 34 INTEGER A . C 35 D I M E N S I O N A ( 5 0 0 ) , C ( 5 0 , 5 0 ) . F ( 5 0 0 ) 36 R E A D ( 5 , 10 ) M,N 37 10 F O R M A T ( 2 I 3 ) 38 R E A D ( 5 , 1 5 ) XM IN,XMAX 39 15 F O R M A T ( 2 F 6 . 0 ) 4 0 R E A D ( 5 , 2 0 ) ( ( C ( I , J ) , d =1 . 1 6 ) , I = 1.N) 41 2 0 F 0 R M A T ( 1 6 I 5 ) 42 K= 1 4 3 DO 3 0 1=1.N 44 DO 3 0 d = 1 , 1 6 4 5 A ( K ) = C ( I , J ) 4 6 K=K+1 47 3 0 CONT INUE 4 8 C A L L A X I S ( 0 . 0 , 0 . 0 , ' R E S I D U E NUMBER ' , - 14 , 7 . 0 , 0 . 0 , X M I N , 1 0 . 0 ) 4 9 C A L L A X I S ( 0 . 0 , 0 . 0 , ' B U L K H Y D R O P H O B I C I T Y ' , 1 9 . 6 . 0 , 9 0 . O , 1 0 . O . 1 . O ) 5 0 DO 4 0 K=1,M 51 I F ( A ( K ) . E Q . 1 ) F ( K ) = 1 2 . 2 8 52 I F ( A ( K ) . E Q . 2 ) F ( K ) = 1 1 . 4 9 53 I F ( A ( K ) . E Q . 3 ) F ( K ) = 1 1 . 0 0 54 I F ( A ( K ) . E Q . 4 ) F ( K ) = 1 0 . 9 7 5 5 I F ( A ( K ) . E Q . 5 ) F ( K ) = 1 4 . 9 3 56 I F ( A ( K ) . E Q . 6 ) F ( K ) = 1 1 . 2 8 57 I F ( A ( K ) . E Q . 7 ) F ( K ) = 1 1 . 1 9 58 I F ( A ( K ) . E Q . 8 ) F ( K ) = 1 2 . 0 1 - 22 7 -5 9 I F ( A ( K ) . E Q . 9 ) F ( K ) = 1 2 . 8 4 6 0 I F ( A ( K ) . E Q . 1 0 ) F ( K ) = 1 4 . 7 7 61 I F ( A ( K ) . E Q . 1 1 ) F ( K ) = 1 4 . 1 0 6 2 I F ( A ( K ) . E Q . 1 2 ) F ( K ) = 1 0 . 8 0 6 3 I F ( A ( K ) . E Q . 1 3 ) F ( K ) = 1 4 . 3 3 64 I F ( A ( K ) . EQ. 14) F ( K ) = 1 3 . 4 3 6 5 I F ( A ( K ) . E Q . 1 5 ) F ( K ) = 1 1 . 1 9 66 I F ( A ( K ) . E Q . 1 6 ) F ( K ) = 1 1 . 2 6 67 I F ( A (K ) . E Q . 1 7 ) F ( K ) = 1 1 . 6 5 6 8 I F ( A ( K ) . EQ. 18 ) F ( K ) = 1 2 . 9 5 6 9 I F ( A ( K ) . E Q . 1 9 ) F ( K ) = 1 3 . 2 9 7 0 I F ( A ( K ) . E 0 . 2 O ) F ( K ) = 1 5 . 0 7 7 1 4 0 CONT INUE 72 L= 1 73 I M I N = I F I T ( X M I N ) 74 I M A X = I F I T ( X M A X ) 75 IM IN= IM IN+1 76 DO 5 0 1 = 3 0 1 , 3 6 9 77 Y = ( F ( I ) - 1 0 . 0 ) 78 X = ( F L 0 A T ( L ) - 0 . 0 ) / 1 0 . 0 79 I F ( L . E Q . 1 ) C A L L S Y M B O L ( X , Y . 0 . 0 7 , 1 1 , 0 . 0 , - 1 ) 8 0 C A L L S Y M B O L ( X , Y . 0 . 0 7 , 1 1 , 0 . 0 , - 2 ) 81 L=L+1 82 5 0 CONT INUE 83 C A L L PLOTND 84 STOP 8 5 END - 22 8 -Appendix 7. L i s t i n g of a Fortran IV computer program for the determina-t i o n of mean residue e l l i p t i c i t y from CD s p e c t r a l data. - 229 -2 C * * 3 C * T H I S PROGRAM C A L C U L A T E S MOLECULAR E L L I P T I C I T Y US ING THE * 4 C * JASCO J - 5 0 0 A SPECTROPOLAR IMETER * 5 C * * 6 C * INPUT FROM THE TEXAS INSTRUMENTS DATA A C Q U I S I T I O N UN IT * 7 C * * 3 Q ******************************************************** ****** 9 C 10 C 11 C V A R I A B L E S : 12 C B A S E L N : B A S E L I N E VOLTAGE READ ING 13 C CDATA : SAMPLE VOLTAGE CORRECTED FOR B A S E L I N E 14 C CONC : CONCENTRAT ION OF THE SAMPLE IN G M / C M * * 3 15 C DATA : VOLTAGE READ ING FOR THE SAMPLE 16 C DPBL : NUMBER OF DATA READ INGS FOR EACH B A S E L I N E 17 C E L L P : E L L I P T I C I T Y IN DEGREES 18 C MOLELP : MOLECULAR E L L I P T I C I T Y IN DEGREES C M * * 2 / D E C I M 0 L E 19 C MOLWT : MOLECULAR WEIGHT I N GM/MOLE 2 0 C NUMBAS : NUMBER OF B A S E L I N E S USED 21 C P A T H L G : PATHLENGTH OF THE CUVETTE IN CM 22 C PEAKHT : PEAK HE IGHT IN CM 2 3 C SENSET : S E N S I T I V I T Y IN M I L L I D E G R E E S / C M 24 C WAVELG : WAVELENGTH IN NM 25 INTEGER D P B L , N U M B A S . M O L W T , I , J , K , N , Y 26 INTEGER P R 0 T N ( 8 O ) 27 REAL B A S E L N , D A T A . C D A T A , P E A K H T , E L L P . M O L E L P , P A T H L G 28 REAL C O N C , S E N S E T , W A V E L G 29 D I M E N S I O N B A S E L N ( 1 1 , 1 0 ) , D A T A ( 1 1 , 1 0 ) , M O L E L P ( 2 0 0 ) 3 0 R E A D ( 5 , 1 0 ) N U M B A S 31 10 F O R M A T ( 1 2 ) 32 P=1 33 15 I F ( P . G T . N U M B A S ) GOTO 2 1 0 34 W R I T E ( 6 , 2 0 ) 35 2 0 F O R M A T ( ' 1 ' , ' I N F O R M A T I O N CONCERNING THE CD RUN : ' / / ) 36 R E A D ( 5 , 3 0 ) S E N S E T 37 3 0 F O R M A T ( F 6 . 2 ) 38 W R I T E ( 6 , 4 0 ) SENSET 39 4 0 FORMAT( ' O ' , ' S E N S I T I V I T Y M I L L I D E G R E E S / C M : ' , F 6 . 3 ) 4 0 R E A D ( 5 , 5 0 ) PATHLG 41 5 0 F O R M A T ( F 6 . 3 ) 42 W R I T E ( 6 , 6 0 ) PATHLG 4 3 6 0 F O R M A T ( ' 0 ' , ' P A T H L E N G T H OF THE CUVETTE IN CM : ' , F 6 . 3 ) 44 M=6 4 5 R E A D ( 5 , 7 0 ) ( ( B A S E L N ( I , v J ) , J = 1 , 1 0 ) , I = 1 , M ) 4 6 7 0 F O R M A T ( 1 0 F 1 0 . 2 ) 47 R E A D ( 5 , 8 0 ) D P B L 4 8 8 0 F O R M A T ( 1 2 ) 4 9 Y=1 5 0 8 5 I F ( Y . G T . D P B L ) GOTO 2 0 0 51 W A V E L G = 2 4 0 . 0 52 R E A D ( 5 , 9 0 ) PROTN 53 9 0 F O R M A T ( 8 0 A 1 ) 54 W R I T E ( 6 , 1 0 0 ) PROTN 55 100 F O R M A T ( ' 0 ' , ' P R O T E I N : ' , 8 0 A 1 ) 56 R E A D ( 5 , 1 1 0 ) MOLWT 57 110 F O R M A T ( 1 6 ) 5 8 W R I T E ( 6 , 1 2 0 ) MOLWT - 230 -5 9 120 F 0 R M A T ( ' 0 ' , ' M O L E C U L A R WEIGHT GM/MOLE : ' , I 6 ) 6 0 R E A D ( 5 , 1 3 0 ) CONC 61 130 F O R M A T ( E 9 . 3 ) 62 W R I T E ( 6 , 1 4 0 ) CONC 6 3 140 F O R M A T ( ' 0 ' , ' C O N C E N T R A T I O N G M / C M * * 3 : ' . E 9 . 3 ) 6 4 W R I T E ( 6 , 1 5 0 ) 6 5 150 F O R M A T ( ' 0 ' , 4 X . ' D A T A ' ,8X , ' B A S E L N ' , 8 X , ' C D A T A ' , 9 X , ' P E A K H T ' , 8 X , 6 6 1 ' E L L P ' , 7 X , ' M O L E L P ' , 8 X . ' W A V E L G ' / / / ) 6 7 R E A D ( 5 , 7 0 ) ( ( D A T A ( I , J ) , J = 1 , 1 0 ) , 1 = 1 , M ) 6 8 K= 1 6 9 DO 170 1=1,M 7 0 DO 160 <J= 1 , 10 71 C D A T A = D A T A ( I , J ) - B A S E L N ( I , J ) 72 P E A K H T = ( C D A T A + 0 . 0 7 ) / 2 . 4 2 73 P E A K H T = P E A K H T / 1 0 . 0 74 E L L P = ( S E N S E T * P E A K H T ) / 1 0 0 0 . 0 75 M O L E L P ( K ) = ( E L L P * M O L W T ) / ( 1 0 . 0 * P A T H L G * C O N C ) 76 W R I T E ( 6 , 1 5 5 ) D A T A ( I , J ) , B A S E L N ( I , J ) , C D A T A , P E A K H T , E L L P . M O L E L P ( K ) 77 1WAVELG 78 155 F O R M A T ( ' 0 ' , 2 X , F 7 . 2 , 7 X , F 7 . 2 , 6 X , F 7 . 2 . 6 X , F 6 . 1 , 4 X , E 1 0 . 4 , 3 X . E 1 0 . 4 . 79 1 7 X , F 5 . 1 ) 8 0 WAVELG=WAVELG -1 . 0 81 K = K+ 1 8 2 160 CONT INUE 8 3 170 CONT INUE 84 N=K- 10 8 5 W R I T E ( 6 , 1 8 0 ) 8 6 180 F O R M A T ( ' 2 ' , ' DATA INPUT FOR PROVENCHER P R O G R A M ' / / / ) 87 W R I T E ( 6 , 1 9 0 ) ( M O L E L P ( K ) , K = 1,N) 8 8 190 F O R M A T ( ' 0 ' , 1 0 F 1 0 . 0 ) 8 9 Y = Y+ 1 9 0 GOTO 8 5 91 2 0 0 CONT INUE 9 2 P = P+1 9 3 GOTO 15 94 2 1 0 CONT INUE 9 5 STOP 9 6 END - 231 -Appendix 8. L i s t i n g of a Fo r t r a n IV computer program to c a l c u l a t e aver-age hydrophobicity based on the algorithm of Bigelow (1967). - 232 -1 c *********************************************** 2 c * * 3 c * B I G E L O W ' S AVERAGE HYDROPHOB IC ITY * 4 c * * 5 c *********************************************** 6 7 C N = NUMBER OF AMINO A C I D S IN THE P R O T E I N 8 C M = ARRAY OF THE AMINO AC IDS IN FORMAT 1 6 I 5 ( R E A D I N G ACROSS ) 9 C ND = NUMBER OF DATA CARDS 10 C A = TEMPORARY ARRAY TO STORE THE AMINO AC ID SEQUENCE 1 1 C X = HYDROPHOB IC I TY VALUE ASS IGNED TO EACH AMINO AC ID 12 C AVG = B I G E L O W ' S AVERAGE HYDROPHOB IC ITY 13 c 14 C THE NUMBER SYSTEM FOR THE AMINO AC IDS FOLLOWS THAT OF PHAM 15 C UN IT 16 MUST BE AS S IGNED A TEMPORARY F I L E INORDER TO RUN 16 C T H I S PROGRAM 17 c 18 c 19 R E A L * 8 X , T O T A L , A V G 2 0 INTEGER A , P R 0 T N ( 8 O ) 21 D I M E N S I O N M ( 3 0 , 3 0 ) . A ( 5 0 0 ) 2 2 R E A D ( 5 , 1 0 ) PROTN 23 10 F O R M A T ( 8 0 A 1 ) 24 W R I T E ( 6 , 1 5 ) PROTN 2 5 15 F O R M A T ( ' 1 ' , 8 0 A 1 ) 26 R E A D ( 5 , 2 0 ) N,ND 27 2 0 F 0 R M A T ( 2 I 3 ) 28 W R I T E ( 6 , 2 5 ) N 2 9 25 F O R M A T ( ' THE NUMBER OF AMINO AC IDS IN THE SEQUENCE = ' , I 3 ) 3 0 R E A D ( 5 , 3 0 ) ( ( M ( I , J ) , d = 1 , 1 6 ) , 1 = 1 , N D ) 31 3 0 F O R M A T ( 1 6 1 5 ) 32 REWIND 16 33 W R I T E ( 1 6 , 4 0 ) ( ( M ( I , d ) , d = 1 , 1 6 ) , I = 1 , N D ) 34 4 0 F O R M A T ( 1 5 ) 35 REWIND 16 36 T O T A L = 0 . 0 3 7 DO 5 0 K = 1 , N 38 R E A D ( 1 6 , 4 0 ) A ( K ) 39 I F ( A ( K ) . E Q . 3 . O R . A ( K ) . E Q . 4 . O R . A ( K ) . E Q . 6 . O R . A ( K ) . E Q . 7 . O R . A ( K ) 4 0 1 . O R . A ( K ) . E Q . 9 . 0 R . A ( K ) . E Q . 16 ) X = 0 . 0 4 1 I F ( A ( K ) . EQ. 18 ) X = 3 . 0 0 4 2 I F ( A ( K ) . EQ. 10 ) X = 2 . 9 5 4 3 I F ( A ( K ) . E Q . 1 9 ) X = 2 . 8 5 44 I F ( A ( K ) . E Q . 1 4 ) X = 2 . 6 5 4 5 I F ( A ( K ) . E Q . 1 5 ) X = 2 . 6 0 46 I F ( A ( K ) . EQ. 11) X = 2 . 4 0 4 7 I F ( A ( K ) . E Q . 2 0 ) X = 1 . 7 0 4 8 I F ( A ( K ) . E Q . 1 2 ) X = 1 . 5 0 4 9 I F ( A ( K ) . E Q . 1 3 ) X = 1 . 3 0 5 0 I F ( A ( K ) . E Q . 5 ) X = 1 . 0 0 51 I F ( A ( K ) . E Q . 1 ) X = 0 . 7 5 52 I F ( A ( K ) . E Q . 2 ) X = 0 . 7 5 53 I F ( A ( K ) . EQ. 17) X = 0 . 4 5 54 TOTAL=TOTAL+X 5 5 5 0 CONT INUE 56 A V G = ( T O T A L * 1 0 0 0 ) / N 57 W R I T E ( 6 , 6 0 ) AVG 58 6 0 F O R M A T ( ' B IGELOW AVERAGE H Y D R O P H O B I C I T Y ' , F 1 2 . 4 ) 5 9 STOP 6 0 END 

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