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Chemical and enzymic assays for available lysine Holguin, Mariluz 1979

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CHEMICAL AND ENZYMIC ASSAYS FOR AVAILABLE LYSINE by MARILUZ HOLGUIN B . S c , U n i v e r s i d a d J a v e r i a n a , 1 9 7 ^ A T h e s i s Submitted i n P a r t i a l F u l f i l l m e n t o f the Requirements f o r the Degree of MASTER OF SCIENCE i n The F a c u l t y o f Graduate S t u d i e s (Department o f Food Science) We accept t h i s t h e s i s as conforming to the r e q u i r e d standard THE UNIVERSITY OF BRITISH COLUMBIA FEBRUARY, 1 9 7 9 (c) M a r i l u z Holguin, 1 9 7 9 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make i t freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the Head of my Department or by his representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. M a r i l u z H o l g u i n _. . . ~ Food Science Department of The University of British Columbia 2075 Wesbrook Place Vancouver, Canada V6T 1W5 Date February 2, 1979 i i ABSTRACT L y s i n e i s o f prime n u t r i t i o n a l s i g n i f i c a n c e s i n c e i t i s the f i r s t l i m i t i n g amino a c i d i n many foods o f p l a n t o r i g i n and i s e a s i l y rendered u n a v a i l a b l e upon heat p r o c e s s i n g o r upon unfavourable storage c o n d i t i o n s . The term " a v a i l a b l e l y s i n e " r e f e r s to forms o f l y s i n e which c o n t a i n f r e e €-amino groups w i t h i n the pepti d e c h a i n . Once the <= -amino group i s blocked, l y s i n e becomes u n a v a i l a b l e s i n c e i t cannot be h y d r o l y z e d by p r o t e o l y t i c enzymes. The a v a i l a b i l i t y o f l y s i n e s i n c a s e i n , lysozyme, | 6 - l a c t o g l o b u l i n , a c i d s o l u b i l i z e d g l u t e n and whole egg was determined by the p e p s i n p a n c r e a t i n d i g e s t i o n t e s t , the d i n i t r o b e n z e n e s u l f o n i c a c i d (DNBS) and the t r i n i t r o -benzene s u l f o n i c a c i d (TNBS) methods. The r e s u l t s were compared to the f l u o r o d i n i t r o b e n z e n e (FDNB) method. Good agreement was obtained between the FDNB o f f i c i a l method and the DNBS technique, with a c o r r e l a t i o n c o e f f i c i e n t o f O . 9 8 9 . When the TNBS method was compared to the FDNB d i f f e -rence technique, a c o r r e l a t i o n c o e f f i c i e n t o f O .988 was found. The p e s i n p a n c r e a t i n d i g e s t i o n t e s t i n d i c a t e d the r e l a t i v e amount o f l y s i n e r e l e a s e d by the enzymes under the c o n d i t i o n s s p e c i f i e d by the t e s t . A c o r r e l a t i o n c o e f -f i c i e n t o f 0.995 was found between the FDNB o f f i c i a l method i i i . and the enzymatic t e s t . The s p e c i f i c i t y o f DNBS f o r the €-amino group o f l y s i n e was determined u s i n g e< - and 6-formyl-lysines» L - l y s i n e , L - l y s y l l y s i n e , L - l y s y l a l a n i n e and r i b o n u c l e a s e -S-peptide. DNBS was found to r e a c t mainly w i t h €-amino group but with a s l i g h t r e a c t i v i t y with oO-amino group. However, i n the case o f p r o t e i n s w i t h s e v e r a l €-amino groups and N-terminal l y s i n e , the c o n t r i b u t i o n o f the c(-amino group to the r e s u l t s becomes n e g l i g i b l e . The DNBS method was found to be the s i m p l e s t and most r e l i a b l e method f o r d e t e r m i n a t i o n o f a v a i l a b l e l y s i n e , f o r the f o l l o w i n g reasons* a) i t does not r e q u i r e a c i d h y d r o l y s i s o f the p r o t e i n s ; b) a l a r g e number of samples can be analyzed s i m u l t a n e o u s l y i n a few hours, and c) i t does not r e q u i r e expensive and l e n g t h y chroma-t o g r a p h i c amino a c i d a n a l y s i s . i v . TABLE OF CONTENTS PAGE ABSTRACT i . ACKNOWLEDGEMENTS i i i . TABLE OF CONTENTS i v . LIST OF FIGURES v i . LIST OF TABLES v i i i . INTRODUCTION 1 LITERATURE SURVEY ON METHODS FOR DETERMINATION OF LYSINE- AVAILABILITY. 8 A. Bioassays 8 B. M i c r o b i o l o g i c a l Assays 12 C. Chemical Methods 16 1 . The f l u o r o d i n i t r o b e n z e n e method (FDNB) 1? 2 . The t r i n i t r o b e n z e n e s u l f o n i c a c i d method (TNBS) 28 3 . G u a n i d i n a t i o n 35 4 . A c r y l o n i t r i l e 36 5 . Methyl a c r y l a t e 36 6. E t h y l v i n y l s u l f o n e 37 7. 2 - C h l o r o - 3 , 5 - d i n i t r o p y r i d i n e 37 8. Sodium borohydride 38 9 . 1 9 F NMR 39 1 0 . The sodium d i n i t r o b e n z e n e s u l f o n a t e method (DNBS) 4 2 1 1 . Fluorescamine 4 8 1 2 . Dye-binding 4 8 1 3 . Chemical methods i n the d e t e r m i n a t i o n o f a v a i l a b l e l y s i n e i n m a t e r i a l s t h a t have undergone M a i l l a r d r e a c t i o n s 51 V . TABLE OF CONTENTS (Continued) PAGE LITERATURE SURVEY ON METHODS FOR DETERMINATION OF LYSINE AVAILABILITY (Continued) D. Enzymatic Methods 54 MATERIALS AND METHODS 65 A. Materials 65 B. Nitrogen Determination 66 C. Amino Acid Analysis 66 D. Lysine Determination "by the Dinitrobenzene Sulfonic Acid Method 67 E. Lysine Determination by the Trinitrobenzene Sulfonic Acid Method 74 F. Lysine Determination by the D i n i t r o f l u o r o -benzene Method 79 G. Enzymatic Digestion Test 85 RESULTS AND DISCUSSION 89 A. The Dinitrobenzene Sulfonic Acid Method 89 B. The Trinitrobenzene Sulfonic Acid Method 96 C. The Fluorodinitrobenzene Method 101 D. The Pepsin Pancreatin Digestion Test 105 GENERAL DISCUSSION 110 CONCLUSION 120 REFERENCES CITED 122 APPENDIX 146 v i . LIST OF FIGURES F i g u r e No. Page No. 1(a) S t r u c t u r e o f f r e e l y s i n e . From: B o d w e l l , 1976. 2 1(b) L y s i n e as i t would e x i s t i n a p e p t i d e c h a i n . From: B o d w e l l , 19 76. 3 2 R e a c t i o n o f FDNB w i t h an amino a c i d . From: H a l l e t a l . , 1974. 21 3 R e a c t i o n o f TNBS w i t h an amino a c i d . From: H a l l e t a l . , 1973. 29 19 4 F NMR spe c t r u m o f the r e a c t i o n p r o d u c t s o f a p r o t e i n and S - e t h y l t r i f l u o r o t h i o a c e t a t e i n d i m e t h y l s u l f o x i d e s o l u t i o n . 41 5 S t r u c t u r e o f t h e r e a c t i v e dye r e m a z o l b r i l l i a n t b l u e R. From: Ney and Wirotama, 1970. 50 6 The d i n i t r o b e n z e n e s u l f o n i c a c i d method f o r d e t e r m i n a t i o n o f a v a i l a b l e l y s i n e . 72 7 S t a n d a r d curve f o r a v a i l a b l e l y s i n e d e t e r m i n a t i o n f o r a sample t r e a t e d w i t h d i n i t r o b e n z e n e s u l f o n i c a c i d . 73 8 The t r i n i t r o b e n z e n e s u l f o n i c a c i d method f o r the d e t e r m i n a t i o n o f a v a i l a b l e l y s i n e . 77 9 S t a n d a r d curve f o r a v a i l a b l e l y s i n e d e t e r m i n a t i o n f o r a sample t r e a t e d w i t h 2 , 4 , 6 - t r i n i t r o b e n z e n e s u l f o n i c a c i d 78 10 R e f l u x i n g system used i n t h e p r e p a r a -t i o n o f p r o t e i n h y d r o l y s a t e s 82 11 The d i n i t r o f l u o r o b e n z e n e method f o r d e t e r m i n a t i o n o f a v a i l a b l e l y s i n e ( d i f f e r e n c e t e c h n i q u e ) . From: Blom e t a l . , 1967. 84 v i i . LIST OF FIGURES (Continued) F i g u r e No. Page No. 12 Comparison between the FDNB d i f f e r e n c e t e c h n i q u e and t h e DNBS method. The % o f l y s i n e i s d e t e r -mined by g l y s i n e / 1 0 0 g p r o t e i n . 112 13 Comparison between the FDNB d i f f e r e n c e t e c h n i q u e and t h e TNBS method. The % o f l y s i n e i s d e t e r m i n e d by g l y s i n e / 100 g p r o t e i n 113 14 Comparison between the FDNB d i f f e r e n c e method and t h e e n z y m a t i c d i g e s t i o n t e s t . The % o f l y s i n e i s d e t e r m i n e d by g l y s i n e / 1 0 0 g p r o t e i n . 114 15 Comparison between the e n z y m a t i c d i g e s t i o n t e s t and the DNBS method. The % o f l y s i n e i s d e t e r m i n e d by g l y s i n e / 1 0 0 g p r o t e i n . 115 16 Comparison between t h e e n z y m a t i c d i g e s t i o n t e s t and the TNBS method. The % of l y s i n e i s d e t e r m i n e d by g l y s i n e / 1 0 0 g p r o t e i n . 116 17 Comparison between t h e TNBS and the DNBS methods. The % o f l y s i n e / 1 0 0 g p r o t e i n . 117 v i i i . LIST OF TABLES Table No. Page No. 1 Reagents used and derivates formed i n Direct Methods. 18 2 Reagents used and derivates formed i n Indirect Methods. 19 3 Protein content of samples assayed for lysine a v a i l a b i l i t y . 90 4 Available lysine content of protein samples as determined by the d i n i t r o -benzene s u l f o n i c acid (DNBS) method. 91 5 Reaction of,amino acids and peptides with dinitrobenzene s u l f o n i c acid (DNBS). 94 6 Available lysine content of protein samples as determined by the t r i n i t r o -benzene s u l f o n i c acid (TNBS) method. 97 7 Available lysine content of protein samples as determined by the fluoro-dinitrobenzene (FDNB) method. 102 8 Amount of lysine released from protein samples subjected to pepsin-pancreatin digestion. 108 9 Lysine a v a i l a b i l i t y determined by the FDNB o f f i c i a l procedure, the DNBS and the TNBS methods, and the enzymatic t e s t . I l l 10 Standard deviation of the random error (in g lysine/100 g protein) i n the a v a i l -able lysine methods, as estimated with Deming's procedure. 119 ix. ACKNOWLEDGEMENTS I wish to express my gratitude to Dr. S. Nakai, Chairman of my committee for his supervision and encourage-ment i n the preparation of t h i s thesis. Also to the other members of my committee: Drs. Powrie, Vanderstoep and Beames my sincere thanks. To Valerie (technician) I am t r u l y g r a t e f u l for her co-operation and assistance. I should also l i k e to thank my husband for his help and encouragement i n the preparing of t h i s t h e s i s . 1 . INTRODUCTION The n u t r i t i o n a l q u a l i t y o f a food p r o t e i n i s dependent not o n l y on the amino a c i d composition and d i g e s t i -b i l i t y o f the p r o t e i n i t s e l f hut a l s o upon the p h y s i o l o g i c a l a v a i l a b i l i t y . A v a i l a b i l i t y Pan be d e f i n e d as the amount or percentage o f a g i v e n amino a c i d i n the food which i s u t i l i z e d f o r p r o t e i n s y n t h e s i s i n the organism (growth and maintenance) when the s a i d amino a c i d i s the o n l y l i m i t i n g f a c t o r o f the d i e t . Growth may t h e r e f o r e be c o n s i d e r e d to be a s u i t a b l e index o f a v a i l a b i l i t y . D i e t a r y l y s i n e i n s u f f i c i e n c y o f t e n l i m i t s growth. In many foods, l y s i n e i s l i m i t i n g not Only because r e l a t i v e l y s m a l l amounts are i n c o r p o r a t e d i n the p r o t e i n s d u r i n g b i o -s y n t h e s i s but a l s o because o f secondary chemical changes due to f a c t o r s such as l i g h t , heat, a l k a l i , and r e d u c i n g sugars, as a r e s u l t o f which l y s i n e becomes n u t r i t i o n a l l y u n a v a i l a b l e . L y s i n e i n i t s f r e e form has a f r e e eC-amino group, a f r e e €-amino group and a f r e e c a r b o x y l group. In most p r o t e i n s , 95 to 100$ o f the l y s i n e i s l i n k e d to oth e r amino a c i d s through p e p t i d e l i n k a g e s i n v o l v i n g the oC-amino and c a r b o x y l groups ( F i g u r e 1 ) . Only the €-amino group i s f r e e to r e a c t with other compounds such as sugars o r amide groups o f other amino a c i d s . 2. FIGURE 1 . (a) S t r u c t u r e o f f r e e l y s i n e . From* Bodwell, 1976 . €-amino group — • NH 2 CH 2 1 1 c CH P 1 c 1 c H~N\- C - GOOH ^ 1 t H U-amino group 3. FIGURE !•• (t>) L y s i n e as i t would e x i s t i n a p o r t i o n o f a pe p t i d e c h a i n . From* Bodwell, 1976 . NH 2 i GH 2 i GHo CH, H-C-OH i N-C-G i i I I H H 0 CH 2 i N-C-G i i I I H H 0 CH. H i N-C-C I I H H 0 S i CH 2 CH 2 i N-C-C i i II H H 0 Threonine L y s i n e G l y c i n e Methionine 4 . L y s i n e i s n u t r i t i o n a l l y a v a i l a b l e i f i t s €-amino group i s f r e e . I f the €-amino group i s bl o c k e d through a chemical bond, the segment of the p r o t e i n near the a f f e c t e d l y s i n e r e s i d u e may not be d i g e s t e d . I f the p r o t e i n i s broken down to amino a c i d s or d i p e p t i d e s , the r e a c t e d l y s i n e may not be absorbed from the g a s t r o i n t e s t i n a l t r a c t . Even i f absorbed, the l y s i n e h a ving a ' d e r i v a t i z e d * €-amino group may not be u t i l i z e d but r a t h e r e x c r e t e d i n the u r i n e . L y s i n e may a l s o be b u r i e d i n a p r o t e i n m a t r i x o f a p a r t i c u l a r sequence or conformation which i s slow to hydro-l y z e o r i s not h y d r o l y z e d a t a l l by animal p r o t e a s e s . Such l y s i n e may or may not appear as c h e m i c a l l y a v a i l a b l e by chemical methods and y e t be t o t a l l y u n a v a i l a b l e n u t r i t i o n a l l y . P r o t e o l y s i s w i t h i n the i n t e s t i n a l lumen i s l a r g e l y c a r r i e d out by the p a n c r e a t i c p r o t e o l y t i c enzymes. T r y p s i n and chymotrypsin account f o r 78% o f the t o t a l p r o t e o l y t i c a c t i v i t y . T r y p s i n a c t s on a p r o t e i n at e i t h e r an a r g i n i n e r e s i d u e or a l y s i n e r e s i d u e which has a f r e e amino group. The a c t i o n o f other p r o t e a s e s i s o n l y p a r t i a l l y i n h i b i t e d when the €-amino group o f l y s i n e i s bl o c k e d . Under severe o r prolonged h e a t i n g c o n d i t i o n s , the €-amino group o f l y s i n e can r e a c t with the )(-carboxyl group o f glutamic a c i d o r the (3-carboxyl group o f a s p a r t i c a c i d on another p r o t e i n o r w i t h i n the same p r o t e i n molecule. 5 . As a r e s u l t , a so c a l l e d i s o p e p t i d e bond i s formed, which i s r e s i s t a n t to h y d r o l y s i s by gut p r o t e a s e s . P r o t e o l y t i c enzy-mes are b e l i e v d to be s p e c i f i c f o r the <£,£-peptide l i n k a g e s . The a s p a r t y l - or g l u t a m y l - l y s i n e remains a f t e r p r o t e o l y s i s . However, Mauron (1972) and Carpenter and Booth (1973) have shown t h a t these d e r i v a t i v e s can be u t i l i z e d as sources o f l y s i n e by the r a t . The d i p e p t i d e s are p r o b a b l y absorbed as such through the i n t e s t i n a l w a l l and h y d r o l y z e d i n the k i d -neys. The presence of numerous such c r o s s - l i n k a g e s might be expected to a t l e a s t reduce the r a t e o f p r o t e i n d i g e s t i o n , p o s s i b l y by s t e r i c hindrance o f the s i t e s o f a t t a c k by d i g e s t i v e enzymes. Another r e a c t i o n o f l y s i n e i s c r o s s - l i n k a g e with dehydroalanine r e s i d u e s . Dehydroalanine i s formed by h e a t i n g s e r i n e or c y s t e i n e under a l k a l i n e c o n d i t i o n s . The dehydro-a l a n i n e thus formed r e a c t s with the €-amino group to form l y s i n o a l a n i n e (LAL). I t appears t h a t l y s i n o a l a n i n e i s not n u t r i t i o n a l l y a v a i l a b l e s i n c e 38 -65% o f the LAL i n g e s t e d by r a t s i n the form o f a l k a l i - t r e a t e d soybean c a s e i n or soybean meal was recovered im the f e c e s (deGroot and Slump, I 9 6 9 ) . A more common reason f o r l o s s e s o f a v a i l a b l e l y s i n e may be r e a c t i o n with carbohydrates. Both r e d u c i n g and non-r e d u c i n g sugars r e a c t with l y s i n e . El-Nockrashy and Frampton (1967) showed t h a t sucrose, r a f f i n o s e and t r e h a l o s e a l l r e a c t with l y s i n e to render i t u n a v a i l a b l e . M a i l l a r d 6 . r e a c t i o n i n which the €-amino group of l y s i n e r e a c t s w i t h aldehyde groups o f r e d u c i n g sugars, or wit h c a r b o n y l groups o f l i p i d s , i s v e r y w e l l known to decrease l y s i n e a v a i l a b i -l i t y . Thompson e t a l . (1976) have s t u d i e d the l o s s r a t e o f a v a i l a b l e l y s i n e d u r i n g thermal p r o c e s s i n g o f a soy model system. A v a i l a b l e l y s i n e l o s s went through three phases. The f i r s t phase was c h a r a c t e r i z e d by a r a p i d l o s s i n which 30 to 60% of the a v a i l a b l e l y s i n e was made u n a v a i l a b l e . Phase two showed a s t a t i s t i c a l l y s i g n i f i c a n t (98$ c o n f i d e n -ce) i n c r e a s e i n a v a i l a b l e l y s i n e when measured by Carpenter's f l u o r o d i n i t r o b e n z e n e procedure (FDNB). The t h i r d phase was c h a r a c t e r i z e d by a s t a b i l i z a t i o n o f FDNB a v a i l a b l e l y s i n e . The r e a c t i o n r e s u l t i n g i n the i n i t i a l r a p i d phase o c c u r r e d a c c o r d i n g to f i r s t o r d e r r e a c t i o n k i n e t i c s . Wolf and Thompson (1977) u t i l i z e d a model system c o n s i s t i n g o f p r o t e i n , glucose,and m i c r o c r y s t a l l i n e c e l l u l o s e . I t was found t h a t glucose and temperature had the dominant e f f e c t on p r e d i c t i n g the s p e c i f i c r e a c t i o n r a t e (k-j.). Water a c t i v i t y and pH a l s o i n f l u e n c e d the p r e d i c t i o n o f k^ but they acted though an i n t e r a c t i o n w i t h g l u c o s e . J o k i n e n e t a l . (1976) developed an eq u a t i o n which p r e d i c t s l y s i n e a v a i l a * b i l i t y as a f u n c t i o n o f pH, water a c t i v i t y , g l u c o s e , s t a r c h , and sucrose i n phase th r e e . Thermal l o s s o f a v a i l a b l e l y s i n e i n f o r t i f i e d r i c e meal was k i n e t i c a l l y assessed by 7. Tsao e t a l . (1978). Because o f the extreme importance o f l y s i n e i n the d i e t , and the a d d i t i o n a l c o n s i d e r a t i o n s o f p r o t e i n damage and i n t e r n a l c r o s s - l i n k a g e s f o l l o w i n g some types o f p r o -c e s s i n g , s e v e r a l methods have "been proposed f o r e s t i m a t i n g n u t r i t i o n a l l y a v a i l a b l e l y s i n e . These methods can be c a t e g o r i z e d i n f o u r groups t B i o l o g i c a l e v a l u a t i o n s t u d i e s , i n v i t r o d i g e s t i o n methods, and m i c r o b i o l o g i c a l and chemi-c a l a n a l y s e s . Much e f f o r t has been made to e s t a b l i s h r e l i a -b l e chemical methods which are qu i c k and s u i t a b l e f o r r o u -t i n e assay. The o b j e c t o f t h i s study was to determine l y s i n e a v a i l a b i l i t y w i t h two o f the most p o p u l a r chemical methods, the d i n i t r o b e n z e n e s u l f o n i c a c i d (DNBS) and the t r i n i t r o -benzene s u l f o n i c a c i d (TNBS) techniques, and to compare the r e s u l t s with the f l u o r o d i n i t r o b e n z e n e (FDNB). AOAC o f f i c i a l method. Enzymatic d i g e s t i o n assays were done i n an attempt to c o r r e l a t e chemical and b i o l o g i c a l a v a i l a b i l i t i e s , 'i For example, i t has been demonstrated t h a t , i n some cases, che-m i c a l d e t e r m i n a t i o n s do not show the extent o f p r o t e i n d i g e s -t i b i l i t y o r the presence o f t r y p s i n i n h i b i t o r s . 8. METHODS FOR DETERMINATION OF LYSINE AVAILABILITY A. BIOASSAYS The ult imate standards fo r the i n v i t r o methods of measuring amino ac id a v a i l a b i l i t y are bioassays with animals. Animal assays are based on the observat ion that the amount and q u a l i t y of a p ro te in can r e s u l t i n a gain or l o s s of body substance. Such gain or l o s s can be i d e n t i f i e d by a change i n body weight or by a change i n a body compon-ent which of ten i s n i t rogen content. The Pro te in E f f i c i e n c y Ratio (PER), which i s the o f f i c i a l AOAC procedure, and the Net Pro te in Ratio (NPR) are based on weight changes. The methods based on body n i t rogen change are the Net P ro te in U t i l i z a t i o n (NPU) where carcass n i t rogen i s measured, and the B i o l o g i c a l Value (BV) where n i t rogen balance i s measured. PER i s the slope of the l i n e r e l a t i n g weight gain of a ra t to p ro te in intake consumed under standardized ex-perimental cond i t ions . Despite i t s long h i s t o r y , wide usage, and o f f i c i a l s ta tus , PER i s not a good assay procedure (Hegsted and Chang, 1965$ Hegsted and Samonds, 1978). Of the c r i t e r i a def ined f o r a v a l i d b ioassay, i . e . , p r e c i s i o n , r e p r o d u c i b i l i t y , s t a t i s t i c a l v a l i d i t y , p r o p o r t i o n a l i t y , s i m p l i c i t y and low cost (Hegsted and Samonds, 1978) PER could be considered as meeting only the c r i t e r i o n of sim-p l i c i t y . Some improvement can be made by i n c l u s i o n of a 9 -group o f animals consuming a n o n - p r o t e i n d i e t f o r a s i m i l a r p e r i o d . When m o d i f i e d i n t h i s way the procedure i s c a l l e d Net P r o t e i n R a t i o (NPR) (Bender and D o e l l , 1 9 5 7 ) . A modi-f i c a t i o n o f NPR c a l l e d R e l a t i v e NPR (RelNPR) i s recommended by the r e c e n t N a t i o n a l Academy o f S c i e n c e s - N a t i o n a l Re-search C o u n c i l Committee on P r o t e i n E v a l u a t i o n (Young and P e l l e t t , 1978) as a u s e f u l procedure i f a m u l t i p l e - p o i n t assay cannot be performed. R e l a t i v e NPR i s performed i n an i d e n t i c a l manner as NPR, but the r e s u l t s are expressed r e l a -t i v e to the value obtained w i t h an 8fo l a c t a l b u m i n d i e t taken as 1 . 0 0 or 1 0 0 . Bioassays are c l a s s e d as e i t h e r two-point or mul-t i p l e - p o i n t assays. With two p o i n t assays, a s t r a i g h t l i -ne c o n n e c t i n g the two p o i n t s must be assumed. S t u d i e s w i t h both r a t s (Hegsted and Neff, 1 9 7 0 j Hegsted, 1971) and humans (Young e t a l . , 1977) have shown t h i s assumption not to be e n t i r e l y v a l i d , e s p e c i a l l y f o r poor q u a l i t y p r o t e i n s . M u l t i p l e - p o i n t procedures i n v o l v e measurements o f the r e s -ponse obtained (weight, body water, or body n i t r o g e n ) i n r e -l a t i o n to p r o t e i n comsumed; at s e v e r a l l e v e l s o f p r o t e i n con-sumption. These l e v e l s must be s e l e c t e d w i t h c a r e , s i n c e o n l y f o r a s m a l l range can a s t r a i g h t l i n e be assumed* at h i g h l e -v e l s more and more p r o t e i n would be deaminated and used f o r energy, while a t v e r y low l e v e l s d e v i a t i o n from l i n e a r i t y c o u l d occur because o f such phenomena as adaptive responses, w i t h 1 0 . consequently more e f f i c i e n t u t i l i z a t i o n o f amino a c i d s . T h i s i s e s s e n t i a l l y t rue when the l i m i t i n g amino a c i d i s l y s i n e . Very good c o r r e l a t i o n s are found between b i o a s -says and amino a c i d score f o r p r o t e i n s having a b i o l o g i c a l v a l u e g r e a t e r than 40 ( P e l l e t t , 1 9 7 8 ) . The r e l a t i o n s h i p va-r i e s w i t h the l i m i t i n g amino a c i d below t h i s l e v e l (Bender, I 9 6 I 5 Hegsted, 1 9 7 1 ) . P r o t e i n s completely l a c k i n g i n l y s i n e can s t i l l have a BV equal to 40, and p r o t e i n s l a c k i n g o t h e r amino a c i d can have v a l u e s s i g n i f i c a n t l y above zero. Young and Scrimshaw (1977) have shown t h a t a l l animals, i n c l u d i n g man, have the a b i l i t y ifeo r e u t i l i z e amino a c i d s f o r the syn-t h e s i s o f p r o t e i n s . F o r the human about 5 - 6 g o f p r o t e i n are s y n t h e s i z e d f o r each g o f p r o t e i n allowance. Not a l l the amino a c i d s behave i n the same way; an absence o f va-l i n e or t o t a l s u l f u r amino a c i d s g i v e s the expected zero response f o r BV, with the o t h e r e s s e n t i a l amino a c i d s beha-v i n g i n an i n t e r m e d i a t e manner. While b i o a s s a y procedures such as the R e l a t i v e P r o t e i n Value (RPV) (Young and P e l l e t t , 1978 ), can g i v e more r e a l i s t i c q u a l i t y estimates f o r poor q u a l i t y p r o t e i n s , no b i o a s s a y i s exempt from t h i s problem. An animal can conserve and r e c y c l e l y s i n e f o r a l i m i t e d pe-r i o d by slowing down l y s i n e o x i d a t i o n (Brooks e t a l . , 1 9 7 2 ) . F o r t u n a t e l y , i n p r a c t i c e v e r y few p r o t e i n s or r e a l d i e t a r i e s have these v e r y low l e v e l s o f e s s e n t i a l amino a c i d s . 1 1 . The content of a v a i l a b l e l y s i n e can he measured by comparing the performance of animals r e c e i v i n g a b a s a l d i e t supplemented w i t h the t e s t m a t e r i a l or with pure l y s i n e . The b a s a l p r o t e i n source has been maize with c y s t i n e and tryptophan ( W i l d e r and K r a y b i l l , 1 9 4 7 ) , wheat (Howard e t a l . , 1958 ; M o r r i s o n e t a l . , , 1 9 6 3 - ; Mauron and Mottu, 1 9 6 2 ), wheat g l u t e n ( P i o n and Rerat, 1962) or a mixture o f wheat and bar-l e y (Bruggemann e t a l . , 1 9 6 9 ) . I t i s e s s e n t i a l t h a t l y s i n e should be a l i m i t i n g f a c t o r i n the d i e t s o f t e s t animals, otherwise they w i l l not be s e n s i t i v e to changes i n the l e v e l o f a v a i l a b l e l y s i n e . Although a b a s a l d i e t may be s p e c i f i c a l l y d e f i c i e n t i n l y s i n e ( i n the sense t h a t t h i s i s the o n l y amino a c i d t h a t w i l l e l i c i t a growth response when added to the d i e t ) , the magnitude of the response to a t e s t supplement can a l s o be a f f e c t e d by the amounts o f o t h e r amino a c i d s t h a t i t c o n t a i n s , through changing the amino a c i d balance o f the d i e t as a whole (Muelenaere e t a l . , 1 9 6 7 ) . D i s c r e p a n c i e s have been found c o n s i s t e n t l y a c c o r d i n g to the dose response r e l a t i o n s used f o r c a l c u l a t i o n o f r e s u l t s which have beeni A. $ l y s i n e i n the d i e t : weight gain/day; B. % l y s i n e i n the d i e t : weight g a i n / g food eaten, and C. g l y s i n e eaten: weight gain/day. In g e n e r a l A has r e s u l t e d i n lower c a l c u l a t e d v a l u e s (Calhoun e t a l . , i 9 6 0 ; Carpenter et a l . , 1963? Guo e t a l . , 1 9 7 1 ) ' B and C have been p r e f e r r e d because they g i v e v a l u e s l e s s a f f e c t e d by amino a c i d balance (Netke e t a l . , 1969) and by other com-pounds i n f l u e n c i n g food consumption (Gupta e t a l . , 1958). Carpenter e t a l . (1963) and Smith and S c o t t (1965) obtained v a l u e s w i t h c h i c k s f o r the potency o f undamaged f i s h p r o t e i n t h a t were h i g h e r than the c o r r e s -ponding t o t a l l y s i n e v a l u e s obtained by chemical a n a l y s i s . With w e l l processed soya meal, Combs e t a l . ( I 9 6 8 ) found n e a r l y 100% a v a i l a b i l i t y , whereas Netke and S c o t t (1970) obtained r e s u l t s e q u i v a l e n t to o n l y 7^ to 80% of the t o -t a l v a l u e s . Thus, i t i s not p o s s i b l e to make r e l i a b l e com-p a r i s o n s between m a t e r i a l s when the p o t e n c i e s are assessed i n d i f f e r e n t l a b o r a t o r i e s and, even f o r wi thin-experiment comparisons there i s a need f o r c a u t i o n and r e p l i c a t i o n . U n f o r t u n a t e l y , growth assays remain the o n l y d i r e c t means to check the v a l i d i t y o f c l a i m s f o r the n u t r i t i o n a l r e l e -vance o f v a l u e s obtained by oth e r procedures. B. MICROBIOLOGICAL ASSAYS A g r e a t advance i n the d e t e r m i n a t i o n o f a v a i l a b l e amino a c i d s was achieved by the i n t r o d u c t i o n by Ford ( i 9 6 0 ) of m i c r o b i o l o g i c a l methods. M i c r o b i o l o g i c a l assays are r a p i d , c o m p a r a t i v e l y cheap and p r o v i d e a means o f t e s t i n g 1 3 . l a r g e number o f samples i n a s h o r t time. They can be used f o r three main purposesi 1) d e t e r m i n a t i o n of amino a c i d a v a i l a b i l i t y a f t e r enzyme h y d r o l y s i s ? 2) d e t e r m i n a t i o n o f t o t a l amino a c i d s a f t e r a c i d h y d r o l y s i s ; and 3) determina-t i o n of p r o t e i n q u a l i t y , expressed as R e l a t i v e N u t r i t i v e Value (RNV), as assessed by the growth of the t e s t organism r e l a t i v e to t h a t w i t h a standard p r o t e i n , u s u a l l y c a s e i n or whole eggs (Bender, 1969 ; Ford, 1965? Szklarska-Cyganska and Kakowska-Lipinska, 1 9 7 0 ) . RNV i s a r e l a t i v e measure o f the Tetrahymena b i o a s s a y and not meant to r e l a t e to the r a t RNV. The microorganisms are c u l t i v a t e d on a medium con-t a i n i n g a l l amino a c i d s except the one to be d e t e c t e d i n an unknown sample. I f a s t r a i n i s used f o r which t h i s amino a c i d s i s i n d i s p e n s a b l e , the c e l l s w i l l not m u l t i p l y . The growth of the c u l t u r e i s measured by the t u r b i d i t y produced by the growing microorganisms, or by d e t e r m i n a t i o n o f the l a c t i c a c i d formed. I f the extent o f growth, or the amount of l a c t i c a c i d i s p l o t t e d a g a i n s t the amount o f amino a c i d added, a standard curve i s obtained which makes i t p o s s i b l e to determine the amino a c i d content o f the unknown sample by g r a p h i c i n t e r p o l a t i o n . The t e s t microorganisms used show a p a t t e r n o f requirement f o r exogenous amino a c i d , b r o a d l y s i m i l a r to t h a t of h i g h e r animals. 14. Ford (1962) has demonstrated the u s e f u l n e s s o f S t r e p t o c o c c u s zymogenes as an assay organism f o r most o f the e s s e n t i a l amino a c i d s , s i n c e i t has c o n s i d e r a b l e pro-t e o l y t i c ^ powers and, when t e s t m a t e r i a l s are g i v e n a p r e -l i m i n a r y p a r t i a l d i g e s t i o n w i t h papain, the v a l u e s o b t a i n -ed f o r a v a i l a b l e methionine" show a g e n e r a l s i m i l a r i t y to va-l u e s from animal assays ( M i l l e r e t a l . , 1965)• Unfortuna-t e l y t h i s organism does not r e q u i r e l y s i n e . D e t ermination o f p r o t e i n q u a l i t y u s i n g the growth of Tetrahymena p y r i f o r m i s as an index was demonstrated by Rockland and Dunn ( 1 9 4 9 ) . Tetrahymena u n l i k e S. zymogenes r e q u i r e s l y s i n e but i t a l s o r e q u i r e s s e r i n e which i s not an i n d i s p e n s a b l e amino a c i d f o r h i g h e r animals. Viswana'tha and L i e n e r (1955) showed a need f o r p r e d i g e s t i o n of the p r o t e i n sample p r i o r to i t s a d d i t i o n to Tetrahymena c u l t u r e medium. They showed t h a t the p r o t e i n a s e s p r e s e n t i n T e t r a -hymena were unable to a t t a c k n a t i v e g l o b u l a r p r o t e i n s and t h a t Tetrahymena was not a f f e c t e d by t r y p s i n i n h i b i t o r or h e m a g l u t i n i n components o f raw soybean. F e r n e l l and Rosen (1956) a l s o s t u d i e d the T e t r a -hymena b i o a s s a y as a method f o r d e t e r m i n i n g p r o t e i n q u a l i -t y . Organism count and ammonia p r o d u c t i o n over a four-day i n c u b a t i o n were used as a means of o b t a i n i n g a R e l a t i v e N u t r i t i v e Value (RNV). No p r e d i g e s t i o n o f the samples was employed, but the p r o t e i n s were d e f a t t e d because of 1 5 . the p o s s i b l e growth i n h i b i t i o n from c e r t a i n f a t t y a c i d s ( R o l l e , 1 9 7 3 ) . Landers (1975) who used a 3-hr p e p s i n p r e d i g e s t i o n and Frank e t a l . (1975) who used a 24-hr t r y p s i n - b r o m e l a i n p r e d i g e s t i o n o f the food samples p r i o r to t h e i r i n c o r p o r a -t i o n i n t o the Tetrahymena growth media, r e p o r t e d h i g h c o r -r e l a t i o n between the Tetrahymena growth and the PER o f the food p r o t e i n . Tetrahymena grows p o o r l y i n media c o n t a i n i n g f r e e amino a c i d s . A s o l u t i o n o f amino a c i d s s i m u l a t i n g the com-p o s i t i o n o f egg p r o t e i n has an RNV o f 50 to 60 as a g a i n s t 100 f o r the i n t a c t p r o t e i n . The microorganism does not produce a c i d , and the response has u s u a l l y been measured by l a b o r i o u s c e l l counts. Although the p r o t o z o a are too heavy to remain i n suspension f o r an o r d i n a r y t u r b i d i t y r e a d i n g , Shorrock (1972) found t h a t with c o n t r o l l e d a u t o c l a v i n g the organisms break up i n a c h a r a c t e r i s t i c way t h a t allows t u r b i d i t y to be used as a measure o f response. Szmelcman and Guggenheim (1967) have p o i n t e d out t h a t the d i s t i n c t i o n between a v a i l a b l e and u n a v a i l a b l e amino a c i d s by m i c r o b i o l o g i c a l assay, i s a r b i t r a r y and r e l a t e s to the p a r t i c u l a r c o n d i t i o n s o f the assay. Much more r e s e a r c h c o n c e r n i n g the a p p l i c a b i l i t y of Tetrahymena b i o a s s a y to a wide range o f foods and i t s s e n s i -t i v i t y to o t h e r n o n - p r o t e i n food i n g r e d i e n t s i s needed before 16 the microorganism can be used as an e f f e c t i v e t o o l to estimate food p r o t e i n q u a l i t y . C. CHEMICAL METHODS Since the f i r s t a p p l i c a t i o n of Sanger's technique (19^5) to food by Carpenter and coworkers ( 1 9 5 5 a , 1 9 5 7 ) t a l l chemical methods f o r e s t i m a t i n g the a v a i l a b l e l y s i n e i n a p r o t e i n have been simple methods intended to estimate, e i t h e r d i r e c t l y or i n d i r e c t l y , how much o f the l y s i n e i s p r e s e n t which has a f r e e €-amino group. The g e n e r a l approach f o r c h e m i c a l l y e s t i m a t i n g n u t r i t i o n a l l y a v a i l a b l e l y s i n e can be summarized as follows« a) A chemical agent i s r e a c t e d w i t h a p r o t e a n and an a c i d s t a b l e d e r i v a t i v e i s formed between f r e e €-amino groups i n the p r o t e i n and the chemical agent. b) The ' d e r i v a t i z e d ' p r o t e i n i s h y d r o l y z e d to y i e l d d e r i v a t i z e d l y s i n e p l u s o t h e r f r e e amino a c i d s ( i n c l u d i n g n o n - d e r i v a t i z e d , u n a v a i l a b l e l y s i n e ) . c) The amount of d e r i v a t i z e d l y s i n e i s determined ( d i r e c t methods) and/or the amount o f unreacted l y s i n e i s determined ( i n d i r e c t methods). The d i r e c t methods c u r r e n t l y i n use or proposed are l i s t e d i n Table 1 , t o g e t h e r with the reagents used and the d e r i v a t i v e s formed. The i n d i r e c t methods are l i s t e d i n Table 2 . In h i g h carbohydrate foods, the d i r e c t methods 1 7 . (not c o n s i d e r i n g m e t h y l i s o u r e a , DNBS and """^ F NMR methods) f a i l to y i e l d s a t i s f a c t o r y a n a l y t i c a l r e s u l t s . Use o f the i n d i r e c t methods appear to circumvent these a n a l y t i c a l problems. However, as r e p o r t e d by Bodwell ( 1 9 7 6 ) , the r e l a t i o n s h i p o f a v a i l a b l e l y s i n e l e v e l s determined by chemical methods and the b i o a s s a y s e s t i m a t e s , i s not always s a t i s -f a c t o r y . F or example, i n wheat p r o t e i n s , 85-95% a v a i l a b i l i t y f o r l y s i n e , o b tained by u s i n g chemical methods, i s pr o b a b l y an overestimate f o r both r a t s and humans. The chemical e s t i m a t e s o f a v a i l a b l e l y s i n e may be overestimated because: a) In the d i r e c t methods, the s i d e r e a c t i o n s c o u l d i n c r e a s e the c o l o r i m e t r i c a l l y determined e s t i m a t e s o f a v a i l a b l e l y s i n e . b) In both d i r e c t and i n d i r e c t methods, some o f the n u t r i -t i o n a l l y a v a i l a b l e l y s i n e may r e a c t with the chemical agents used to form a c i d s t a b l e d e r i v a t i v e s and, i f so, esti m a t e s o f the a v a i l a b l e l y s i n e l e v e l would be exaggerated. In the f o l l o w i n g pages, the chemical methods l i s t e d i n T a b l e s 1 and 2 w i l l be d e s c r i b e d . When p o s s i b l e , advantages and disadvantages f o r each one w i l l be g i v e n . 1 . The f l u o r o d i n i t r o b e n z e n e method (FDNB). The f i r s t good chemical method f o r the e s t i m a t i o n o f n u t r i t i o n a l l y a v a i l a b l e l y s i n e was developed by Carpenter and h i s a s s o c i a t e s i n 1955 - The procedure was based on the work o f Sanger who used the reagent l - f l u o r o - 2 , TABLE 1. Reagents used and d e r i v a t i v e s formed i n d i r e c t methods Method Reagent Used D e r i v a t i v e Formed FDNB or Carpenter TNBS G u a n i d i n a t i o n 1 9 F NMR A v a i l a b l e l y s i n e A v a i l a b l e l y s i n e A v a i l a b l e l y s i n e DNBS A v a i l a b l e l y s i n e Dye-binding FDNB ( l - f l u o r o - 2 , 4 - d i n i t r o b e n z e n e ) TNBS ( <2 ,4 ,6-trinitrobenzene s u l f o -n i c a c i d ) O-Methylisourea S - E t h y l - t r i f l u o r o t h i o a c e t a t e A c r y l o n i t r i l e 2 T C h l o r o - 3 1 5 - d i n i t r o p y r i d i n e Sodium borohydride DNBS (Sodium d i n i t r o b e n z e n e s u l f o -nate) Fluorescamine ( 4-phenylspiro-[furan-2-(3H)-1'-phthalan] - 3 , 3 '-dione) Remazol b r i l l i a n t blue R DNP-lysine ( € , N - d i n i t r o p h e n y l - l y s i n e ) TNP-lysine ( € T N - t r i n i t r o p h e n y l - l y s i n e ) Homoarginine € , N - t r i f l u o r o a c e t y l - l y s i n e € , N - c y a n o e t h y l - l y s i n e 6 , N - d i n i t r o p y r i d y l - l y s i n e €-N,N-dimethyl-lysine DNP-lysine Fluorescamine complex Dyed p r o t e i n co TABLE 2. Reagents used and d e r i v a t i v e s formed i n i n d i r e c t methods Method Reagent Used D e r i v a t i v e Formed D i f f e r e n c e o r S i l c o c k T o t a l L y s i n e Minus U n a v a i l a b l e L y s i n e T o t a l L y s i n e Minus U n a v a i l a b l e L y s i n e T o t a l L y s i n e Minus Unavailab<be .Lys ine FDNB TNBS Methyl a c r y l a t e E t h y l v i n y l s u lfone DNP-lysine TNP-lysine € , € , N , N - d i c a r b o x y e t h y l -l y s i n e and € , N-monocarboxyethyl-lysine € , N - e t h y l s u l f o n y l e t h y l -l y s i n e , or € ,€ , N , N t > b i s e t h y l s u l f o n y l -e t h y l - l y s i n e 20. ^ - d i n i t r o b e n z e n e to determine the i d e n t i t i e s and number o f the d i f f e r e n t N-terminal amino groups i n p r o t e i n s and p e p t i d e s . Each f r e e amino group became l a b e l l e d , w i t h a d i n i t r o p h e n y l (DNP) group t h a t r e s i s t e d subsequent d i g e s t i o n with a c i d , so t h a t each N-terminal amino a c i d u n i t Ithat had o r i g i n a l l y r e a c t e d was converted to an <£-DNP-amino a c i d . These y e l l o w compounds were separated and i d e n t i f i e d by column chromatography. In a d d i t i o n N-terminal l y s i n e gave oC,€-di-DNP-lysine, and where l y s i n e was p r e s e n t i n o t h e r p o s i t i o n s Sanger obtained €-DNP-lysine ( a b b r e v i a t e d as DNP-l y s i n e ) which c o u l d a l s o be separated by chromatography. The r e a c t i o n of FDNB with an amino a c i d i s i l l u s t r a -t e d i n F i g u r e 2. In Carpenter's o r i g i n a l method the p r o t e i n i s d i n i t r o -phenylated and then h y d r o l y z e d . A p o r t i o n o f the h y d r o l y s a t e i s e x t r a c t e d with e t h e r and the e x t i n c t i o n o f the aqueous phase i s read d i r e c t l y . Another p o r t i o n i s t r e a t e d with methoxycarbonyl c h l o r i d e to c o n v e r t DNP-lysine to an e'ther-s o l u b l e d e r i v a t i v e which i s then e x t r a c t e d . The e x t i n c t i o n o f the remaining aqueous phase i s read and the d i f f e r e n c e between the d i r e c t r e a d i n g and the r e a d i n g on the t r e a t e d sample i s taken as measuring €-DNP-lysine. . A disadvantage o f the method i s t h a t i t estimates €-DNP-hydroxylysine, d C-DNP-ornithine arid X-DNP-argiriine, • as. l y s i n e . On the o t h e r hand, the method does not estimate N-terminal l y s i n e or f r e e l y s i n e . FIGURE 2 . R e a c t i o n o f l - f l u o r o - 2 , 4 - d i n i t r o b e n z e n e (FDNB) with an amino a c i d . (From H a l l e t a l . , 1 9 7 3 ) . R N H 2 - C H - C O O H Amino acid R i N H - C H - C O O H N a H C 0 3 + HCI hydro lys is l-Fluoro -2,4-dini trobenzene D in i t ropheny la ted amino acid 2 2 . C a r p e n t e r ' s method i s s u b j e c t t o i n t e r f e r e n c e by c a r b o h y d r a t e , e s p e c i a l l y i n v e g e t a b l e p r o t e i n c o n c e n t r a t e s . I n t e r f e r e n c e r e s u l t s i n l o w and v a r i a b l e r e c o v e r i e s o f € - D N P - l y s i n e . The e f f e c t i s due t o the d e s t r u c t i o n o f D N P - l y s i n e by c a r b o h y d r a t e s d u r i n g a c i d h y d r o l y s i s . The p r o c e d u r e d e s c r i b e d by C a r p e n t e r and E l l i n g e r i n 1955 was m o d i f i e d by C a r p e n t e r ( i 9 6 0 ) t o remove c e r t a i n i n t e r f e r i n g compounds when a p p l i e d t o d i f f e r e n t t y p e s o f f e e d s t u f f s . The major c r i t i c i s m o f C a r p e n t e r ' s FDNB i s t h a t a c o r r e c t i o n f a c t o r i s r e q u i r e d f o r samples t h a t c o n t a i n ap-p r e c i a b l e amount o f c a r b o h y d r a t e s , and i t becomes d i f f i c u l t t o a p p l y w i t h i n c r e a s i n g c o n c e n t r a t i o n s o f c a r b o h y d r a t e s . The c o r r e c t i o n f a c t o r has been a s s e s s e d by a d d i n g €-DNP-l y s i n e t o d u p l i c a t e samples a t the b e g g i n i n g o f the h y d r o -l y s i s s t a g e and t a k i n g i t t h r o u g h the r e s t o f the p r o c e d u -r e . W i t h a n i m a l m a t e r i a l s the r e c o v e r y was found t o be 92%. A c c o r d i n g l y , a l l v a l u e s d e t e r m i n e d s h o u l d be m u l t i p l i e d by a f a c t o r 6f 1 0 0 / 9 2 = 1 . 0 9 t o c o r r e c t f o r l o s s e s d u r i n g d i g e s t i o n . F o r v e g e t a b l e f o o d s the r e c o v e r y o f added €-DNP-l y s i n e was 60 -85%. The use o f t h e c o r r e c t i o n f a c t o r depends on the a s s u m p t i o n t h a t t h e € - D N P - l y s i n e t h a t a r i s e s from the h y d r o l y s i s o f p r o t e i n i s d e s t r o y e d t o the same e x t e n t as the added € - D N P - l y s i n e . Booth (1971) found t h a t t h i s a s s u m p t i o n i s i n v a l i d ; € - D N P - l y s i n e i n a p r o t e i n i s des-t r o y e d t o a l e s s e r e x t e n t t h a n € - D N P - l y s i n e t h a t has been 2 3 . added as a free compound. Thus, the correction factor i n the Carpenter method i s l e s s than formerly supposed. For materials that contain no carbohydrates a factor of 1 . 0 5 i s more suitable than Carpenter's o r i g i n a l 1.09, although very soluble albumins need the l a t t e r . For wheat and some other cereals the r e s u l t s need to be corrected by multiplying by 1 . 2 . Between these two groups, l i e beans, ground nut, and maize, requiring a factor of approximately 100/88 = 1.14. The presence of carbohydrates r e s u l t s i n very low recoveries of DNP-lysine, due to reduction of n i t r o groups i n the dinitrophenylated amino acid to amino groups, which give de-r i v a t i v e s without the c h a r a c t e r i s t i c s used to measure DNP-l y s i n e . Handwerck e t . a l . ( i 9 6 0 ) reported that sugars are l e s s destructive i f they have previously been i n contact with FDNB. Starch can be equally destructive and may not be inactivated by previous contact with FDNB (Erbersdobler and Zucker, 1964 ; El-Nockrashy, 1 9 6 5 ) . Destruction due to carbohydrates i s lessened when the r e f l u x i n g i s c a r r i e d out i n the presence of another aromatic n i t r o compounds such as dinitrophenol, p i c r i c acid or FDNB i t s e l f (Blom et a l . , 1 9 6 ? ; Matheson .1968; Ruderus and Kihlberg, 1970 ; Booth, 1 9 7 D . presumably by competition with the n i t r o groups from DNP-l y s i n e . This protection may be achieved by adding these compounds to the suspension before r e f l u x i n g begins or by leaving the dinitrophenol and FDNB from the f i r s t stage of 24. the r e a c t i o n i n the r e a c t i o n f l a s k ( C a rpenter and E l l i n g e r , 1 9 5 5 a ; Carpenter, i 9 6 0 ) . Matheson (1968) has shown t h a t the q u a n t i t i e s l e f t i n t h i s way are s u f f i c i e n t to g i v e the p r o t e c t i v e e f f e c t . With sugars c a r r i e d through t h i s p r o -cedure the d e s t r u c t i o n o f DNP-lysine added immediately before h y d r o l y s i s i s no more than 5 - 1 0 $ but i t can be l a r g e r w i t h s t a r c h (Matheson, 1968) or w i t h s t r u c t u r a l carbohydrates (Booth, 1 9 7 D • Because o f the t e c h n i c a l d i f f i c u l t i e s encountered i n the complete r e c o v e r y o f €-DNP-lysine, an a l t e r n a t i v e approach has been to measure a v a i l a b l e l y s i n e i n d i r e c t l y . The d i f f e r e n c e between the t o t a l l y s i n e content of the sample measured a f t e r d i r e c t a c i d h y d r o l y s i s , a!nd, t h e ; r e s i d u a l l y s i n e content measured a f t e r a c i d h y d r o l y s i s o f the sample a f t e r blockage of the f r e e €-amino groups by r e a c t i o n with DNFB, i s assumed to be theiav.aiibable l y s i n e o f the sample. The main advantages of t h i s technique, a l s o known as the D i f f e r e n c e technique or S i l c o c k method, are t h a t p a r t i a l d e s t r u c t i o n o f the d e r i v a t i v e d u r i n g a c i d h y d r o l y s i s does not cause d i f f i c u l t i e s , and t h a t l y s i n e i t s e l f i s extremely s t a b l e to d e s t r u c t i o n by a c i d . However, automatic i o n -exchange chromatography equipment i s n e c e s s a r y f o r ease i n e s t i m a t i o n o f l y s i n e c o n c e n t r a t i o n . Rao e t a l . (1963) f i r s t used the D i f f e r e n c e technique as a check on the procedure they developed f o r d e t e r m i n a t i o n 25 -of a v a i l a b l e l y s i n e i n o i l s e e d meal p r o t e i n s . Roach e t a l . (1967) were the f i r s t to use the D i f f e r e n c e procedure as a working replacement f o r the Carpenter FDNB method. They compared the methods o f Carpenter ( i 9 6 0 ) and Rao e t a l . (1963) w i t h the S i l c o c k method f o r the e s t i m a t i o n o f a v a i l a b l e l y s i n e i n f i s h m e a l and groundnut meal, and showed t h a t the S i l c o c k method i s not s u b j e c t i v t o i n t e r f e r e n c e by carbo-h y d r a t e s . Compared to Carpenter's procedure, the S i l c o c k method gave lower v a l u e s f o r a v a i l a b l e l y s i n e i n groundnut meal. S i m i l a r r e s u l t s v e r e r e p o r t e d by Ostrowski e t a l . (1970) r e l a t i n g to soybean meal. Carpenter and co-workers (1957) d i r e c t l y measured the absorbance o f the y e l l o w e t h e r - e x t r a c t e d a c i d h y d r o l i s a t e s o f d i n i t r o p h e n y l a t e d p r o t e i n s i n t h e i r e s t i m a t i o n o f a v a i l a b l e l y s i n e . In a l a t e r m o d i f i c a t i o n ( i 9 6 0 ) Carpenter reduced some of the e r r o r i n the a n a l y s i s by r e a c t i n g the e - d i n i t r o p h e n y l h y d r a z o n e - l y s i n e with methyl chloroformate to produce an e t h e r s o l u b l e l y s i n e d e r i v a t i v e . The d i f f e r e n c e i n c o l o r i n t e n s i t y of the h y d r o l y s a t e b e f o r e r e a c t i o n with methyl-chloroformate and a f t e r r e a c t i o n and e x t r a c t i o n with e t h e r was taken as a measure o f a v a i l a b l e l y s i n e . D i n i t r o p h e n o l i s the major by-product o f the d i n i t r o -p h e n y l a t i o n o f p r o t e i n s . Conkerton and Frampton ( 1 9 5 9 ) 1 hy t a k i n g advantage o f the d i f f e r e n c e i n absorbance o f d i n i t r o -phenol a t 360 nm i n a c i d and a l k a l i n e media, were able to 2 6 . c o r r e c t f o r the q u a n t i t y o f d i n i t r o p h e n o l p r e s e n t i n the p r o t e i n h y d r o l y s a t e . Other y e l l o w substances might occur i n the h y d r o l y s a t e o f some d i n i t r o p h e n y l a t e d p r o t e i n s (Handwerck e t a l . , i 9 6 0 ) and i n t r o d u c e e r r o r s i n a c o l o r i -m e t r i c e s t i m a t i o n o f a v a i l a b l e l y s i n e . In a d d i t i o n , the brown humin pigments t h a t i n v a r i a b l y occur i n a c i d hydro-l y s a t e s may a l s o be expected to c o n t r i b u t e to the e r r o r . Many o f the sources o f e r r o r found i n the e a r l i e r methods, are e l i m i n a t e d i n the procedure d e s c r i b e d by Rao e t a l (1963) where €-DNP-lysine i s separated from d i n i t r o p h e n o l and o t h e r y e l l o w d e r i v a t i v e s o f the r e a c t i o n between DNFB and the meal p r o t e i n s , as w e l l as from the brown humin products o f a c i d h y d r o l y s i s , through the use o f an ion-exchange column t h a t i s developed w i t h a mixture o f methyl e t h y l ketone and aqueous HC'l. Blom e t a l . (1967) a l s o made use of a chromatographic s e p a r a t i o n technique to p u r i f y the r e s u l t i n g €-DNP-lysine. The a c i d h y d r o l y z e d FDNB-treated sample was passed through a n y l o n powder column to separate €-DNP-lysine from a number of i n t e r f e r i n g compounds. Blom and h i s a s s o c i a t e s o u t l i n e d t hree d e t e c t i o n procedures f o r d e t e r m i n a t i o n o f DNP-lysine: a) D i r e c t photometry o f the y e l l o w c o l o u r . b) Photometry o f the r e a c t i o n product w i t h n i n h y d r i n . c) P o l a r o g r a p h i c measurement based on r e d u c i n g the n i t r o group a t the dropping mercury e l e c t r o d e . 27 . In the o r i g i n a l S i l c o c k method the s e p a r a t i o n o f o r n i t h i n e and l y s i n e was poor, thus c a u s i n g some e r r o r i n the e s t i m a t i o n o f l y s i n e . W i l l i a m s (1967) r e p o r t e d t h a t the sm a l l shoulder on the l y s i n e peak c o u l d be t o t a l l y r e s o l v e d by l e n g t h e n i n g the column, although a h i g h e r p r e s s u r e would then be necessary to m a i n t a i n a s u f f i c i e n t l y , r a p i d flow r a t e . In cases where the o r n i t h i n e peak i s s m a l l and not c l e a r l y separated from the l y s i n e peak, but i s c l e a r l y d i s t i n g u i s h -able as p a r t o f i t , i t i s p o s s i b l e to compute the t r u e a r e a due to l y s i n e . Sometimes, however, the o r n i t h i n e l e v e l i s h i g h i n comparison to the are a o f the r e s i d u a l l y s i n e peak, and so there c o u l d be a considerable- e r r o r i n the e s t i m a t i o n o f the tru e a v a i l a b l e l y s i n e o f the analyzed sample. Ostrowski e t a l . (1970) used a m o d i f i e d s h o r t column technique to improve the r e s o l u t i o n o f o r n i t h i n e and l y s i n e . By u s i n g t h e i r technique, no automatic a n a l y z e r i s r e q u i r e d to determine t o t a l and u n a v a i l a b l e l y s i n e . The DNFB D i f f e r e n c e technique p u b l i s h e d by Couch i n 1975 has been adopted as o f f i c i a l - A O A C f i r s t a c t i o n . The t e c h n i c a l advantages o f the D i f f e r e n c e technique are t h a t N-terminal l y s i n e and f r e e l y s i n e are i n c l u d e d , and t h a t there i s no problem from p a r t i a l r e d u c t i o n o f DNP-lysine d u r i n g h y d r o l y s i s . The disadvantages are t h a t l y s i n e i s more d i f f i c u l t to estimate than DNP-lysine u n l e s s one has 28. automated ion-exchange chromatography equipment, and t h a t FDNB i s not water s o l u b l e and has a v e s i c a n t e f f e c t on hu-man s k i n . 2. The t r i n i t r o b e n z e n e s u l f o n i c a c i d method (TNBS) A s i m p l e r t e s t based on the same p r i n c i p l e as t h a t o f Carpenter (i960) i n which use i s made o f the reagent 2,4,6-trinitrobenzene s u l f o n i c a c i d (TNBS), i n s t e a d o f l- f l u o r o - 2 , 4 - d i n i t r o b e n z e n e (FDNB), has been suggested by Kakade and L i e n e r (1969). The r e a c t i o n o f TNBS with an ami-no a c i d i s i l l u s t r a t e d i n F i g u r e 3. The o r i g i n a l work with t h i s compound on pure amino a c i d s and p e p t i d e s was c a r r i e d out by Okuyama and Satake (i960) and Satake e t a l . (i960). The T N P - d e r i v a t i v e s o f amino a c i d s and p e p t i d e s were c h a r a c t e r i z e d by paper chroma-tography and spectroscopy. No r e a c t i o n was observed with the s i d e chains o f h i s t i d i n e , t y r o s i n e , t hreonine and s e r i n e a f t e r treatment f o r three days a t e l e v a t e d temperatures. T h i s f a v o u r a b l e s p e c i f i c i t y o f TNBS prompted i t s use i n the m o d i f i c a t i o n o f cytochrome c (Takemori e t a l . , I962), haemoglobin (Shinoda, 1965), and xanthine oxidase (Greenlee and Handler, 1964). The SH groups of c y s t e i n e and mercapto-e t h a n o l were observed to r e a c t w i t h TNBS ( K o t a k i e t a l . , 1964). Gold'farb (I966) d e s c r i b e d the r e a c t i o n o f TNBS with human serum albumin and analyzed i t s course i n terms o f three c l a s s e s o f amino groups o f d i f f e r e n t r e a c t i v i t i e s . FIGURE 3. R e a c t i o n o f 2 , 4 , 6-trinitrobenzene s u l f o n i c a c i d (TNBS) w i t h an amino a c i d . (From H a l l e t a l . , 1973)' S0 2 O H R N H g - C H - C O O H Amino acid N H - C H - C O O H NaHC0 3 + HCL i hydrolysis , 4 , 6 - T r i n i t r o b e n z e n e sul fonic acid Tr in i t ropheny la ted amino acid 30 . The same reagent was used to e s t a b l i s h the f u n c t i o n a l r o l e o f amino groups i n some n a t u r a l t r y p s i n i n h i b i t o r s (Haynes e t a l . , 1967) with a mathematical procedure suggested by Ray and Koshland ( 1 9 6 1 ) . Freedman and Radda (1968) examined q u a n t i t a t i v e l y the r e a c t i v i t i e s o f a v a r i e t y o f amino a c i d s and p e p t i d e s towards TNBS. K i n e t i c s t u d i e s were performed by m o n i t o r i n g the e x t i n c t i o n o f the -NH-TNP group a t 3^0 nm on a r e c o r d i n g spectrophotometer. In agreement with G o l d f a r b ' s r e s u l t s they found t h a t the r e a c t i o n o f the TNBS w i t h amino a c i d s and p e p t i d e s i s a second order r e a c t i o n . The pH dependence showed t h a t o n l y the unprotonated amino group i>s the r e a c t i v e s p e c i e s . The SH group o f N - a c e t y l c y s t e i n e was found to be more r e a c t i v e to TNBS than most amino groups. However, the -S-TNP group i s u n s t a b l e a t a l k a l i n e pH and has a much lower e x t i n c t i o n c o e f f i c i e n t a t 340 nm than the -NH-TNP group. I t was a l s o found t h a t both amino groups o f l y s i n e r e a c t w i t h TNBS. However, at pH 7 . 4 and a t room temperature, the €-amino groups were 30 times more r e a c t i v e to TNBS than the cC-amino groups. By u s i n g TNBS, Habeeb (1966) was able to study the r e a c t i o n o f f r e e amino groups i n p r o t e i n w i t h sodium dodecyl s u l f a t e , potassium t h i o c y a n a t e and formaldehyde. I t was found t h a t p r o t e i n s such as bovine serum albumin, ovalbumin and human gamma-globulin have the p r o p e r t y o f b i n d i n g sodium dodecyl sulfate through the e-amino group. The amino group involved at this binding s i t e became unreactive to TNBS. P a r t i a l reaction of potassium thiocyanate with the free amino groups i n bovine serum albumin, ovalbumin, human a-globulin and lactalysate was demonstrated by reaction with TNBS. I t was also found that the reaction of bovine serum albumin with formaldehyde involves the formation of methylene cross links between the amino group on one hand and the amide, guanyl, indole, and imidazole groups on the other hand. The method developed by Habeed suffers the disadvantage that i t does not d i f f e r e n t i a t e between free e-amino and N-terminal amino groups of proteins. This could be c r i t i c a l i n the case of peptides, low molecular weight proteins such as i n s u l i n , or proteins containing several peptide chains or subunits. Kakade and Liener (1969) developed a method to deter-mine s p e c i f i c a l l y the available lysine content of protein foodstuffs. The s p e c i f i c i t y of the technique for the e-amino groups of proteins resides i n the fact that, subsequent to acid hydrolysis of the TNP-protein, the ct-TNP amino acids may be extracted with ether, whereas e-TNP-lysine remains i n the aqueous phase where i t i s determined speetrophoto-metr i c a l l y . A hydrolysis period of 1 hr at 120°C with 6 N HCI seems s u f f i c i e n t for quantitative recovery. Complete hydrolysis of the protein i s not necessary since small 32. TNP-peptides p o s s e s s e s s e n t i a l l y the same s o l u b i l i t y and s p e c t r o p h o t o m e t r i c p r o p e r t i e s as the f r e e TNP-amino a c i d s . The amount o f e - T N P - l y s i n e i s c a l c u l a t e d from a s t a n d a r d curve o b t a i n e d w i t h v a r i o u s l e v e l s o f e - T N P - l y s i n e w h i c h has been s u b j e c t e d t o t h e same p r o c e d u r e as t h e p r o t e i n s . Kakade and L i e n e r found c l o s e agreement between the a v a i l a b l e l y s i n e v a l u e s d e t e r m i n e d w i t h TNBS and the v a l u e s o b t a i n e d w i t h t h e DNFB method o f C a r p e n t e r . H o l s i n g e r e t a l . (1970) used the TNBS method o f Kakade and L i e n e r t o e v a l u a t e the a v a i l a b l e l y s i n e o f a c i d p r e c i p i t a t e d c a s e i n s . a g^-,and 3 - c a s e i n y i e l d e d a v a i l a b l e l y s i n e amounts c o n s i s t e n t w i t h t o t a l l y s i n e . However, a v a i l a b l e l y s i n e o f the K - f r a c t i o n was 15% h i g h e r than t h e t o t a l l y s i n e c o n t e n t . An e x p l a n a t i o n f o r t h e s e h i g h v a l u e s c o u l d be the presence o f D-glucosamine and D - g a l a c t o s a m i n e , and p r o b a b l y o t h e r u n c h a r a c t e r i z e d g l y c o p r o t e i n f ragments. Amino s u g a r s r e a c t w i t h TNBS t o form c o l o r e d d e r i v a t i v e s w h i c h are not removed by e t h e r e x t r a c t i o n . H o l s i n g e r e t a l . c o n c l u d e d t h a t a l t h o u g h e x a c t v a l u e s o f a v a i l a b l e l y s i n e c o u l d n o t be d e t e r m i n e d by TNBS i n foods c o n t a i n i n g l a r g e amounts o f g l y c o p r o t e i n s , the p r o c e d u r e c o u l d s t i l l f i n d a p p l i c a t i o n i n the d e t e r m i n a t i o n o f r e l a t i v e v a l u e s o f l y s i n e i n a c t i v a t i o n d u r i n g p r o c e s s i n g o f p r o d u c t s o f i d e n t i c a l c o m p o s i t i o n . Posati et a l . (1972) noticed that the presence of lactose i n cheese whey int e r f e r e d with the determination of available lysine i n whey protein when TNBS was used. Loss of e-TNP-lysine was dependent on the amount of lactose i n the test material. The sample weight used for analysis, also influenced the r e s u l t s . The larger the sample of eithe pure protein or carbohydrate containing material, the smalle the amount of available lysine found. H a l l et a l . (1973) proposed a method for the deter-mination of available lysine with TNBS i n animal p r o t e i n s T which d i f f e r s i n several aspects from that of Kakade and Liener. The necessity of accurate weighing of a very small sample, was eliminated by the use of agar suspensions of the f i n e l y ground material. They also modified the reaction conditions used by Kakade and Liener (0.1% TNBS solution for 2-hr at 60°C; 2-hr hydrolysis period with 6 N HC1 i n an autoclave at 120°C) and suggested, 0.5% TNBS solution reacting with the protein for 75 min at 40°C, followed by a 2-hr hydrolysis with 11 N HCl i n a b o i l i n g water, bath. The b o i l i n g water bath overcomes the serious corrosive action of the HCl upon the m e t a l l i c i n t e r i o r of the autoclave. Their results for available lysine i n animal protein concen-trates were in close agreement with those measured by the Carpenter procedure. H a l l et a l . (1973) i n agreement with Ousterhout and Wood (1970) proposed the use of pure lysine as a standard instead of e-TNP-lysine, since i t has been shown that a, e - d i T N P - l y s i n e i n i t i a l l y formed i s u n s t a b l e i n h o t 6N HCl and i s e a s i l y c o n v e r t e d i n t o e - T N P - l y s i n e ( K o t a k i and S a t a k e , 1 9 6 4 ) . H a l l e t a l . (1973) i n v e s t i g a t e d t h e r e a c t i o n o f TNBS w i t h h y d r o x y l y s i n e , c a d a v e r i n e and o r n i t h i n e , and found t h a t t h e s e compounds r e a c t t o g i v e TNP-products t h a t remain i n t h e aqueous phase a f t e r e t h e r e x t r a c t i o n and have absorbance v a l u e s s i m i l a r t o e - T N P - l y s i n e . TNBS i s s e r i o u s l y a f f e c t e d by the pr e s e n c e o f c a r b o -h y d r a t e , p a r t l y because l y s i n e i s r a p i d l y c o u p l e d w i t h a l d o s e g r o u p s , t h u s making t h e t e r m i n a l amino l i n k a g e u n a v a i l a b l e t o form e - T N P - l y s i n e , and p a r t l y because o f the a d s o r p t i o n o f the e - T N P - l y s i n e on t o the carbon p a r t i c l e s (from c h a r r i n g ) and t o the i n t e r f e r i n g absorbance o f the browning p r o d u c t s . I n consequence, f a l s e h i g h l e v e l s o f a v a i l a b l e l y s i n e can be found i n samples c o n t a i n i n g c a r b o h y d r a t e compounds. F u r t h e r m o r e , t h e amines agmatine, spermine, s p e r m i d i n e and t a u r i n e found i n numerous p l a n t s p e c i e s (Smith, 1972) c o u l d e x p l a i n why t h e v a l u e s f o r a v a i l a b l e l y s i n e i n c e r t a i n samples are h i g h e r than the t o t a l l y s i n e c o n t e n t ( H a l l e t a l . 1 9 7 5 ) . -H a l l e t a l . (1975) c o n c l u d e d t h a t TNBS can be used f o r d e t e r m i n i n g a v a i l a b l e l y s i n e i n c a r b o h y d r a t e - r i c h m a t e r i a l , p r o v i d e d t h a t the sample mass does n o t exceed 5 mg and t h a t the r e a c t i o n i s m a i n t a i n e d f o r o n l y 30 min a t 30°C, i n s t e a d o f 75 min a t 40°C. Under t h e s e c o n d i t i o n s the 'blank' absorbance, i . e . the value of the unreacted sample simply heated with HCI i s very high i n r e l a t i o n to the absorbance of the actual e-trinitrophenylated product. Attempts to reduce this value to an a n a l y t i c a l l y more acceptable l e v e l have had li m i t e d success. Eklund (1976) s l i g h t l y modified the method of Kakade and Liener (1969) by increasing the sample size as well as by subjecting the TNP-proteins to hydrolysis at 110°C for 90 min. Eklund determined available lysine i n casein and rapeseed with 'his procedure, that of Kakade and Liener and the method of Rao et a l . (1963). Throughout, he obtained higher values with his modified method, but the degree of v a r i a b i l i t y between repeated analyses of the same material was lower as compared to the v a r i a b i l i t y obtained with the method of Kakade and Liener. 3. Guanidination Due to the r e l a t i v e i n s t a b i l i t y of e-DNP-lysine upon acid hydrolysis (Carpenter, 1960), Mauron and Bujard (1963) suggested the use of O-methyl-isourea for determina-ti o n of available l y s i n e . The guanidation with O-methyl-isourea transforms the e-amino group of lysine into a guanidine derivative, which on hydrolysis y i e l d s homoarginine. The hydrolysate i s analyzed by column chromatography and homoarginine i s eluted with the basic amino acids, where i t appears after arginine. No homoarginine i s formed when the lysine units are combined with sugar i n a M a i l l a r d reaction (Finot and Mauron, 1972) .' Although methyl-isourea appears t o be a very s p e c i f i c reagent f o r the e-amino groups of l y s i n e , probably, the long r e a c t i o n time (3 days) needed has precluded any extensive use of the approach. 4„ A c r y l o n i t r i l e A e r y l o i i t r i l e w i l l a l s o r e a c t , though again s l o w l y , w i t h the e-amino groups of l y s i n e t o form a cyanoethylated d e r i v a t i v e t h a t i s s t a b l e under the ordinary c o n d i t i o n s f o r the a c i d h y d r o l y s i s of the p r o t e i n s (Riehm and Scheraga, 1 9 6 6 ) . When the reagent was used by Pisano et a l . (1968) and by Harding and Rogers (1971) to measure the r e a c t i v e l y s i n e u n i t s i n c r o s s - l i n k e d f i b r i n and h a i r p r o t e i n s , the measurements agreed wi t h the values f o r e-N- (X-glutairiyl) l y s i n e obtained by enzymic d i g e s t i o n i n v i t r o . 5. Methyl a e r y l a t e Methyl a c r y l a t e i s an a l t e r n a t i v e t o a c r y l o n i t r i l e (Cavins and Friedman, 1 9 6 7 ) , and i t s use f o r n u t r i t i o n a l studies has been proposed by F i n l e y and Friedman ( 1 9 7 3 ) . The methyl a c r y l a t e method i s based on condensing an a, 3-unsaturated compound wit h a v a i l a b l e amino groups. The r e a c t i o n of the a c c e s s i b l e amino groups (non-terminal) of l y s i n e i n p r o t e i n s w i t h excess methyl a c r y l a t e at pH 9.1 y i e l d s , a f t e r h y d r o l y s i s , mainly e,e,N,N ;-dicarboxyethyl-l y s i n e and a small amount of e,N-monOcarboxyethyllysine. The r e a c t i o n t ime can be c u t down t o 4 h r by u s i n g a 75% DMSO ( d i p o l a r a p r o t i c s o l v e n t ) : 25% (v/v) pH 9.1 B u f f e r . A v a i l a b l e l y s i n e i s measured by comparing l y s i n e c o n t e n t b e f o r e and a f t e r a l k y l a t i o n . F i n l e y and Friedman r e p o r t e d good agreement between the m e t h y l a e r y l a t e method and TNBS and DNFB p r o c e d u r e s on a s e r i e s o f pure p r o t e i n s . 6. E t h y l v i n y l s u l f o n e The e t h y l v i n y l s u l f o n e method (Friedman and F i n l e y , 197 5) may be used t o measure n u t r i t i o n a l l y a v a i l a b l e l y s i n e as t h e mono- and d i s u b s t i t u t e d e t h y l s u l f o n y l e t h y l d e r i v a -t i v e s o f l y s i n e and a l s o u n a v a i l a b l e l y s i n e t h a t i s l i b e r a t e d d u r i n g a c i d h y d r o l y s i s as l y s i n e r e m a i n i n g a f t e r e t h y l v i n y l s u l f o n e t r e a t m e n t . A v a i l a b l e l y s i n e i s measured as t h e d i f f e r e n c e between t h e l y s i n e c o n t e n t o f the s t a r t i n g m a t e r i a l b e f o r e and a f t e r e t h y l v i n y l s u l f o n e t r e a t m e n t . I n p r i n c i p l e , e t h y l v i n y l s u l f o n e can a l k y l a t e a l l f u n c t i o n a l groups c o n t a i n i n g a c t i v e hydrogens i n p r o t e i n s ; f o r i n s t a n c e , the e-amino group o f l y s i n e , s i d e c h a i n s and i m i d a z o l e groups o f h i s t i d i n e r e s i d u e s , by M i c h a e l - t y p e n u c l e o p h i l i c a d d i t i o n r e a c t i o n s . 7. 2 - C h l o r o - 3 , 5 - d i n i t r o p y r i d i n e S e l i m (1965) employed Sanger's r e a g e n t 1 - f l u o r o - 2 , 4 -d i n i t r o b e n z e n e (FDNB) t o e s t i m a t e the l y s i n e c o n t e n t o f p r o t e i n ' h y d r o l y z a t e s a f t e r b l o c k i n g t h e ot-amino groups o f the f r e e amino a c i d s w i t h copper. 38. A s i m i l a r p r i n c i p l e was used by T s a i e t a l . (1972) f o r t h e s c r e e n i n g o f l y s i n e c o n t e n t i n maize seeds. I n t h e i r method the d e f a t t e d p r o t e i n sample i s h y d r o l y z e d w i t h pronase o r a m i x t u r e o f a l c a l a s e and p a n c r e a t i c t r y p s i n , t o y i e l d f r e e amino a c i d s o r low m o l e c u l a r weight p e p t i d e s . The a - c a r b o x y l and a-amino groups o f t h e f r e e amino a c i d s are b l o c k e d w i t h c u p r i c i o n s (a copper phosphate s u s p e n s i o n ) , l e a v i n g the e-amino groups o f l y s i n e f r e e t o r e a c t . An e - d i n i t r o - p y r i d y l d e r i v a t i v e o f l y s i n e i s th e n formed on r e a c t i o n w i t h 2 - c h l o r o - 3 , 5 - d i n i t r o p y r i d i n e . E x t r a c t i o n o f the r e a c t i o n m i x t u r e w i t h e t h y l a c e t a t e removes the e x c e s s 2 - c h l o r o - 3 , 5 - d i n i t r o p y r i d i n e , l e a v i n g an aqueous s o l u t i o n o f e - D N P y r - l y s i n e . The absorbance o f t h i s s o l u t i o n i s then d e t e r m i n e d a t 400 nm. L y s i n e c o n t e n t as e s t i m a t e d by t h i s c o l o r i m e t r i c method i s u s a l l y h i g h e r than t h e e s t i m a t e o f l y s i n e c o n t e n t from the amino a c i d a n a l y z e r , but i t i s i n good g e n e r a l agreement. The h i g h e r l y s i n e v a l u e s found i n t h e c o l o r i m e t r i c assays may be a consequence o f the r e l a t i v e l y m i n o r c o n t r i -b u t i o n made t o absorbance by the a r g i n i n e p r e s e n t . 8. Sodium b o r o h y d r i d e Thomas (1970) used NaBH^ f o r the d e t e r m i n a t i o n o f t h e a v a i l a b l e l y s i n e c o n t e n t o f c o t t o n s e e d meal, a m a t e r i a l i n which l y s i n e u n i t s may combine w i t h t h e aldehyde groups o f 39. the g o s s y p o l pigment, u n l e s s p r o c e s s i n g i s c a r e f u l l y con-t r o l l e d . M a i l l a r d - t y p e compounds o f aldehyde and l y s i n e u n i t s b r e a k down on a c i d h y d r o l y s i s , t o g i v e a v a r i a b l e , b u t o f t e n h i g h r e c o v e r y o f l y s i n e . However, when th e y are t r e a t e d w i t h sodium b o r o h y d r i d e t h e y a re reduced t o a c i d - s t a b l e compounds. Thus, when p r o t e i n s are t r e a t e d w i t h f o r m a l d e -hyde f o l l o w e d by b o r o h y d r i d e t r e a t m e n t , e-N,N-dimethy1-l y s i n e u n i t s are formed (Means and Feeney, 1968). The ' t o t a l ' l y s i n e r e l e a s e d on a c i d h y d r o l y s i s f o l l o w i n g such t r e a t m e n t may, t h e r e f o r e , be a measure o f those l y s i n e u n i t s i n a t e s t m a t e r i a l t h a t had n o t r e a c t e d w i t h a l d e h y d e s . Thomas (1970) and Couch and Thomas (1976) r e p o r t e d c l o s e agreement between a v a i l a b l e l y s i n e d e t e r m i n e d by t h e FDNB- D i f f e r e n c e t e c h n i q u e and the sodium b o r o h y d r i d e method. The c h e m i c a l d a t a compared v e r y f a v o r a b l y w i t h t h e d a t a from c h i c k assay. The advantage o f the sodium b o r o h y d r i d e method, as compared w i t h the o f f i c i a l D N F B - D i f f e r e n c e t e c h n i q u e , i s t h a t i t e l i m i n a t e s t h e n e c e s s i t y o f making two h y d r o l y s e s and two d e t e r m i n a t i o n s on t h e amino a c i d a n a l y z e r i n o r d e r t o o b t a i n the a v a i l a b l e l y s i n e d a t a . 9. 1 9 F NMR Ramirez e t a l . (1975) have d e v e l o p e d a method t o determine the e-amino group o f l y s i n e by F NMR s p e c t r o s c o p y . F r e e e-amino groups o f l y s i n e are t r i f l u o r o a c e t y l a t e d w i t h 40. the r e a g e n t S - e t h y l t r i f l u o r o t h i o a c e t a t e i n d i m e t h y l s u l f o x i d e s o l u t i o n and t h e number o f such groups i s quan-19 t i t a t i v e l y d e t e r m i n e d u s i n g s t a n d a r d F NMR t e c h n i q u e . D i m e t h y l s u l f o x i d e i s used as t h e r e a c t i o n medium i n o r d e r t o improve t h e homogeneity o f t h e system, When the m i x t u r e becomes homogeneous, a p o r t i o n i s added d i r e c t l y t o 19 an NMR sample tube and the F NMR spectrum i s o b t a i n e d . A t y p i c a l spectrum i s shown i n F i g u r e 4. The resonance a t l o w e s t f i e l d (A) c o r r e s p o n d s t o t r i f l u o r o a c e t i c a c i d , a h y d r o l y s i s p r o d u c t o f t h e t h i o l e s t e r . The peak a t i n t e r m e d i a t e f i e l d (B) c o r r e s p o n d s t o the t r i f l u o r o a c e t y l a t e d e-amino groups o f l y s i n e , and t h e peak at h i g h e s t f i e l d ( C ) , t o the t r i f l u o r o a c e t y l group o f the t h i o l e s t e r ( u n r e a c t e d r e a g e n t ) . The r e l a t i v e i n t e g r a t e d i n t e n s i t i e s a re c a l c u l a t e d from the w e i g h t s o f t h e a p p r o p r i a t e l y c u t - o f f peaks. The m o l a r r a t i o o f l y s i n e t o t h e o r i g i n a l t h i o l e s t e r i s g i v e n by the r a t i o o f t h e w e i g h t o f the i n t e r m e d i a t e peak t o the t o t a l w e i g h t o f a l l peaks. The molar optimum r a t i o o f t h i o l r e a g e n t t o l y s i n e i s 4:1. H i g h e r c o n c e n t r a t i o n s o f t h i o l r e a g e n t l e a d t o double s u b s t i t u t i o n o f some l y s i n e s i t e s . W i t h lower con-c e n t r a t i o n s , n o t a l l the e-amino groups are a t t a c k e d . E x c e l l e n t agreement was found between th e composi-t i o n a l d a t a f o r pure m i l k p r o t e i n s and the l y s i n e c o n t e n t 19 d e t e r m i n e d w i t h F NMR. 1+1. FIGURE 4 . yF NMR spectrum o f the r e a c t i o n products o f a p r o t e i n and S - e t h y l t r i f l u o r o t h i o a c e t a t e i n dimethyl s u l f o x i d e s o l u t i o n . H 42. F NMR s p e c t r o s c o p y o f f e r s a r e a s o n a b l y a c c u r a t e , f a s t and r e l a t i v e l y s i m p l e p r o c e d u r e , w i t h o u t t h e d i f f i c u l t y o f v a r i a b i l i t y of r e s u l t s from v a r y i n g amounts o f p r o t e i n 19 p r e s e n t . I t does r e q u i r e a c c e s s t o a F NMR s p e c t r o m e t e r and f a i r l y c o n c e n t r a t e d p r o t e i n s o l u t i o n s . 10. The sodium d i n i t r o b e n z e n e s u l f o n a t e method (DNBS). The DNBS t e c h n i q u e f o r d e t e r m i n a t i o n o f a v a i l a b l e l y s i n e , u n l i k e most o f t h e c h e m i c a l methods so f a r d e s c r i b e d 19 ( 2 - c h l o r o - 3 , 5 - d i n i t r o p y r i d i n e and F NMR a r e t h e e x c e p t i o n ) does n o t r e l y on a c i d h y d r o l y s i s o f t h e t r e a t e d p r o t e i n i n o r d e r t o dete r m i n e a v a i l a b l e l y s i n e . P r e v i o u s methods f o r l y s i n e d e t e r m i n a t i o n u s i n g u n h y d r o l y z e d p r o t e i n s i n c l u d e t h e Van S l y k e n i t r o u s a c i d method (Van S l y k e , 1911, 1912) and t h e Hofman r e a c t i o n and i o d o m e t r i c back t i t r a t i o n method (Baraud, 1957). These methods however were n ot s p e c i f i c f o r t h e e-amino group o f l y s i n e . The work o f E i s e n e t a l . ( 1 9 5 3 ) , L i (1956) , I k e n a k a (1959)/ Hosoya (1960) and Sugae (196 0) s u g g e s t e d t h a t d i n i t r o b e n z e n e s u l f o n a t e c o u l d be used as a s p e c i f i c r e a g e n t f o r d e t e r m i n i n g l y s i n e i n p r o t e i n s w i t h o u t h y d r o l y s i s . E i s e n e t a l . (1953) demonstrated t h a t DNBS r e a c t s s p e c i f i c a l w i t h o n l y amino groups. L i (1956) r e a c t e d i n s u l i n w i t h DNBS f o r 216 h r a t 3°C i n 0.1 M sodium c a r b o n a t e and found t h a t the e-amino group o f l y s i n e was t h e o n l y r e a c t i v e species; N-terminal glycine and phenylalanine, and the residue tyrosine did not react with DNBS. When DNBS was reacted with takaamylase-A at pH 10.7, 37°C, only 11 of 22 lysine residues i n the enzyme molecule were dinitrophenylated after 50 hr (Ikenaka, 1959). S i m i l a r l y , 3 out of 25 lysine residues i n b a c t e r i a l amylase reacted a f t e r 74 hr (Sugae, 1960). The calcium acetate, used to minimize denaturation of the enzyme, and the low pH used by these authors could render many of the lysine residues unreactive by s t e r i c and e l e c t r o -s t a t i c factors. Concon (1971) confirmed the v a l i d i t y of DNBS as a reagent for lysine determination i n unhydrolyzed proteins, by reacting p u r i f i e d proteins with DNBS. The number of lysine residues per mole of protein was i n f u l l agreement with the reported values. Concon attempted to spe'ed up the DNBS reaction by increasing the pH, temperature and reaction time. I t became evident that the difference i n reaction rate between the a- and e-amino groups of lysine i s such that reaction conditions could be chosen i n which only the e-amino group w i l l react. The optimum combination of factors for s i g n i f i -cant reaction of the e-amino group with DNBS, lay within the following l i m i t s : pH 9.0 - 13.0; temperature 30 - 60°C; reaction time 0.5 - 2.0 hr. The r e a c t i v i t y of the e-amino groups with DNBS, increased almost l i n e a r l y with pH at 40°C. No reaction was observed at pH 9. The maximum r e a c t i v i t y appeared t o be between pH 12 and 13. A t pH above 12, t h e r e a c t i v i t y o f the a-amino group was a p p a r e n t l y absent due t o i n s t a b i l i t y o f the a~NH2-DNP d e r i v a t i v e . e-NB^-DNP d e r i v a t i v e was s t a b l e a t pH 12 even a f t e r 3 h r a t 40°C. There was no advantage t o be g a i n e d i n terms o f s p e c i f i c i t y by i n c r e a s i n g the temperature t o 45°C and a t the same time d e c r e a s i n g t h e pH t o 11. W i t h most p r o t e i n s a w o r k a b l e c o m b i n a t i o n o f pH 12.3, 40°C and 1 h r r e a c t i o n t i m e (pH 10.5, 60°C, 1 h r f o r r i c e ) gave q u i t e s a t i s f a c t o r y r e s u l t s . These v a l u e s were n e i t h e r optimum n o r i d e a l , b ut w i t h p ure l y s i n e t h e s e c o n d i t i o n s gave absorbance r e a d i n g s w i t h no d e t e c t a b l e c o n t r i b u t i o n from t h e a-amino groups. The s p e c i f i c i t y o f the DNBS under t h e a f o r e m e n t i o n e d c o n d i t i o n s , was shown by paper chromatography o f t h e a c i d h y d r o l y z e d , DNBS-treated p r o t e i n s . Only e-N-DNP-lysine was d e t e c t e d (Concon, 1975a). E v i d e n c e o f s p e c i f i c i t y was a l s o o b t a i n e d from a comparison o f the s p e c i f i c r e a c t i o n r a t e c o n s t a n t s f o r pure l y s i n e and c e r e a l p r o t e i n s . F o r g r a i n s a n a l y z e d a t pH 12.3, 40°C, 1 h r t h e average v a l u e o f K was -3 -1 2.5 x 10 min . The same v a l u e was found f o r pure l y s i n e . F o r r i c e , a n a l y z e d a t pH 10.5, 60°C, 1 h r t h e average v a l u e -3 -1 o f K was 6.2 x 10 min , whi c h was a l s o e x a c t l y the v a l u e o f l y s i n e under t h e s e c o n d i t i o n s . Large p o s i t i v e e r r o r s (20-40%) r e s u l t when l y s i n e i n r i c e i s d e t e r m i n e d a t pH 12.3 and 40°C. I t i s b e l i e v e d t h a t t h i s i s due t o the h i g h a r g i n i n e c o n t e n t o f r i c e . Under h i g h a l k a l i n e c o n d i t i o n s and e l e v a t e d t e m p e r a t u r e , a r g i n i n e may degrade t o o r n i t h i n e . O r n i t h i n e (pK^ = 10.8) r e a c t s w i t h DNBS s i m i l a r l y as l y s i n e (pK^ = 1 0 . 5 ) . I n a d d i t i o n , a r g i n i n e w i t h a pK^ = 12.5 may r e a c t s i g n i f i c a n t l y w i t h DNBS a t pH 12.3. Lo w e r i n g the pH t o 10.5 a p p a r e n t l y m i n i -m i z e d t h e s e r e a c t i o n s , even a t 60°C. The drop i n pH n e c e s s i t a t e d a change i n s u l f h y d r y l masking agent t o m e r c u r i c c h l o r i d e (HgCl^) s i n c e p h e n y l m e r c u r i c c h l o r i d e (PMC) p r e c i p i t a t e s below pH 12. Other g r a i n s g i v e l o w e r l y s i n e v a l u e s , by as much as 30-40%, when a n a l y z e d under the same c o n d i t i o n s used f o r t h e r i c e samples. I t i s p o s s i b l e t h a t because o f the h i g h a r g i n i n e c o n t e n t , r i c e p r o t e i n s have i n t e r n a l c o n d i t i o n s a l k a l i n e enough t o p e r m i t more complete d i s s o c i a t i o n o f the e-amino groups even though the pH o f the s u r r o u n d i n g s o l u t i o n i s lo w e r t h a n t h a t p e r m i t t e d by the Henderson-Hasselbach e q u a t i o n . A t pH 12.3, 40°C, 1 h r , t h e DNBS r e a c t i o n i s i n c o m p l e t e ; o n l y about 14% o f the t o t a l l y s i n e i s r e a c t e d . However, i t i s p o s s i b l e t o dete r m i n e d i r e c t l y t he amount o f l y s i n e from the f r a c t i o n r e a c t i n g i n 1 h r . K i n e t i c s t u d i e s i n d i c a t e t h a t i n t h e pr e s e n c e o f ex c e s s DNBS, t h e r e a c t i o n f o l l o w s f i r s t o r d e r k i n e t i c s as d e s c r i b e d by e q u a t i o n 1. The f r a c t i o n o f l y s i n e r e a c t i n g a t any g i v e n t i m e i s d i r e c t l y p r o p o r t i o n a l t o the t o t a l l y s i n e o r i g i n a l l y p r e s e n t . L t = [ ( e k t - l ) / e k t ] L-0 ( E q u a t i o n 1), where = t h e f r a c t i o n o f l y s i n e r e a c t i n g a t time t ; L ? 0 = t h e t o t a l l y s i n e o r i g i n a l l y p r e s e n t ; k - the s p e c i f i c r e a c t i o n r a t e c o n s t a n t ; and e = the base o f the n a t u r a l l o g a r i t h m . L-j. i s d i r e c t l y p r o p o r t i o n a l t o L Q a t any g i v e n t i m e . T h e r e f o r e , from t h e absorbance due t o L ^ , L Q may be de t e r m i n e d d i r e c t l y . A p l o t o f t h e absorbance due t o L^-v e r s u s L C Q conforms r e m a r k a b l y t o Beer and Lambert's law. S i n c e t h e DNBS r e a c t i o n i s h i g h l y dependent on a d i r e c t p r o p o r t i o n a l i t y between t o t a l l y s i n e and t h e f r a c t i o n r e a c t i n g i n any g i v e n t i m e , e v e r y p r o t e i n - b o u n d e-amino group must be e q u a l l y a c c e s s i b l e and r e a c t i v e . These r e q u i r e m e n t s are s a t i s f i e d by c o n d u c t i n g the d i n i t r o p h e n y -l a t i o n r e a c t i o n a t h i g h pH and i n h i g h c o n c e n t r a t i o n s o f ur e a . The DNBS r e a c t i o n d e t e r m i n e s t h e e-amino group o f l y s i n e i n u n h y d r o l y z e d p r o t e i n s . On the o t h e r hand, f r e e amino a c i d s may a l s o r e a c t w i t h DNBS. Tryptophan and c y s t e i n e are among the most r e a c t i v e . C y s t e i n e i s seven t i m e s more r e a c t i v e than f r e e l y s i n e because t h e SH group a l s o r e a c t s w i t h DNBS. The DNBS d e r i v a t i v e s o f f r e e amino a c i d s , e x c e p t f o r c y s t e i n e , a r g i n i n e and h i s t i d i n e , can be removed by e t h e r e x t r a c t i o n . A major p o r t i o n o f the c y s t e i n e i n t e r f e r e n c e i s e l i m i n a t e d by e t h e r e x t r a c t i o n . However, the s u l f h y d r y l group s h o u l d be masked by m e r c u r a t i o n w i t h PMC o r H g C l 2 . The s l i g h t r e a c t i v i t i e s o f the o t h e r b a s i c amino a c i d s are reduced by m e r c u r a t i o n . Mercury forms 47. complexes w i t h b a s i c amino a c i d s i n a l k a l i n e medium ( K a i , 1967) . The r e a c t i v i t i e s o f a r g i n i n e and h i s t i d i n e are q u i t e s m a l l when compared t o l y s i n e . T h e i r i n t e r f e r e n c e can be e l i m i n a t e d t o a c e r t a i n e x t e n t w i t h m e r c u r i c c h l o r i d e (but n o t PMC) and by use o f a s m a l l e r sample s i z e . When the r a t i o o f f r e e a r g i n i n e o r h i s t i d i n e t o l y s i n e approaches 1:2, u n r e l i a b l e r e s u l t s become more e v i d e n t even w i t h t h e use o f m e r c u r i c c h l o r i d e . The N - t e r m i n a l amino groups o f p e p t i d e s are even l e s s r e a c t i v e t o DNBS than the a-amino groups o f f r e e amino a c i d s . F o r example, the r e a c t i v i t y o f t r y p t o p h a n as t r y p t o p h a n y l - t r y p t o p h a n i s reduced by as much as 9 3%. I n p r o t e i n s , a t pH 12, 40°C, 1 h r r e a c t i o n t i m e , the N - t e r m i n a l amino group i s v i r t u a l l y u n r e a c t i v e t o DNBS. Once r e a c t e d w i t h DNBS r e a g e n t , the t e s t p r o t e i n i s s u b j e c t e d t o e t h e r e x t r a c t i o n , and the o p t i c a l d e n s i t y i s measured a t 385 nm. The maximum wavelength o f e-DNP-lysine i s between 36 0 and 365 nm. U n f o r t u n a t e l y , the components of the r e a c t i o n media (PMC, DNBS, phosphates) and the v a r i o u s c e r e a l p i g m e n t s , absorb s t r o n g l y a t these w a v e l e n g t h s . The DNBS method appears t o be r a p i d and s e n s i t i v e f o r the e-amino groups o f l y s i n e , w i t h t h e advantage t h a t i t does n o t r e q u i r e a c i d h y d r o l y s i s o f t h e p r o t e i n s l i k e most o f the o t h e r c h e m i c a l methods used f o r d e t e r m i n a t i o n o f l y s i n e a v a i l a b i l i t y . 48. 11. F l u o r e s c a m i n e Puree11 e t a l . (1976) have d e v e l o p e d a r a p i d method o f the d e t e r m i n a t i o n o f f r e e amino groups i n i n t a c t pure p r o t e i n s , where p r o t e i n , h y d r o l y s i s and e x t r a c t i o n o f a l a b e l e d l y s i n e d e r i v a t i v e i s n o t n e c e s s a r y . The method r e q u i r e s o n l y two s t e p s : l a b e l i n g the p r i m a r y amino groups w i t h 4 - p h e n y l - s p i r o - [ f uran-2- (3H) ~ 1 ' - p h t h a l a n ]-3 ; 3"' - d i o n e , commonly r e f e r r e d t o as f l u o r e s c a m i n e , and measuring the l a b e l e d groups by a b s o r p t i o n s p e c t r o s c o p y i n the range 375-390 nm. F l u o r e s c a m i n e , f i r s t s y n t h e s i z e d by W e i g e l e e t a l . ( 1972), r e a c t s s p e c i f i c a l l y w i t h p r i m a r y and secondary amino groups t o y i e l d a f l u o r e s c e n t and a n o n f l u o r e s c e n t p r o d u c t r e s p e c t i v e l y . The r e s u l t o f r e a c t i n g f l u o r e s c a m i n e w i t h p r o t e i n s y i e l d s a d i r e c t e s t i m a t e of t h e u n s u b s t i t u t e d e-amino groups and hence, o f t h e a v a i l a b l e l y s i n e c o n t e n t o f p r o t e i n s . However, the a-amino groups o f t h e p r o t e i n c h a i n i s a l s o l a b e l e d , and i s a minor c o n t r i b u t i o n t o t h e r e s u l t s o b t a i n e d . 12. D y e - b i n d i n g . D y e - b i n d i n g p r o c e d u r e s are r a p i d , i n e x p e n s i v e methods whi c h have been s u c c e s s f u l l y adapted t o a u t o m a t i c and semi-a u t o m a t i c systems o f a n a l y s i s . Three c l a s s e s o f d y e s t u f f s have been used f o r p r o t e i n q u a l i t y e v a l u a t i o n : p h t h a l e i n dyes, r e a c t i v e dyes and a c i d azo dyes ( L a k i n , 1973). Ney and V7irotama (1970) have r e p o r t e d 49. the use o f t h e r e a c t i v e dye, remazol b r i l l i a n t b l u e R f o r the d e t e r m i n a t i o n o f a v a i l a b l e l y s i n e i n m i l k and cheese. The dye, whose s t r u c t u r e i s shown i n F i g u r e 5 , when con-v e r t e d t o the v i n y l ( a c t i v e ) form by h e a t i n g i n s t r o n g a l k a l i , w i l l form a c o v a l e n t bond w i t h the f r e e €-amino group o f the l y s i n e r e s i d u e s and w i t h t h e t h i o l group o f c y s t e i n e . No r e a c t i o n s were o b s e r v e d w i t h the h y d r o x y l groups o f s e r i n e and t y r o s i n e . The a u t h o r s c l a i m e d t h a t r e a c t i o n w i t h c y s t e i n e can be d i s r e g a r d e d i n the case o f m i l k p r o t e i n s . F o r d e t e r m i n i n g a v a i l a b l e l y s i n e t h e p r o t e i n i s r e a c t e d w i t h r e m a z o l b r i l l i a n t b l u e R, and the m i x t u r e i s p a s s e d o v e r Sephadex G-25 c o a r s e . The dyed p r o t e i n i s e l u t e d a f t e r 10 minutes and d i s c a r d e d . The unbound dye i s e l u t e d o v e r the n e x t 2 h r and i t s c o n c e n t r a t i o n i s d e t e r m i n e d s p e c t r o p h o m e t r i c a l l y . The amount o f bound dye i s c a l c u l a t e d by comparing t h e 280 nm e x t i n c t i o n o f t h e e l u a t e w i t h t h a t o f d i f f e r e n t s o l u t i o n s o f the o r i g i n a l a c t i v a t e d dye s o l u t i o n i n pH 8 b u f f e r . T h i s v a l u e was found t o d e c r e a s e w i t h t i m e when m i l k was m a i n t a i n e d a t 95°C; t h a t i s , t h e m i l k p r o t e i n s bound l e s s dye because o f t h e i r c o n t e n t o f a v a i l a b l e l y s i n e f e l l . FIGURE 5 i S t r u c t u r e o f the r e a c t i v e dye remazol b r i l l i a n t blue R. (From Ney and Wirotama, 1 9 7 0 ) . 51. Although Pruss and Ney (1972) demonstrated no i n t e r -ference from lactose and other sugars, the method suffers from the disadvantage that the reaction i s dependent on the quantity of protein present i n the sample. As the quantity of protein increases, the amount of dye bound de-creases. Most of the dye-binding r e s u l t s reported i n the l i t e r a t u r e are given i n terms of "dye-binding capacity" or "DBC". DBC i s an almost pseudo-scientific term which implies that a given amount of protein has an a f f i n i t y for a given amount of dye. This value depends upon the experimental conditions, the structure of the dye molecule, the composition' of the buffer system and the purity of the dye. The tempera-ture of the system and i t s pH are also important factors. Therefore, unless these parameters are s p e c i f i e d , a state-ment of dye-binding capacity has no r e a l meaning and may even prove to be misleading (Lakin, 1973). Although quantitative determination of available ly s i n e i s d i f f i c u l t with t h i s technique, dye-binding pro-cedures could be used to monitor losses of lysine during the processing of food proteins, as long as the protein content of the material remains constant. 13. Chemical methods i n the determination of available lysine i n materials that have undergone Mai H a r d reactions. The type of damage which some of the chemical pro-cedures f a i l to measure adequately i s that occurring i n m a t e r i a l s where l y s i n e and r e d u c i n g sugars have been i n p r o l o n g e d c o n t a c t a t r e l a t i v e l y low te m p e r a t u r e s (37°C), and i n w h i c h ' e a r l y ' M a i l l a r d compounds, o f which a - N - f o r m y l -( e - N - d e o x y f r u c t o s y 1 ) - l y s i n e (FFL) i s a model ( F i n o t and Mauron, 1972), would be e x p e c t e d . F i n o t and Mauron (1972) r e a c t e d d e o x y - k e t o s y l d e r i v a t i v e s o f l y s i n e w i t h FDNB ( C a r p e n t e r , 196 0 ) , w i t h TNBS (Kakade and L i e n e r , 1969), and w i t h O - m e t h y l - i s o u r e a (Mauron and B u j a r d , 1963). When C a r p e n t e r ' s method was a p p l i e d t o FFL i t was found t h a t 14-2 8% o f t h i s u n a v a i l a b l e form was d e t e r m i n e d as a v a i l a b l e l y s i n e . G u a n i d i n a t i o n u s i n g 0-methyl-iso.urea was the most s p e c i f i c ( 0 % ) , whereas t h e TNBS method was c o m p l e t e l y n o n - s p e c i f i c (82-89%). Presumably, TNBS r e a c t s w i t h the b a s i c s e c o n d a r y amine groups o f t h e e a r l y M a i l l a r d compound and t h e n on a c i d d i g e s t i o n t h e p r e s e n c e o f the TNP groups weakens the s u g a r l i n k a g e which s p l i t s o f f t o g i v e a h i g h y i e l d o f T N P - l y s i n e . O - m e t h y l - i s o u r e a appears t o be a h i g h l y s e n s i t i v e i n d i c a t o r o f a l l t y p e s o f l y s i n e b i n d i n g , i n c l u d i n g t h e f o r m a t i o n o f e a r l y M a i l l a r d compounds. A l t h o u g h i t cannot be recommended i n i t s p r e s e n t form as a r o u t i n e q u a l i t y -c o n t r o l measure because o f t h e t i m e t a k e n (3 days) f o r an i n d i v i d u a l d e t e r m i n a t i o n , and a l s o because d i f f e r e n t pH v a l u e s are needed f o r d i f f e r e n t m a t e r i a l s , i t i s o f s p e c i a l i n t e r e s t as a r e f e r e n c e method. When FFL was s u b j e c t e d to FDNB-difference treatment ( F i n o t and Mauron, 1972; H u r r e l l and Carpenter, 1974) , t o t a l l y s i n e minus FDNB-reactive l y s i n e was c o n s i d e r a b l y g r e a t e r than a v a i l a b l e l y s i n e . T h i s f i n d i n g showed t h a t FDNB v/ould s t i l l r e a c t with a l y s i n e u n i t i n which the e p s i l o n amino group was bound t o a sugar. The FDNB-lysine-sugar complex breaks down on a c i d h y d r o l y s i s t o y i e l d n e i t h e r l y s i n e nor DNP-lysine t o any s i g n i f i c a n t e x t e n t . I t can be concluded that Carpenter's FDNB, O-methyl-i s o u r e a and sodium borohydride methods ( H u r r e l l and Carpenter, 1974) g i v e a good estimate o f the a v a i l a b l e l y s i n e i n ' e a r l y M a i H a r d 1 damage as seen i n r o l l e r - d r i e d m i l k powders (Mottu and Mauron, 1967). With 'advanced M a i l l a r d ' and p r o t e i n - p r o t e i n damage, where there i s a decrease i n o v e r a l l p r o t e i n d i g e s t i b i l i t y , these procedures may s t i l l over-estimate a v a i l a b l e l y s i n e , but they are, of course, more s e n s i t i v e i n d i c a t o r s of damage than i s t o t a l - l y s i n e d e t e r m i n a t i o n . F i n o t (1973) suggested a method f o r the e v a l u a t i o n o f a v a i l a b l e l y s i n e i n heat-damaged milk powders based on the d e t e r m i n a t i o n o f t o t a l l y s i n e and f u r o s i n e content measured a f t e r a c i d h y d r o l y s i s of the p r o t e i n . When pure e-N-deoxy-k e t o s y l - l y s i n e d e r i v a t i v e s are h y d r o l y z e d , three main compounds are o b t a i n e d : l y s i n e , f u r o s i n e and p y r i d o s i n e . These compounds are always present i n the same proportions, the r e s u l t being dependent only on the concentration of the acid used for hydrolysis (Finot and Mauron, 1972). The quantity of lysine blocked i n the deoxy-ketosyl form, that i s , unavailable l y s i n e , can be calculated d i r e c t l y from an acid hydrolysate of the protein. H u r r e l l and Carpenter (1974) reported that autoclaving protein with sucrose for 2 hr resulted i n less furosine production than did 1 hr. Presumably t h i s i s because the more complex polymers formed i n the advanced stages of the M a i l l a r d reaction do not break down to furosine on acid hydrolysis. Sulser (1973) has reported s i m i l a r r e s u l t s . While the presence of furosine i n the acid-hydrolysate of a test material indicates that some of i t s lysine has been involved i n M a i l l a r d reactions, i t cannot be used as a quantitative i n d i c a t o r i n a l l instances of protein-sugar damage. D. ENZYMATIC METHODS Even though the amino acid p r o f i l e i s important i n evaluating the n u t r i t i v e q u a l i t y of a protein, the d i g e s t i -b i l i t y of that protein i s the primary determinant of the a v a i l a b i l i t y of i t s amino acids. The d i g e s t i b i l i t y of a food protein may be obtained by using a rat bioassay but t h i s i s an expensive and time comsuming procedure. Several in vitro-enzymic digestion methods for the measurement of protein a v a i l a b i l i t y have been developed. One of the e a r l i e s t procedures was that of Melnick et a l . (1946) . I n h i s p r o c e d u r e food p r o t e i n s were d i g e s t e d w i t h p a n c r e a t i n . At i n t e r v a l s , a l i q u o t s o f t h e i n c u b a t i o n m i x t u r e were withdrawn and t h e degree o f h y d r o l y s i s was measured by f o r m o l t i t r a t i o n . I t was found t h a t t h o s e f a c t o r s known t o i n c r e a s e t h e n u t r i t i v e v a l u e o f soy p r o t e i n a l s o i n c r e a s e d the s u s c e p t i b i l i t y o f t h e p r o t e i n t o enzyma-t i c d i g e s t i o n . I t was a l s o found t h a t m e t h i o n i n e i s r e l e a s e d ' e a r l i e r from heat p r o c e s s e d soy meal than from raw soy meal. M e l n i c k and co-workers (1946), and R i e s e n e t a l . (1947) p r o p o s e d t h a t i n a d d i t i o n t o the t o t a l amino a c i d c o m p o s i t i o n , the r a t e o f r e l e a s e o f amino a c i d s from p r o t e i n by p a n c r e a t i c d i g e s t i o n was an i m p o r t a n t f a c t o r i n t h e n u t r i t i o n a l q u a l i t y o f a p r o t e i n . T h i s c oncept was u t i l i z e d by Horn e t a l . (195 3) t o e v a l u a t e t h e n u t r i t i o n a l q u a l i t y o f food p r o t e i n s by m e a s u r i n g m i c r o b i o l o g i c a l l y t h e i n d i v i d u a l amino a c i d s made a v a i l a b l e by p e p s i n , t r y p s i n , and hog mucosa. T h i s method gave good c o r r e l a t i o n w i t h the b i o l o g i c a l v a l u e o f c o t t o n s e e d meal w h i c h had been s u b j e c t e d t o v a r i o u s degrees o f p r o c e s s i n g ; however, t h e r e was no i n d i c a t i o n t h a t i t c o u l d be used t o compare p r o t e i n s from d i f f e r e n t s o u r c e s . Evans and B u t t s (1948) found t h a t enzymic h y d r o l y s i s o f soybean p r o t e i n b r o u g h t about some l o s s o f a l l e s s e n t i a l amino a c i d s , i n the sense t h a t they had been r e n d e r e d u n a v a i l a b l e f o r l i b e r a t i o n by d i g e s t i v e enzymes, though t h e y were e v i d e n t l y r e g e n e r a t e d by a c i d h y d r o l y s i s . When h e a t i n g i n t h e p r e s e n c e o f s u c r o s e , l a r g e amounts o f the b a s i c and s u l f u r amino a c i d s had becQme u n a v a i l a b l e and the l o s s e s were much g r e a t e r than i n t h e absence o f s u c r o s e . S h e f f n e r e t a l . (1956) by c o m b ining the p a t t e r n o f e s s e n t i a l amino a c i d s r e l e a s e d by i n v i t r o p e p s i n d i g e s t i o n w i t h the amino a c i d p a t t e r n o f the remainder o f t h e p r o t e i n , d e s c r i b e d an i n t e g r a t e d i n d e x , t h e P e p s i n - D i g e s t - R e s i d u e (PDR) amino a c i d i n d e x . D i v i s i o n o f t h e PDR i n d e x by the d i g e s t i b i l i t y c o e f f i c i e n t o f the r e s p e c t i v e p r o t e i n s , y i e l d e d v a l u e s w h i c h a c c u r a t e l y p r e d i c t e d the b i o l o g i c a l v a l u e o f the p r o t e i n s s t u d i e d . S i n c e e n z y m a t i c h y d r o l y s i s may be i n h i b i t e d by the h y d r o l y s i s p r o d u c t s w h i c h i n v i v o are r a p i d l y a b s o r b e d , d i a l y s i s (Mauron e t a l . , 1955) and g e l f i l t r a t i o n (Ford and S a l t e r , 1966) have been used t o remove the amino a c i d s as they are r e l e a s e d . Mauron e t a l . (1955) employed an i n -v i t r o d i g e s t i o n p r o c e d u r e i n w h i c h the t e s t p r o t e i n was s i m u l t a n e o u s l y d i g e s t e d and d i a l y z e d , f i r s t w i t h p e p s i n and then w i t h p a n c r e a t i n . A l t h o u g h the method has been employed o c c a s i o n a l l y f o r o t h e r f o o d p r o t e i n s , i t has been used e x t e n s i v e l y i n the q u a l i t y c o n t r o l o f h e a t p r o c e s s e d m i l k . I n m i l k , the l o s s o f l y s i n e a v a i l a b i l i t y i s due t o a M a i l l a r d type r e a c t i o n o f t h e l y s i n e s i d e c h a i n w i t h l a c t o s e . T h i s l i n k a g e a t the e-amino group i s r e s i s t a n t t o the a c t i o n o f the d i g e s t i v e enzymes. A comparison o f the v a l u e s f o r a v a i l a b l e l y s i n e o b t a i n e d by t h i s e n z y m a t i c p r o c e d u r e w i t h those a c h i e v e d w i t h C a r p e n t e r ' s c h e m i c a l method (1960) and those from b i o l o g i c a l evaluation based on growth, showed good conformity between the three methods, on a series of milk samples (Bujard et a l . , 1 9 6 7 ; Mottu and Mauron, I 9 6 7 ) . Ford and Salter (1966) attempted the continuous removal of reaction products from the i n v i t r o digestion system, by causing the enzyme-substrate mixture to pass through a calibrated column of Sephadex gel G-25. Each digest was thus resolved into four f r a c t i o n s : the residue and three soluble f r a c t i o n s , 'soluble protein', 'peptide', and 'free amino acids'. Freeze-dried cod f i l l e t s subjected to d i f f e r e n t heat treatments were used as t e s t proteins. Microbiological assay of a l l the fractions and the o r i g i n a l test protein after prolonged digestion with pepsin, pan-creatin and erepsin, gave broadly s i m i l a r r e s u l t s . With increasing temperature, the 'free amino aci d 1 component i n the digests became increasingly d e f i c i e n t i n several amino acids r e l a t i v e to t h e i r content i n the o r i g i n a l unheated meal notably i n lysine and the sulphur-containing amino acids. Mauron (197 0) calculated the i n - v i t r o amino acid a v a i l a b i l i t y from the data of Ford and Salter, by d i v i d i n g the value in the 'free amino acid' f r a c t i o n of the heated samples by the corresponding value i n the unheated sample. He found good agreement between his i n - v i t r o digestion pro-cedure aft e r d i a l y s i s and the results of Ford and Salter. 58. A n other way t o s e p a r a t e t h e u n d i g e s t e d r e s i d u e from the low m o l e c u l a r w e i g h t s o l u b l e f r a c t i o n s was e x p e r i m e n t e d by P r a h l and T a u f e l (1966). These a u t h o r s r e p l a c e d d i a l y s i s , by f i l t r a t i o n t h r o u g h a membrane under p r e s s u r e . Amino a c i d d a t a are however n o t y e t a v a i l a b l e w i t h t h i s method, s i n c e o n l y n i t r o g e n was measured. Akeson and Stahmann (1964) used p e p s i n f o l l o w e d by p a n c r e a t i n d i g e s t i o n t o e s t i m a t e t h e d i g e s t i b i l i t y and p r o t e i n q u a l i t y o f p r o c e s s e d f o o d s . Upon d i g e s t i o n o f the p r o t e i n , the u n d i g e s t e d p r o t e i n and p e p t i d e s were p r e c i p i t a -t e d w i t h p i c r i c a c i d and the amino a c i d s were d e t e r m i n e d by a u t o m a t i c amino a c i d a n a l y s i s . U s i n g whole egg as a s t a n d a r d they found e x c e l l e n t c o r r e l a t i o n between t h e p e p s i n -p a n c r e a t i n i n d e x v a l u e s f o r 12 p r o t e i n s and the b i o l o g i c a l v a l u e s r e p o r t e d i n the l i t e r a t u r e , from f e e d i n g t r i a l s . I n 1975, Stahmann and W o l d e g i o r g i s s l i g h t l y m o d i f i e d the p r o c e d u r e o f Akeson and Stahmann by i n t r o d u c i n g s u l f o -s a l i c y l i c a c i d , i n s t e a d o f p i c r i c a c i d , as a p r e c i p i t a n t o f the u n d i g e s t e d p r o t e i n and p e p t i d e s . S u l f o s a l i c y l i c a c i d i s n i n h y d r i n n e g a t i v e ( P e r r y and Hansen, 1969) and can be d i r e c t l y a p p l i e d t o the columns f o r amino a c i d a n a l y s i s . Menden and Cremer (19 66) r e p o r t e d t h a t h y d r o l y s i s by p a n c r e a t i n a l o n e o r i n c o m b i n a t i o n w i t h a c i d h y d r o l y s i s g i v e s a b e t t e r i n d i c a t i o n o f p r o t e i n q u a l i t y o f p r o c e s s e d c a s e i n and meat p r o d u c t s than a c i d h y d r o l y s i s a l o n e . They s u b j e c t e d the p r o t e i n t o a s h o r t d i g e s t i o n t i m e w i t h a l a r g e amount of pancreatin. The authors argue that the i n i t i a l steps i n digestion are of importance and that i t i s pre-ferable to digest with a high concentration of enzyme for a shorter period, so as to avoid auto-hydrolysis of the enzymes used. Buchanan (1969) and Buchanan and Byers (1969) des-cribed an i n v i t r o system for measuring protein d i g e s t i b i l i t y on a v<heat leaf protein concentrate with an enzymatic digestion u t i l i z i n g papain. Examination of the data shows a f a i r degree of correlation between the i n vivo feeding t r i a l s and in v i t r o figures on a freeze-dried material, but a serious difference i n the case of the same material after moist heat treatment. Buchanan was unable to obtain a correlation of protein d i g e s t i b i l i t y by Akeson and Stahmann's procedure, and his res u l t s with papain showed poor c o r r e l a -tion between i n vivo and i n v i t r o data. Saunders et a l . (1973) employed the pepsin-pancreatin system described by Akeson and Stahmann (1964), and the papain method of Buchanan and Byers (1969) to determine the protein d i g e s t i b i l i t y of a l f a l f a protein concentrates. They found excellent c o r r e l a t i o n between the values obtained from the enzyme system used by Akeson and Stahmann and i n vivo data (0.874) , whereas very poor c o r r e l a t i o n was found between the papain digestion and i n vivo data. The papain system may have produced low values compared to the i n vivo data 60. because o f i n t e r f e r e n c e i n t h e d i g e s t i o n by t h e h i g h l i p i d l e v e l i n a l f a l f a p r o t e i n c o n c e n t r a t e s . Saunders e t a l . (1973) d e v e l o p e d a p e p s i n - t r y p s i n system, t h e i n v i t r o r e s u l t s o f which c o r r e l a t e d w e l l ( r = 0.914) w i t h i n v i v o d i g e s t i b i l i t y . A f t e r the p e p s i n -t r y s i n d i g e s t i o n , the p r o t e i n r e m a i n i n g i n s o l u b l e was a n a l y z e d f o r n i t r o g e n c o n t e n t and the p r o t e i n d i g e s t i b i l i t y was c a l c u l a t e d as the d i f f e r e n c e between t h e n i t r o g e n i n the p r o t e i n c o n c e n t r a t e and the n i t r o g e n i n t h e u n d i g e s t e d f r a c t i o n . Rayner and Fox (1976) have c r i t i s i z e d the f a c t t h a t most o f t h e i n v i t r o e n z y m a t i c methods do n o t r e s u l t i n maximum d i g e s t i b i l i t y d u r i n g e n z y m o l y s i s , so t h a t the d e t e r m i n a t i o n o f amino a c i d s r e l e a s e d from a p r o t e i n i s n o t an a b s o l u t e method o f measuring a v a i l a b i l i t y . A r e f e r e n c e s t a n d a r d r e p r e s e n t e d by an o p t i m a l l y t r e a t e d t e s t m a t e r i a l i s t h e r e f o r e n e c e s s a r y . Rayner and Fox (1976) used t h e enzyme pronase t o d i g e s t a u t o c l a v e d r a p e s e e d meals. A f t e r i n c u b a t i o n , t h e u n d i g e s t e d p r o t e i n was p r e c i p i t a t e d w i t h p i c r i c a c i d and the amino a c i d s were d e t e r m i n e d on an amino a c i d a n a l y z e r . A c o r r e l a t i o n c o e f f i c i e n t o f 0.991 was o b t a i n e d when comparing i n • v i t r o a v a i l a b l e l y s i n e w i t h S i l c o c k a v a i l a b l e l y s i n e . The r e c o v e r y o f amino a c i d s from the pronase i n - v i t r o e n z y m o l y s i s showed r e c o v e r i e s a p p r o a c h i n g 10 0% f o r most amino a c i d s , b u t low r e c o v e r i e s f o r c y s t e i n e , m e t h i o n i n e and t r y o s i n e , p r o b a b l y due t o o x i d a t i o n by p i c r i c a c i d . An i n t e r e s t i n g r e s u l t was the h i g h r e c o v e r y o f t r y p t o p h a n . Akeson and Stahmann (1964) r e p o r t e d t h a t t r y p t o p h a n i s d e s t r o y e d by p i c r i c a c i d , however t h e s e r e s u l t s i n d i c a t e d t h a t t r y p t o p h a n can be d e t e r m i n e d by t h i s p r o c e d u r e . The a p p l i c a t i o n o f the pronase i n v i t r o p r o c e d u r e , i a l s o a l l o w s t o d i s c o v e r the p r e s e n c e o f ."trypsin i n h i b i t o r s . Trop and B i r k (19 70) r e p o r t e d t h a t 90% of n a t u r a l t r y p s i n i n h i b i t o r s i n a c t i v a t e p r o n a s e . The method d e v e l o p e d by Dvorak (196 8) a l s o uses t o t a l e n z y m a t i c d i g e s t i o n t o determine t h e maximum t h e o r e t i -c a l amount o f a v a i l a b l e amino a c i d s . A v a i l a b l e e s s e n t i a l amino a c i d s from n a t i v e and h e a t - t r e a t e d beef serum a l b u m i n , i n the p r e s e n c e of g l u c o s e o r w i t h o u t i t , were d e t e r m i n e d m i c r o b i o l o g i c a l l y , a f t e r t o t a l enzymic h y d r o l y s i s o f t h e p r o t e i n by p a p a i n , l e u c i n e a minopeptidase and p r o l i d a s e . T h i s enzyme c o m b i n a t i o n was p r e v i o u s l y used by H i l l and Schmidt (1962). I n t h i s system l e u c i n e a minopeptidase i s used t o h y d r o l y z e a l l p e p t i d e bonds t h a t remain i n t a c t i n p a p a i n h y d r o l y s a t e s , e x c e p t those w h i c h c o n t a i n the i m i n o n i t r o g e n o f p r o l i n e . P r o l i n e p e p t i d e s are h y d r o l y z e d by p r o l i d a s e . R e s u l t s o f t h i s s t u d y showed t h a t the a v a i l -a b i l i t y o f amino a c i d s i n n a t i v e serum a l b u m i n , m i c r o -b i o l o g i c a l l y d e t e r m i n e d a f t e r t o t a l enzyme d i g e s t i o n , corresponds to the t h e o r e t i c a l amount i n the protein. Maga et a l . (1973) pointed out that the i n i t i a l rates of hydrolysis by trypsin on some commonly used protein sources were good indicators of the protein d i g e s t i b i l i t y . After incubation of the protein with trypsin, incubation mixtures were withdrawn at one minute i n t e r v a l s and the pH was immediately recorded. In a l l products, most of the proteolysis occurred during the f i r s t few minutes and then remained constant. Steaming of the products resulted i n faster hydrolysis rates. I t became apparent that although certain vegetable proteins may have a high n u t r i t i o n a l and biochemical values, t h e i r digestive a c c e p t a b i l i t y may be quite poor since they are not rapidly hydrolyzed i n the digestive system. Rhinehart (1975) modified Maga's procedure and examined several enzyme systems which included t r y p s i n , pepsin-trypsin, trypsin-chymotrypsin, and t r y p s i n -chymotrypsin-peptidase combinations. The results were encouraging, with co r r e l a t i o n c o e f f i c i e n t s of 0.79, 0.72, 0.80 and 0.74, respectively. These aforementioned i n v i t r o methods have not been widely accepted, either because no corresponding i n vivo data were presented or because the procedures were complicated and time consuming, consequently/ d i f f i c u l t for application i n r o u t i n e i n - p l a n t q u a l i t y c o n t r o l . Hsu e t a l . (1977) d e v e l o p e d a multienzyme system f o r the e s t i m a t i o n o f p r o t e i n d i g e s t i b i l i t y . The method was based on t h e d i g e s t i o n o f an aqueous p r o t e i n s u s p e n s i o n a t 37°C, pH 8, u s i n g a c o m b i n a t i o n o f t r y p s i n , c h y m o t r y p s i n and p e p t i d a s e . As t h e p r o t e o l y t i c enzymes a t t a c k and break the p e p t i d e bonds w i t h i n t h e p r o t e i n ' s p r i m a r y s t r u c t u r e , the f r e e d c a r b o x y l groups t h a t f o rmed,immediately r e l e a s e d H +, w h i c h , i n t u r n , l o w e r e d the pH o f the p r o t e i n s u s p e n s i o n . The pH drop was m o n i t o r e d w i t h a r e c o r d i n g pH meter. The pH o f t h e p r o t e i n s u s p e n s i o n a f t e r 10 minutes d i g e s t i o n w i t h the multienzyme s o l u t i o n was h i g h l y c o r r e l a t e d w i t h t h e i n v i v o apparent d i g e s t i b i l i t y o f r a t s . Hsu e t a l . were a b l e t o demonstrate t h a t the m u l t i -enzyme t e c h n i q u e was u n a f f e c t e d by f o o d l i p i d s and b u f f e r i n g s a l t s commonly found i n f o o d s , and was s e n s i t i v e enough t o be a b l e t o d e t e c t the p r e s e n c e o f soybean t r y p s i n i n h i b i t o r i n soy f l o u r s , and c h l o r o g e n i c a c i d i n l e a f p r o t e i n concen-t r a t e . The method was a l s o s e n s i t i v e enough t o d e t e c t changes i n p r o t e i n d i g e s t i b i l i t y t h a t o c c u r r e d d u r i n g t h e heat p r o c e s s i n g o f s p e c i f i c f o o d s . A multienzyme system c o u l d reduce the e f f e c t caused by a s p e c i f i c enzyme i n h i b i t o r . C o n s e q u e n t l y , u s i n g a com-b i n a t i o n o f enzyme i n s t e a d o f t r y p s i n a l o n e , c o u l d a v o i d u n d e r - p r e d i c t i n g the d i g e s t i b i l i t y o f p r o t e i n s c o n t a i n i n g trypsin i n h i b i t o r . A single enzyme system that attacks at a s p e c i f i c peptide bond may give d i f f e r e n t r e s u l t s for proteins containing d i f f e r e n t concentrations of the s p e c i f i c amino acid. A multienzyme system reduces the l i m i t a t i o n s that are evident for a single enzyme system, and gives a better approximation of protein d i g e s t i b i l i t y . The most s i g n i f i c a n t advantage of this i n v i t r o method for pre-d i c t i n g apparent protein d i g e s t i b i l i t y i s that i t can be completed within one hour and with a high degree of s e n s i t i v i t y . MATERIALS AND METHODS A. M a t e r i a l s B - l a c t o g l o b u l i n was o b t a i n e d from N u t r i t i o n a l B i o c h e m i c a l C o r p o r a t i o n , C l e v e l a n d , Ohio. I t was c r y s t a l -l y z e d and l y o p h i l i z e d powder from m i l k . S a l t - f r e e lysozyme was p u r c h a s e d from W o r t h i n g t o n B i o c h e m i c a l C o r p o r a t i o n , F r e e h o l d , N.J. 1.5 N HCI s o l u b i l i z e d g l u t e n was p r e p a r e d a c c o r d i n g t o the method o f Wu e t a l . (1976). C a s e i n (pH 7.5). and d r i e d whole egg s o l i d s were p r e p a r e d i n t h e l a b o r a t o r y . R i b o n u c l e a s e - S - p e p t i d e (bovine p a n c r e a s , grade X I I - P E , MW 2166.35), p a n c r e a t i n ( p o r c i n e p a n c r e a s , grade V I ) , and p e p s i n (hog stomach mucosa, 2x c r y s t a l l y z e d and l y o p h i l i z e d powder) were the p r o d u c t s o f Sigma C h e m i c a l Company, S a i n t L o u i s , Mo. The amino a c i d d e r i v a t i v e s N - e - f o r m y l - L - l y s i n e (MW 174.2), L - l y s y l - L - l y s i n e d i h y d r o c h l o r i d e (MW 347.3) and L - l y s y l - L - a l a n i n e d i h y d r o b r o m i d e (MW 379.1) were from Sigma C h e m i c a l Company. N - a - f o r m y l - L - l y s i n e (MW 174.2) was s y n t h e s i z e d a c c o r -d i n g t o the method d e s c r i b e d by Hofmann e t a l . (1960). A c e t i c a n h y d r i d e was added t o a s o l u t i o n o f L - l y s i n e formate i n 9 8% f o r m i c a c i d . L - l y s i n e formate was p r e p a r e d from l y s i n e m o n o h y d r o c h l o r i d e w i t h A m b e r l i t e IR-4B i n the formate c y c l e . The s o l v e n t s were e v a p o r a t e d . The r e s u l t i n g o i l was d i s s o l v e d i n e t h a n o l and the c r y s t a l s were c o l l e c t e d and r e c r y s t a l l i z e d from e t h a n o l . The p u r i t y o f the compound was a s s e s s e d on an amino a c i d a n a l y z e r . a-and e - c a r b o b e n z o x y - l y s i n e s were o b t a i n e d from Eastman O r g a n i c C h e m i c a l s , D a l l a s , Texas. £-TNP-L-lysine. HCI. H 20 (MW;'. 411.:8) was from ICN P h a r m a c e u t i c a l I n c . , L i f e S c i e n c e Group, C l e v e l a n d , Ohio. l - f l u o r o - 2 , 4 - d i n i t r o b e n z e n e (FDNB)was from Calbiochem, San Diego, CA. 2 , 4 - d i n i t r o b e n z e h e s u l f o n i c a c i d (DNBS) and 2 , 4 , 6 . - t r i n i t r o b e n z e n e s u l f o n i c a c i d (TNBS) i n t h e form o f t r i h y d r a t e (80% min. anhydrous by t i t r a t i o n ) were o b t a i n e d from Eastman Kodak Company, R o c h e s t e r , N.Y. 5 - s u l f o s a l i -c y l i c . a c i d (98%) was from A l d r i c h C h e m i c a l Company, I n c . B. N i t r o g e n d e t e r m i n a t i o n The n i t r o g e n c o n t e n t o f $ - l a c t o g l o b u l i n , l y s o z y m e , c a s e i n , a c i d s o l u b i l i z e d g l u t e n and eggs was d e t e r m i n e d by the r a p i d m i c r o - K j e l d a h l p r o c e c u r e o f Concon and S o l t e s s (1973). The p r o t e i n c o n t e n t was c a l c u l a t e d by m u l t i p l y i n g the n i t r o g e n conten by a f a c t o r o f 6.25 f o r egg and lysozyme; 6.38 f o r c a s e i n and ( 3 - l a c t o g l u b i n ; 5.7 f o r g l u t e n . C. Amino a c i d a n a l y s i s . The d i g e s t e d p r o t e i n was a n a l y z e d on a s i n g l e column system (Durrum C h e m i c a l C o r p o r a t i o n , P a l o A l t o , CA) a t t a c h e d t o a P h o e n i x Model M6 800 Amino A c i d A n a l y z e r (Phoenix P r e c i s i o n I n s t r u m e n t Company). D. L y s i n e d e t e r m i n a t i o n by the d i n i t r o b e n z e n e s u l f o n i c a c i d method. The l y s i n e a v a i l a b i l i t y o f v a r i o u s p r o t e i n s was de t e r m i n e d a c c o r d i n g t o the method d e v e l o p e d tfoy Concon (1971). The r e a g e n t 2 , 4 - d i n i t r o b e n z e n e s u l f o n i c a c i d i s used t o com-p l e x t h e f r e e e-amino groups o f l y s i n e . Reagents Reagent A 50 g o f u r e a and 7 g o f Na^PC^ ,. I 2 H 2 O were d i s s o l v e d i n 36 ml o f hot wat e r w i t h a magnetic s t i r r i n g b a r , then 1 0 0 mg o f p h e n y l m e r c u r i c c h l o r i d e was added. The s o l u t i o n was a l l o w e d t o c o o l and t h e volume was made up t o 1 0 0 m l . The s o l u t i o n was f i l t e r e d t h r o u g h Whatman N o . l and the pH was a d j u s t e d t o 1 2.30 - 1 2.35 w i t h IN NaOH. DNBS re a g e n t 6 g a c t i v a t e d c h a r c o a l was added t o 6 g o f DNBS powder d i s p e r s e d i n 1 0 0 ml o f wa t e r . I t was then s t i r r e d f o r 1 0 minutes and f i l t e r e d t h r o u g h Whatman No. 1 . The r e f r a c t i v e i n d e x was measured and the s o l u t i o n was d i l u t e d t o o b t a i n the f i n a l c o n c e n t r a t i o n o f 3% DNBS. The rea g e n t was k e p t f r o z e n i n s m a l l v i a l s . (See appendix 1 ) . E t h e r e x t r a c t a n t E q u a l volumes o f p e r o x i d e - f r e e e t h y l e t h e r and d i s t i l l e d water were mixed i n an e x t r a c t i o n f u n n e l , a l l o w i n g the gas t o escape from time t o t i m e . The aqueous l a y e r was 68. discarded and the water-saturated ether was used. Other reagents and materials 90% formic acid; 0.07 5 N NaOH solution; b o i l i n g chips (Hengar granules, Hengar Co., Philadelphia, PA), 10 mesh. The chips were soaked i n concentrated HCl f o r 30 minutes, washed with water and dried. Lysine standard 2.5 mM lysine solution was prepared by d i s s o l v i n g 45.66 mg of ly s i n e monohydrochloride (MW = 146.20) in d i s -t i l l e d water and d i l u t i n g to 100 ml. From t h i s stock solution, f i v e l e v e l s of lysine standards were prepared as f ollows: Tube No. 2.5 mM lysine soln. 0.075 N NaOH (mis) (mis) 1 5 20 2 10 15 3 15 10 4 20 5 5- 25 0 These d i l u t i o n s contained 0.125, 0.250, 0.375, 0.500 and 0.625 micromoles of lysine per 0.25 ml, respectively. They were used along with each experiment for the construc-tion of the standard curve. The d i l u t i o n s were kept frozen i n small v i a l s . P r o t e i n s o l u t i o n s The t e s t p r o t e i n was d i s s o l v e d i n 25 mis o f 0.075 N NaOH t o g i v e a f i n a l c o n c e n t r a t i o n range o f 30 - 100 ug o f l y s i n e p e r 0.25 ml o f p r o t e i n s o l u t i o n . The m i x t u r e was shaken on a v o r t e x m i x e r and c e n t r i f u g e d a t 2,500 x g f o r 20 mi n u t e s . The s u p e r n a t a n t was c o l l e c t e d and used f o r a n a l y s i s . P r e p a r a t i o n o f w o r k i n g p r o t e i n s o l u t i o n s L i t e r a t u r e v a l u e D e s i r e d l e v e l mg p r o t e i n P r o t e i n (mg l y s i n e p e r (ug l y s i n e i n 25 ml 100 mg p r o t e i n ) p e r 0.25 ml) 0.075N NaOH G l u t e n 1.6 40 250 C a s e i n 7.6 76 100 Lysozyme 5.7 57 100 3 - l a c t o g l o b u l i n 11. 3 56.5 50 Egg 6.2 62 100 RNase-S-peptide 10. 3 51. 5 50 C a l c u l a t i o n f o r g l u t e n 100 mg g l u t e n 40 yg lysine x 2 5 m l , J a 0 H 1.6 mg l y s i n e 0.2 5 ml = 250 mg g l u t e n d i l u t e d t o 25 ml w i t h 0.075 N NaOH. Pr o c e d u r e 0.5 ml o f r e a g e n t A was added t o a l l tubes w i t h o u t t o u c h i n g the s i d e s ; 0.2 5 ml o f p r o t e i n s o l u t i o n was t h e n p i p e t t e d t o the s u r f a c e o f sample and sample b l a n k t u b e s . To the r e a g e n t b l a n k , i n s t e a d o f p r o t e i n , 0.25 ml o f 0.075 N NaOH were p i p e t t e d ; t o the s t a n d a r d s , 0.25 ml o f two t o t h r e e l e v e l s o f l y s i n e s t a n d a r d s o l u t i o n s were added t o t h e s u r f a c e o f re a g e n t A. The s o l u t i o n was mixed t h o r o u g h l y by f i r m w r i s t m o t i o n , and a l l o w e d t o s t a n d f o r a few mi n u t e s . 0.25 ml o f 3% DNBS was v e r y c a r e f u l l y added t o a l l t u b e s , e x c e p t f o r sample b l a n k s w h i c h r e c e i v e d 0.25 ml o f water. The DNBS was added, f i r s t t o r e a g e n t b l a n k s and then t o s t a n d a r d s and samples i n i n c r e a s i n g l y s i n e c o n c e n t r a t i o n . A f t e r t h o r o u g h l y m i x i n g , t h e tubes were immersed i n a water bath a t 40°C and i n c u b a t e d f o r 1 h r . The tubes were then c o o l e d i n an i c e - w a t e r b a t h . 2.0 ml HCL was added t o a l l tubes e x c e p t f o r sample b l a n k s , t o which 1.75 ml HCL and 0.25 ml 3% DNBS were added. E t h e r e x t r a c t i o n was c a r r i e d out t h r e e t i m e s w i t h 3.0, 2.5 and 2.5 ml o f w a t e r - s a t u r a t e d e t h e r . A v o r t e x m i x e r was used f o r e f f e c t i v e b l e n d i n g and e x t r a c t i o n . Each tube was shaken f o r a few seco n d s , t o a v o i d the f o r m a t i o n o f a g e l o r e m u l s i o n a t the e t h e r phase. The e t h e r l a y e r was c a r e f u l l y removed w i t h a P a s t e u r p i p e t by means o f a g e n t l e s u c t i o n . A c l e a n P a s t e u r p i p e t was used f o r each s o l u t i o n . 0.5 ml of 90% f o r m i c a c i d and one Hengar b o i l i n g g r a n u l e were added t o a l l t u b a s . The r e s i d u a l e t h e r was e l i m i n a t e d i n a water b a t h a t 5 5°c f o r no more than 15 mi n u t e s . The s o l u t i o n was c o o l e d dowi t o room t e m p e r a t u r e and the absorbance o f samples,. sample b l a n k s and s t a n d a r d s were measured a t 385 nm i n a Beckman S p e c t r o p h o t o m e t e r DB a g a i n s t the r e a g e n t b l a n k . I t was e a s i e r and more r e l i a b l e t o t a k e the absorbance r e a d i n g s on t h e f o l l o w i n g day, when the d r o p l e t s on the s i d e s o f the tubes had s e t down. I n t h i s c ase t h e t u b e s were k e p t i n a d a r k p l a c e t o p r o t e c t them from l i g h t . shown i n F i g u r e 6. The amount o f e D N P - l y s i n e was c a l c u l a t e d from t h e s t a n d a r d c u r v e o b t a i n e d w i t h d i f f e r e n t l e v e l s o f l y s i n e , as shown i n F i g u r e 7. e - f o r m y l - l y s i n e s , a- and e-carbobenzoxy l y s i n e s , r i b o n u c l e a s e -S - p e p t i d e and the d i p e p t i d e s l y s y l - l y s i n e and l y s y 1 - a l a n i n e , i n an attempt t o determine t h e s p e c i f i c i t y o f t h e DNBS method f o r the e p s i l o n - a m i n o group o f l y s i n e . C a l c u l a t i o n C a s e i n absorbance, ave. o f 3 d e t e r m i n a t i o n s = 0.300. From the s t a n d a r d c u r v e , an absorbance o f 0.300 c o r r e s p o n d s t o 0.450 ymoles o f l y s i n e / 3 ml. A v a i l a b l e l y s i n e c o n t e n t i n c a s e i n i s c a l c u l a t e d as f o l l o w s : The f l o w diagram o f t h e e x p e r i m e n t a l p r o c e d u r e i s The same p r o c e d u r e was c a r r i e d out on a- and 0.45,0 ymoles l y s / 3 ml 1 mol X 7= 0.0010 g c a s e i n / 3 m l 10 ymol x 146.2 g 1 mol l y s x 100 g c a s e i n = 6.58 g o f l y s i n e / 1 0 0 g c a s e i n . FIGURE 6. The d i n i t r o b e n z e n e s u l f o n a t e (DNBS) method f o r d e t e r m i n a t i o n o f a v a i l a b l e l y s i n e . P r o t e i n DNBS treatment e-DNP-protein E t h e r e x t r a c t i o n Aqueous l a y e r CE-DNP-lysine Read a t 385 nm E t h e r l a y e r -DNP-free amino a c i d s 73. FIGURE 7. Standard curve f o r a v a i l a b l e l y s i n e d e t e r m i -n a t i o n f o r a sample t r e a t e d w i t h d i n i t r o b e n z e n e s u l f o n i c a c i d . £ c to . 125 .250 .375 .500 .625 LYSINE (umoles / 3 ml reaction mixture) F o r the o t h e r p r o t e i n s , the amount o f sample p e r 3 ml o f s o l u t i o n was; 0.0005 g o f $ - l a c t o g l o b u l i n ; 0.0010 g f o r eggs and lysozyme; 0.0025 g f o r g l u t e n , and 0.00135 g f o r RNase-S-peptide. E. L y s i n e d e t e r m i n a t i o n by t h e t r i n i t r o b e n z e n e s u l f o n i c a c i d method. The TNBS method t o determine a v a i l a b l e l y s i n e i n p r o t e i n s was d e r i v e d from the p r o c e d u r e o f E k l u n d (1976). The r e a g e n t 2 , 4 , 6 - t r i n i t r o b e n z e n e s u l f o n i c a c i d i s used t o complex t h e f r e e e-amino groups o f l y s i n e . The s c a l e o f the e x p e r i m e n t s was reduced t o one t e n t h o f t h e o r i g i n a l p r o c e d u r e . Reagents 34 mM TNBS s o l u t i o n : 590.26 mg o f 2 , 4 , 6 - t r i n i t r o -benzene s u l f o n i c a c i d (MW 347.21) were d i s s o l v e d i n d i s t i l l e d w ater and the volume made up t o 50 ml. The s o l u t i o n was s t o r e d i n a dark b o t t l e and kept f r o z e n . 0.4 8 M sodium b i c a r b o n a t e s o l u t i o n : pH 8.5 and 8.07 g o f sodium b i c a r b o n a t e were d i s s o l v e d i n d i s t i l l e d water and d i l u t e d t o 200 ml. W a t e r - s a t u r a t e d d i e t h y l e t h e r : was p r e p a r e d as d e s c r i b e d f o r the DNBS t e c h n i q u e . P r o t e i n s o l u t i o n s 9 0 mg o f t e s t p r o t e i n was d i s s o l v e d i n 15 ml o f 0.47 M NaHCO,, pH 8.5 so t h a t t h e p r o t e i n e x t r a c t c o n t a i n e d 6 mg p r o t e i n p e r m l . The p r o t e i n e x t r a c t s were p r e p a r e d f r e s h f o r each e x p e r i m e n t , s i n c e t h e s t a b i l i t y o f the p r o t e i n i n sodium b i c a r b o n a t e i s unknown. e - T N P - l y s i n e s t a n d a r d s ' 62 mg o f e-TNP-lysine-HCL.I^O were made up t o 50 ml w i t h 0.4 8 M NaHCO^ s o l u t i o n . A s e r i e s o f tubes c o n t a i n i n g graded l e v e l s o f e - T N P - l y s i n e were s e t up as f o l l o w s : Tube No. e-TNP-lysine/NaHC0 3 s o l n . 0.48 M NaHC0 3 (mis) (mis) 1 0.10 0.40 2 0.20 0.30 3 0.30 0.20 4 0.40 0.10 5 0.50 0.00 These d i l u t i o n s c o n t a i n e d 0.301, 0.602, 0.903, 1.204 and 1.505 ymoles o f e - T N P - l y s i n e p e r f i n a l volume o f 25 ml. They were used f o r t h e c o n s t r u c t i o n o f t h e s t a n d a r d c u r v e . P r o c e d u r e -0.5 ml o f p r o t e i n e x t r a c t was p l a c e d i n a h y d r o l y s i s tube and shaken i n a water b a t h a t 40°C f o r 10 m i n u t e s . 0.5 ml o f 34 mM TNBS s o l u t i o n was added t o t h e p r o -t e i n e x t r a c t . To a v o i d c r y s t a l l i z a t i o n o f t h e s u p e r s a t u r a t e d TNBS s o l u t i o n i n the p i p e t , the s o l u t i o n was warmed t o 40°C p r i o r t o use. I n s t e a d o f TNBS, 1.5 ml HCL was added t o t h e sample b l a n k s . 76. After an incubation period of 2 hr at 40 C, the tubes were immersed i n an ice-water bath, and 1.5 ml of concen-trated HCL was added to samples; 0.5 ml of TNBS was added to sample blanks. The tubes were sealed and the contents were hydrolyzed for 90 minutes i n a Reacti-Therm heating module at 110°C. The tubes were allowed to cool at room temperature and the hydrolysates were f i l t e r e d through Whatman 1 and d i l u t e d to 25 ml with d i s t i l l e d water. 5 ml-aliquots were transferred to stoppered t e s t tubes and extracted four, times with 5 ml of water-saturated ether. Residual ether was evaporated by placing the tubes i n a shaking water-bath at 55°C u n t i l no ether smell could be detected. The absorbance of the aqueous protein solution was measured at 346 nm i n a Beckman Spectrophotometer DB against the sample blank. The flow diagram of the experimental procedure can be seen i n Figure 8. The amount of e-TNP-lysine (or i t s lysine equivalent) was calculated from the standard curve obtained with various l e v e l s of e-TNP-lysine, measured at 346 nm, as shown i n Figure 9. FIGURE 8. The t r i n i t r o b e n z e n e s u l f o n i c a c i d method f o r the d e t e r m i n a t i o n of a v a i l a b l e l y s i n e . P r o t e i n TNBS treatment a- and e-TNP-protein A c i d h y d r o l y s i s a-TNP-amino a c i d s + e-TNP-lysine E t h e r e x t r a c t i o n E t h e r l a y e r Aqueous l a y e r a-TNP-amino ac i d s e-TNP-lysine Read at 346 nm 78. FIGURE 9. Standard curve f o r a v a i l a b l e l y s i n e d e t e r m i n a t i o n f o r a sample t r e a t e d w i t h 2,4,6 - t r i n i t r o b e n z e n e s u l f o n i c a c i d . • 301 .602 .903 1.204 1.505 E - T N P - L Y S I N E . H C L . H 2 0 ( u m o l e s / 2 5 ml ) 79\ Calculation Gluten absorbance, ave. of 3 determinations = 0.10 0. From the e-TNP-lysine standard curve, an absorbance of 0.10 corresponds to 0.240 ymoles of e-TNP-lysine. Available lysine content i n gluten i s calculated as follows: 0.240 ymoles e-TNP-lys./25 ml 1 mol 146.2 g 0.003 g gluten/25 ml 10 ymol 1 mol l y s . = 1.17 g lysine/100 g gluten. F. Available lysine determination by the d i n i t r o f l u o r o -benzene method. The 'difference' technique described by Couch (1975) has been used to determine available lysine of proteins. This method has been adopted as o f f i c i a l - f i r s t action, AOAC. The reagent 1-fluoro-2,4-dinitrobenzene (DNFB) i s used to complex the e-amino groups of l y s i n e . Available lysine i s calculated as the difference between t o t a l and unavailable lysines. Reagents The reagent DNFB, s o l i d at room temperature, was warmed up p r i o r to use. 10% N a H C 0 3 solution: 2 g of sodium bicarbonate was dissolved i n deionized water and the volume made up to 20 ml. 6 N HCL: 495.87 ml of concentrated hydrochloric acid (12.1 N) were d i l u t e d to 1 l i t e r with deionized water. 80. \ Sodium c i t r a t e buffer pH 2.2: 137.26 g Na c i t r a t e 2^0 was dissolved i n water, and 26 ml HCI added. 40 g Brij-35 was dissolved i n water by heating and added to the solution, followed by 0.4 ml n-caprylic acid. The solution was d i l u t e d to 4 l i t e r s and f i l t e r e d . Procedure 1. Preparation of protein hydrolysate with DNFB (Unavailable l y s i n e ) . 0.1 g of protein was placed i n a dry 2 - l i t e r b o i l i n g flask, with a few glass beads. Care was taken to deposit the sample at the bottom. 10 ml freshly prepared 10% NaHCO^ solution was added, care being taken that no meal adhered to the sides of the flask. The contents were thoroughly mixed by gentle swirling and the suspension was allowed to stand for 10 minutes. A solution of 0.4 ml DNFB i n 10 ml ethanol was then added and mixed by gentle swirling. The side of the flask was rinsed with 3-5 ml ethanol. The flask was stoppered and the contents were shaken under subdued l i g h t for 3 hr on a mechanical shaker. 2 ml of 6 NrHCl was added. The mixture was concentra-ted to o i l y dryness i n a vacuum rotary evaporator at 40°C, 81. and the residue was extracted with four-5 0 ml portions of anhydrous ether. In each case, the ether was mixed thoroughly with the semisolid mass and the two phases were allowed to separate completely before the ether was decanted. F i n a l traces of ether were removed by aeration i n the rotary evaporator/without vacuum. 125 ml of 6 N HCI was added and the mixture boiled under reflux for 18 hr, maintaining a constant stream of prep u r i f i e d nitrogen through a c a p i l l a r y tube which reached about 2.5 cm above the surface of the solution (Figure 10). The mixture was allowed to cool down to room temperature and the condensor was washed down with 6 N HCI. If time permitted at the cooling stage, the flask was kept overnight i n the r e f r i g e r a t o r , since i t helped to pre-c i p i t a t e the excess DNFB and reduce the subsequent work of extraction (Carpenter et a l . , 1957). The mixture was then evaporated to a sticky paste i n a vacuum rotary evaporator at 50°C. The paste was washed by adding 100 ml of deionized water and drying back to a paste on the vacuum rotary evaporator at 5 0°C. This procedure was repeated 5 times. The l a s t evaporation was carried on u n t i l the sample was completely dry. 100 ml of pH 2.2 sodium c i t r a t e buffer was added. The flask was stoppered, shaken for 5 minutes and f i l t e r e d through Whatman 43 into a storage bottle that was kept in 8 2 . FIGURE 1 0 . R e f l u x i n g system used i n the p r e p a r a t i o n o f p r o t e i n h y d r o l y s a t e s -83. a c o l d room a t 5°C; 1.0 ml f i l t r a t e c o n t a i n i n g 0.72-0.88 mg p r o t e i n was used f o r amino a c i d a n a l y s i s . 2. P r e p a r a t i o n o f p r o t e i n h y d r o l y s a t e w i t h o u t DNFB ( T o t a l l y s i n e ) 0.025 g of p r o t e i n sample was p l a c e d i n a 2 l i t e r round-bottom f l a s k w i t h 4-5 g l a s s beads. 200 ml o f 6 N HCI was added and the m i x t u r e a l l o w e d t o b o i l on a h e a t i n g m a n t l e , u n t i l 100 ml HCI had e v a p o r a t e d . The m i x t u r e was h y d r o l y z e d under r e f l u x f o r 24 h r , as d e s c r i b e d under p r e p a r a t i o n o f sample w i t h DNFB. The h y d r o l y s a t e was e v a p o r a t e d t o s t i c k y p a s t e i n a vacuum r o t a r y e v a p o r a t o r a t 50°C. I t was washed 5 t i m e s w i t h 100 m l - p o r t i o n s o f d e i o n i z e d w a t e r and e v a p o r a t e d t o d r y n e s s d u r i n g the l a s t e v a p o r a t i o n . A f t e r 100 ml o f "sodium c i t r a t e b u f f e r , pH 2.2 was added, the c o n t e n t s were shaken and f i l t e r e d t h r o u g h Whatman No. 43. 1.0 ml f i l t r a t e c o n t a i n i n g 0.18-0.22 mg p r o t e i n was used f o r a n a l y s i s on an amino a c i d a n a l y z e r . The f l o w d i a g r a m o f t h e e x p e r i m e n t a l p r o c e d u r e i s shown i n F i g u r e 11. C a l c u l a t i o n , The peaks on t h e chromatograms were i n t e g r a t e d by m u l t i p l y i n g the h e i g h t o f t h e peak (absorbance r e a d i n g a t the maximum peak h e i g h t , minus absorbance r e a d i n g a t the b ase-84. FIGURE 11. The d i n i t r o f l u o r o b e n z e n e method f o r d e t e r m i n a -t i o n o f a v a i l a b l e l y s i n e ( d i f f e r e n c e t e c h n i q u e ) . From: Blom e t a l . , (1967) . P r o t e i n A c i d h y d r o l y s i s DNFB t r e a t m e n t Column' chromatography T o t a l l y s i n e Aqueous l a y e r A c i d h y d r o l y s i s e - D NP-lysine U n r e a c t e d l y s i n e E v a p o r a t e t o d r y n e s s C i t r a t e b u f f e r Column chromatography U n r e a c t e d l y s i n e T o t a l l y s i n e - U n r e a c t e d l y s i n e = A v a i l a b l e l y s i n e . 85. l i n e o f t h e p e a k ) fay t h e w i d t h a t h a l f t h e h e i g h t ( d o t c o u n t ) . The a r e a u n d e r t h e p e a k was t h e n d i v i d e d b y t h e c o n s t a n t f o r t h e g i v e n amino a c i d s o t h a t , m i c r o m o l e s o r m i c r o g r a m s o f amino a c i d p e r v o l u m e a p p l i e d t o t h e a n a l y z e r , vcere o b t a i n e d . T h i s was v e r y e a s i l y done b y u s i n g a p r o -g r a m on t h e Monroe c a l c u l a t o r . The v a l u e s w e r e a d j u s t e d s o t h a t t h e sum o f a l l t h e amino a c i d s was 100%. A v a i l a b l e , l y s i n e c o n t e n t i n c a s e i n was c a l c u l a t e d as f o l l o w s : 1. T o t a l l y s i n e 14.035 y g l y s i n e / m l ( v o l u m e a p p l i e d ) = 1403.5 y g l y s i n e / 1 0 0 m l / 2 5 mg c a s e i n = 5.614 mg l y s i n e / 1 0 0 mg c a s e i n . 2. U n a v a i l a b l e l y s i n e 2.632 y g l y s i n e / m l = 263.2 yg l y s i n e / 1 0 0 m l / 1 0 0 mg c a s e i n = 0.2632 mg l y s i n e / 1 0 0 mg c a s e i n . 3. A v a i l a b l e l y s i n e = T o t a l l y s i n e - U n a v a i l a b l e l y s i n e A v a i l a b l e l y s i n e = 5.3508 mg l y s i n e / 1 0 0 mg c a s e i n . G. E n z y m a t i c d i g e s t i o n t e s t The d i g e s t i b i l i t y o f p r o t e i n s was d e t e r m i n e d b y t h e p e p s i n - p a n c r e a t i n d i g e s t i o n m e t h o d o f A k e s o n a n d Stahmann (1964) , as m o d i f i e d b y Stahmann and W o l d e g i o r g i s ( 1 9 7 5 ) . 86. Reagents P e p s i n s o l u t i o n : 10 mg o f p e p s i n was p l a c e d i n a 100 ml v o l u m e t r i c f l a s k and brought t o volume w i t h 0.1 N HCI. P a n c r e a t i n s o l u t i o n : 26.6 7 mg o f p a n c r e a t i n was p l a c e d i n a 50 ml v o l u m e t r i c f l a s k and brought t o volume w i t h pl-l 8.0 phosphate b u f f e r . pH 8.0 sodium phosphate b u f f e r (0.1M) : 21.92 g o f K 2HP0 4.3H 20 and 0.523 g o f anhydrous K H 2 P 0 4 were d i s s o l v e d i n w a t e r and d i l u t e d t o one l i t e r . 14% s u l f o s a l i c y l i e a c i d s o l u t i o n : 14.29 g o f s u l f o s a l i c y l i e a c i d was d i s s o l v e d i n d i s t i l l e d w a t e r and d i l u t e d t o 100 ml. The s o l u t i o n was f i l t e r e d t h r o u g h Whatman No. 1 i n t o a dark b o t t l e . C i t r a t e sample d i l u t e r pH 2.2 (See DNFB r e a g e n t s ) . 50 ppm m e r t h i o l a t e s o l u t i o n . P r o c e d u r e 50 mg o f p r o t e i n sample was i n c u b a t e d w i t h 7.5 ml o f p e p s i n s o l u t i o n i n a s h a k i n g w a t e r b a t h a t 37°C f o r 3 h r . A f t e r n e u t r a l i z a t i o n w i t h 3.75 ml o f 0.2 N NaOH, 3.75 ml of p a n c r e a t i n s o l u t i o n and 0.25 ml o f 50 ppm m e r t h i o l a t e was added. The d i g e s t i o n m i x t u r e was i n c u b a t e d f o r an a d d i t i o n a l 2 4 h r s i n a s h a k i n g w a t e r b a t h a t 37°C. To p r e c i p i t a t e the u n d i g e s t e d p r o t e i n and p e p t i d e s , 5.0 ml o f d i g e s t i o n m i x t u r e .was mixed w i t h 5.0 ml o f 14% 87. s u l f o s a l i c y l i c acid solution, to bring the f i n a l concentra-tion of s u l f o s a l i c y l i c acid to 4.7%. The flasks were shaken for 30 minutes and the pH was adjusted to 2.2 by adding drops of 6 N NaOH. The digests were then transferred to a 15 ml gradua-ted tube and brought to volume with pH 2.2 c i t r a t e buffer. The solution was centrifuged at 1,000 x g for 30 minutes, f i l t e r e d through Whatman No.42 and stored i n the freezer at -2 0°C for amino acid analysis. The same procedure was carr i e d out for an enzyme blank that was prepared by incubation with the protein sample omitted. Calculation: 15.2 5 ml of digestion mixture ( f i n a l volume) con-tained 50 mg protein; thus, 5 ml of digestion mixture contained 16.39 mg protein. 5.00 ml of digestion mixture mixed with s u l f o s a l i c y l i c acid and made up to 15 ml with c i t r a t e buffer, contained 16.39 mg protein; 0.5 ml used for amino acid analysis, contained 0.5 46 mg protein. The areas under the peaks of the ef f l u e n t curve were calculated by multiplying the height of the peak by the width at half the height, and di v i d i n g by the constant for the given amino acid. Micromoles, or micrograms of amino acid per volume applied, v/ere obtained. 88. I f c a s e i n had 13.899 yg l y s i n e / 0 . 5 ml: 13.899 yg l y s i n e / 0 5 ml 1 mg 0.546 mg p r o t e m / 0 . 5 ml 1000 yg ^ 2.54 4 mg l y s i n e / 1 0 0 mg c a s e i n . 89. RESULTS AND DISCUSSION The p r o t e i n c o n t e n t o f the samples, as d e t e r m i n e d by the r a p i d m i c r o - K j e l d a h l method o f Concon and S o l t e s s (1973) i s shown i n T a b l e 3. A. The d i n i t r o b e n z e n e s u l f o n i c a c i d (DNBS) method The method d e s c r i b e d by Concon (1975a) employs t h e reag e n t sodium d i n i t r o b e n z e n e s u l f o n a t e (DNBS) i n a u r e a -p h o s p h a t e - p h e n y l m e r c u r i c c h l o r i d e s o l u t i o n , t o complex the e-amino groups o f l y s i n e . A f t e r c o l o u r development, t h e i n t e r f e r e n c e s are e l i m i n a t e d by e t h e r e x t r a c t i o n . The o p t i c a l d e n s i t y i s the n measured a t 385 nm and the amount o f a v a i l a b l e l y s i n e i s c a l c u l a t e d from a s t a n d a r d c u r v e . Table 4 shows the a v a i l a b l e l y s i n e c o n t e n t o f p r o t e i n samples as d e t e r m i n e d by t h e DNBS method. From the samples s u b j e c t e d t o the DNBS p r o c e d u r e , o n l y 1.5 N FICL g l u t e n and whole egg s o l i d s had a h i g h b l a n k v a l u e . T h i s i s o f t e n e n c o u n t e r e d i n u n d e f a t t e d m a t e r i a l s o r i n t h o s e c e r e a l g r a i n s w i t h h i g h l y pigmented o u t e r l a y e r s . A c c o r d i n g t o Concon (1975b), f o r t h e DNBS method, l i p i d s need not be removed e x c e p t i n h e a v i l y pigmented v a r i e t i e s o f b a r l e y , o a t s , r y e , and wheat. However, s u b j e c t i n g t h e g l u t e n and egg samples t o f a t e x t r a c t i o n might have produced l o w e r b l a n k v a l u e s . The sample b l a n k s f o r B - l a c t o g l o b u l i n , l y sozyme, c a s e i n and RNase-S-peptide gave l o w e r absorbance r e a d i n g s TABLE 3. Protein content of samples assayed for lysine a v a i l a b i l i t y g protein/100 g sample 35.20 63.43 89.20 72.30 37.26 Sample 1.5 N HCl Gluten 3 - l a c t o g l o b u l i n Lysozyme Casein Whole Egg (freeze-dried) 9 1 . TABLE 4. A v a i l a b l e l y s i n e content o f p r o t e i n samples as determined by the d i n i t r o b e n z e n e s u l f o n i c a c i d (DNBS) method. (Values i n g l y s i n e / 1 0 0 g p r o t e i n ) . P r o t e i n Experiment No. 1 2 3 Ave G l u t e n 1 . 8 0 1 1 . 6 6 8 1 .712 1.818 1 . 6 6 8 1 .715 1 . 8 3 8 1 . 7 0 2 1.764 Average 1.819 1.679 1 . 7 3 0 1.748 C a s e i n 7.413 7.575 7 . 5 5 0 7 . 4 0 8 7 . 5 0 4 7 . 5 0 1 7 . 4 9 6 7 . 5 7 5 7 .618 Average 7 . 4 3 9 7 . 5 5 1 7 . 5 5 6 7 . 5 1 5 Lysozyme 5 . 4 6 3 5.327 5 . 5 0 7 5 . 5 4 5 5 . 3 9 6 5 . 4 0 2 5.414 5 . 4 3 0 5.402 Average 5.474 5 . 3 8 4 5 . 4 3 7 5 . 4 3 2 /3 - l a c t o g . 1 2 . 3 5 4 11 .837 12.723 1 2 . 4 2 8 11.837 12.797 1 2 . 2 8 3 11 .543 1 2 . 5 0 3 Average 1 2 . 3 5 5 11.739 12.674 1 2 . 2 5 6 Egg 5 . 4 9 0 5 . 3 6 8 5.610 5 . 5 2 3 5-340 5 . 6 2 2 5.346 5 . 3 2 7 5 . 7 2 2 Average 5 . 4 5 3 5 . 3 4 5 5 . 6 5 1 5 . 4 8 3 RNase-S-nfintide 10.670 10.618 10.644 Reported v a l u e s 5 . 4 J 1 0 . 5 1 0 . 2 J " A v a i l a b l e l y s i n e content, based on known amino a c i d sequence, from P u r c e l l e t a l . , 1976. 92. than the reagent blank. In t h i s case, the negative value was ignored; obviously, no absorbance i s contributed by the sample without dinitrophenylation. The higher absorbance of the reagent blank may come from the reaction of DNBS with impurities i n the reagents or simply contaminations (Concon, 1971). The time of dinitrophenylation greatly a f f e c t s the precision of the DNBS results since the absorbance of the solution during color development increases with time. A change of about 0.006 absorbance units per minute per ml with a lysine concentration of 0.4 moles per ml at pH 12 and 40°C, can be expected (Concon, 1971). In t h i s t h e s i s , about 8 minutes elapsed between addition of DNBS to the f i r s t tube and addition of the reagent to the l a s t tube, before incubation at 40°C. Thus, an absorbance error of 0.048 units or less, since at room temperature very l i t t l e dinitropheny-l a t i o n occurs, can be expected i n the r e s u l t s . Studying the r e a c t i v i t y of TNBS with amino acids and peptides, Freedman and Radda (1968) showed that, at room temperature, pH 7.4, the e-amino groups of lysines were 30 times more reactive to TNBS than were the a-amino groups. When the neighboring carboxyl group was. amidated or joined i n a peptide bond, the a-amino groups became even less reactive. These authors concluded that the decreased n u c l e o p h i l i c i t y of the amino group as a r e s u l t of amidation or peptide bond formation of the carboxyl r a d i c a l , accounted for the decreased r e a c t i v i t y of TNBS. I t was then suggested that DNBS, a reagent less e l e c t r o p h i l i c than TNBS, might be more s p e c i f i c for the e-amino group. The absence of one extra ortho n i t r o group i n DNBS i s expected to diminish the corres-ponding e l e c t r o p h i l i c i t y of the sulfonate r a d i c a l . S t e r i c and other e l e c t r o s t a t i c factors may also influence the r e a c t i v i t y of both DNBS and primary amino groups. Therefore, under the reaction conditions used, a v i r t u a l disappearance of the absorbance, due to a-amino groups may r e s u l t . However, using model lysine derivatives with d i f f e r e n t numbers of a- and e-amino groups, i t has been found that DNBS also reacts with the a-amino groups to a certain extent. Table 5 indicates the results of t h i s experiment. e-formyl-lysine which has one free a-amino group, when reacted with DNBS, shows a 385 nm absorbance value of 0.091, but a-formyl-lysine that has one free e-amino group, shows an absorbance value of 0.2 89. The expected values of l y s i n e , l y s y l - l y s i n e , lysyl-alanine and ribonuclease-S-peptide, have been calculated assuming DNBS s p e c i f i c i t y for the e-amino group. The difference between the experimental and the expected values, shows the extent to which the a-amino group reacts with DNBS. The difference i s 0.095 for lysine, 0.069 for the dipeptide l y s y l - l y s i n e , and only 0.026 for RNase-S-peptide. A difference of 0.001 was TABLE 5. R e a c t i o n o f amino a c i d s and p e p t i d e s w i t h d i n i t r o b e n z e n e s u l f o n i c a c i d (DNBS) Amino a c i d o r p e p t i d e (1) Number e-NH 2 a-o f -NH2 E x p e r i m e n t a l E x p e c t e d (2) D i f f e r e n c e e - f o r m y l - l y s i n e 0 1 0.091 0.000 0.091 a - f o r m y l - l y s i n e 1 0 0.289 0.289 0.000 L y s i n e 1 1 0.384 0.289 0.095 L y s y l - l y s i n e 2 1 0.647 0.578 0.069 A l a n i n e 0 1 0.000 0.000 0.000 L y s y l - a l a n i n e 1 1 0.290 0.289 0.001 RNase-S-peptide 2 1 0.604 0.578 0.026 (1) 2.5 mM- s o l u t i o n s . (2) I f DNBS i s s p e c i f i c f o r e-NH 95. found f o r the d i p e p t i d e l y s y 1 - a l a n i n e , i n d i c a t i n g t h a t the a-amino group o f a l a n i n e i s n o t measured by t h e DNBS t e c h n i q u e . From the above f i g u r e s i t can be seen t h a t when l y s i n e i s a t the N - t e r m i n a l p o s i t i o n i n a p r o t e i n , the r e -s u l t w i l l be s l i g h t l y h i g h e r than t h e t r u e c o n t e n t o f a v a i l a b l e l y s i n e , s i n c e the a-amino group i s c o n t r i b u t i n g t o the r e s u l t s . RNase-S-peptide i s a 2 0 - r e s i d u e component w h i c h has two l y s i n e u n i t s , one o f them l o c a t e d a t the N - t e r m i n a l p o s i t i o n ( R i c h a r d s and V i t h a y a t h i l , 1 9 5 9 ) . I n t h i s p e p t i d e , t h e r a t i o o f e- t o a-amino groups i s 2 t o 1, and t h u s , t h e c o n t r i b u t i o n o f the a-amino group t o the r e s u l t i s compara-t i v e l y h i g h ( 0 . 0 2 6 ) . I f t h e e x p e c t e d v a l u e f o r RNase-S-p e p t i d e , t h a t i s 0.578, i s used t o c a l c u l a t e t h e a v a i l a b l e l y s i n e c o n t e n t i n g p e r 100 g o f p r o t e i n , t h e r e s u l t i s 10.2. The same f i g u r e was c a l c u l a t e d by P u r c e l l e t a l . ( 1 9 7 6 ) , b ased on known amino a c i d sequence. I n t h i s work, t h e a v a i l a b l e l y s i n e c o n t e n t o f RNase-S - p e p t i d e c a l c u l a t e d from an absorbance v a l u e o f 0.604, was found t o be 10.64 g p e r 100 g o f p r o t e i n . P u r c e l l e t a l . u s i n g a r e a g e n t l e s s s p e c i f i c t h a n DNBS, such as f l u o r e s c a m i n e , w h i c h l a b e l s the e- as w e l l as t h e a-amino groups o f p r o t e i n s , o b t a i n e d a v a l u e o f 15.3 g l y s i n e p e r 100 g o f RNase-S-peptide. I n p r o t e i n s and h i g h e r p o l y p e p t i d e s t h a t have N-, t e r m i n a l l y s i n e , t h e r a t i o would be t h a t o f s e v e r a l e-amino groups t o one a-amino group. I n t h i s c a s e , the c o n t r i b u t i o n o f t h e a-amino group t o the r e s u l t s , becomes n e g l i g i b l e . Thus, the l y s i n e a v a i l a b i l i t y as d e t e r m i n e d by t h e DNBS method, w i l l g i v e r e l i a b l e d a t a f o r p r o t e i n s c o n t a i n i n g a s u b s t a n t i a l number o f e-amino groups p e r c h a i n . a- and e - c a r b o b e n z o x y - l y s i n e s were a l s o used f o r the DNBS s p e c i f i c i t y s t u d y . These d e r i v a t i v e s d i d n o t show any absorbance v a l u e when r e a c t e d w i t h DNBS. T h i s b e h a v i o r c o u l d be e x p l a i n e d i n terms o f a s t e r i c b e n z y - b e n z y l h i n d r a n c e . B. The t r i n i t r o b e n z e n e s u l f o n i c a c i d method (TNBS) The r e a g e n t 2 , 4 , 6 - t r i n i t r o b e n z e n e s u l f o n i c a c i d was used t o complex w i t h t h e f r e e e-amino group o f l y s i n e , and the r e s u l t i n g e - T N P - l y s i n e was d e t e r m i n e d s p e c t r o p h o t o -m e t r i c a l l y a f t e r 90. min o f a c i d h y d r o l y s i s . T h i s method gave v a l u e s f o r a v a i l a b l e l y s i n e , i n f a i r agreement w i t h t h o s e r e p o r t e d i n t h e l i t e r a t u r e , as shown i n Table 6. TNBS has been found t o r e a c t w i t h v a r i o u s i n t e r -f e r i n g compounds; t h u s , t h e r e p o r t e d v a l u e s are sometimes c o n t r a d i c t o r y . H o l s i n g e r e t a l . (1970) and H o l s i n g e r and P o s a t i (1975) r e p o r t e d anomalously h i g h r e s u l t s when t h e TNBS pr o c e d u r e o f Kakade and L i e n e r (1969) was a p p l i e d t o c a s e i n s . The a v a i l a b l e l y s i n e c o n t e n t f o r k - c a s e i n was 15% h i g h e r than t h e t o t a l l y s i n e c o n t e n t , and whole c a s e i n v a l u e s were 10-15% h i g h . They a s c r i b e d t h i s f i n d i n g t o the p r e s e n c e o f d-glucosamine and d - g a l a c t o s a m i n e , as w e l l as t o o t h e r 9 7 . TABLE 6 . A v a i l a b l e l y s i n e content o f p r o t e i n samples as determined by the t r i n i t r o b e n z e n e s u l f o n i c a c i d (TNBS) method. (Values i n g l y s i n e / 1 0 0 g p r o t e i n ) . Experiment No. -Protein 1 2 3 Reported v a l u e s G l u t e n 1 . 3 8 8 1 .370 1 . 5 8 5 1.241 1.428 1 .376 I . 6 0 7 Average 1 . 4 0 8 1 . 3 7 3 1 . 5 9 6 Lysozyme 5-734 5 . 5 8 4 5 . 7 9 3 5 . 7 4 2 5 . 7 8 1 5 . 8 5 8 5 . 6 0 3 5.777 Average 5 . 7 9 6 5 . 5 9 ^ 5-785 C a s e i n 7-59^ 6 . 9 1 3 6 . 9 0 0 6 . 7 0 3 8 . 5 4 - 0 . 2 1 ^ 7 .620 6 . 8 3 7 6.957 8 . 2 3 5 9 . 6 5 5 8 . 0 6 1 Average 7.607 6 . 8 7 4 6 . 9 2 9 / 3 - l a c t o g l o b . 1 2 . 9 8 0 14 .024 14 .564 9.793 1 1 . 6 5 2 1 3 . 0 0 2 14 .228 14.759 Average 1 2 . 9 9 1 14 .126 14 .662 Egg 6.134 7*028 6.797 6 . 1 9 2 7 . 2 5 3 6.857 Average 6 . 1 6 3 7.141 6 . 8 2 7 A v a i l a b l e l y s i n e as determined by the TNBS method1 1 . From F i n l e y and Friedman, 1973. 2 . From Kakade and L i e n e r , I 9 6 9 . 3 . From H o l s i n g e r e t a l . , 1970. 4 . From Eklund, 1976. 5 . From H a l l et a l . , 1973. 98. u n c h a r a c t e r i z e d g l y c o p r o t e i n fragments i n the c a s e i n m i c e l l e . Amino sugars r e a c t w i t h TNBS t o form c o l o r e d d e r i v a t i v e s w h i c h are n o t removed by e t h e r e x t r a t i o n . oi - and B - c a s e i n s known t o be c a r b o h y d r a t e - f r e e , gave a v a i l a b l e l y s i n e c o n t e n t s , e i t h e r e q u a l t o o r l e s s t h a n t h e t o t a l l y s i n e p r e s e n t . On t h e o t h e r hand, low TNBS v a l u e s have been r e p o r -t e d by P o s a t i and h e r a s s o c i a t e s (1975) i n p r o d u c t s c o n t a i n i n g l a c t o s e . These a u t h o r s found t h a t t h e pr e s e n c e o f l a c t o s e i n l e v e l s a p p r o a c h i n g t h e l y s i n e : l a c t o s e r a t i o i n d e h y d r a t e d whey powders, r e s u l t e d i n s e v e r e d e s t r u c t i o n o f t h e e-TNP-l y s i n e d e r i v a t i v e . H a l l e t a l . (1975) and P o s a t i e t a l . (1972) have s t r e s s e d the im p o r t a n c e o f a s m a l l sample s i z e i n o r d e r t o o b t a i n a r e l i a b l e d a t a w i t h TNBS. A sample o f 0.05 mg o f bo v i n e serum albumin gave about 89% a v a i l a b l e l y s i n e , whereas a sample s i z e o f 0.7 mg gave o n l y 75% a v a i l a b l e l y s i n e ( P o s a t i e t a l . , 1972). On t h e o t h e r hand, E k l u n d (1976) o b t a i n e d r e p r o d u c i b l e r e s u l t s by emp l o y i n g a 30 mg sample. There i s a l s o some c o n f l i c t i n the l i t e r a t u r e r e -g a r d i n g the r e a c t i v i t y o f f r e e l y s i n e towards TNBS. L y s i n e r e a c t s w i t h TNBS t o form i n i t i a l l y a , e - d i T N P - l y s i n e , which K o t a k i and Satake (1964) showed t o be u n s t a b l e i n h o t 6 N h y d r o c h l o r i c a c i d ; 81% e - T N P - l y s i n e and 17% f r e e l y s i n e was r e c o v e r e d a f t e r a c i d h y d r o l y s i s o f a , e - d i T N P - l y s i n e (Okuyama and S a t a k e , 1960). O u s t e r h o u t and Wood (1970) fo u n d , however, t h a t f r e e l y s i n e was d i r e c t l y c o n v e r t e d t o e-TNP-l y s i n e . H a l l and associates (197 3) found that recoveries of free lysine and e-TNP-lysine added as i n t e r n a l standards to protein suspensions which were then subjected to the TNBS reaction and acid hydrolysis were variable. Furthermore, since a, e-diTNP-lysine i s ether soluble, i n proteins where lysine i s at the N-terminal p o s i t i o n , t h i s derivative or small peptides containing i t , would be extracted into the ether phase and would escape detection. H a l l et a l . (1975) found variable results for available lysine i n carbohydrate-rich materials. According to them,browning and charring produced during acid hydrolysis accounted for the low results f o r available l y s i n e . e-TNP-lysine was adsorbed on to the carbon p a r t i c l e s (from charring) which were then removed during the ether extraction process. When the sample size was decreased, H a l l et a l . found that the•available lysine measured by TNBS reaction exceeded the value for the t o t a l lysine. In materials r i c h i n starches, the p u r i f i e d starches themselves reacted with TNBS to give an apparent lysine content. From these findings, H a l l et a l . recommended that the TNBS reaction be ca r r i e d out at 30°C for 30 minutes, for determining available l y s i n e , i n feeds and cereals r i c h i n carbohydrates. Not only carbohydrates and sugars i n t e r f e r e i n the determination of lysine a v a i l a b i l i t y with TNBS, but nat u r a l l y occurring amines such as agmatine, spermine, spermidine and taurine, react with TNBS and can be measured as available 100. l y s i n e . C a d a v e r i n e , h y d r o x y l y s i n e , o r n i t h i n e and p u t r e s c i n e , i f p r e s e n t i n samples o f t i s s u e s t h a t have undergone some d e c o m p o s i t i o n , can g i v e m i s l e a d i n g l y h i g h r e s u l t s . There i s some disag r e e m e n t i n r e g a r d t o t h e s t a n d a r d used t o draw the s t a n d a r d c u r v e f o r the TNBS method. Kakade and L i e n e r (1969) and E k l u n d (1976) used e - T N P - l y s i n e as a s t a n d a r d . Graded l e v e l s o f e - T N P - l y s i n e were s u b j e c t e d t o t h e same p r o c e d u r e as t h e p r o t e i n samples. S i n c e e-TNP-l y s i n e was n o t a v a i l a b l e i n the U.K., H a l l e t a l . (1973) p r o -posed t h e use o f l y s i n e as a s t a n d a r d . They demonstrated t h a t t h e p r o d u c t from p u r e D L - l y s i n e a f t e r r e a c t i o n w i t h TNBS and e t h e r e x t r a c t i o n had v i r t u a l l y t h e same adsorbance v a l u e p e r u n i t mass as a s o l u t i o n o f s y n t h e t i c e - T N P - l y s i n e . However, when l y s i n e was r e a c t e d w i t h TNBS and s u b j e c t e d t o a c i d h y d r o l y s i s and e t h e r e x t r a c t i o n , we found a molar a b s o r p t i v i t y 4 -1 -1 of 1.04 x 10 ( m o l e s / l i t e r ) cm . e - T N P - l y s i n e , s u b j e c t e d 4 t o the same p r o c e d u r e , had a m o l a r a b s o r p t i v i t y o f 0.89 x 10 ( m o l e s / l i t e r ) ^ cm B u t , when e - T N P - l y s i n e was r e a d d i r e c t l y a t 346 nm, the m o l a r a b s o r p t i v i t y was found t o be 1.46 x 10^ ( m o l e s / l i t e r ) - 1 c m - 1 which i s i n agreement w i t h the v a l u e r e p o r t e d by Kakade and L i e n e r ( 1 9 6 9 ) . I n t h e p r e s e n t work, T N B S - a v a i l a b l e l y s i n e was de t e r m i n e d from a s t a n d a r d c u r v e which was b u i l t by r e a d i n g d i r e c t l y the 346 nm absorbance o f graded l e v e l s o f e-TNP-l y s i n e . A c c o r d i n g t o H a l l e t a l . (1973) about 100% o f e - T N P - l y s i n e can be r e c o v e r e d from e i t h e r a) e - T N P - l y s i n e d i r e c t l y i n 1 N. HCI; b) e - T N P - l y s i n e i n 1 N HCI a f t e r e t h e r 101. e x t r a c t i o n , o r c) e - T N P - l y s i n e e t h e r - e x t r a c t e d a f t e r 2 h r i n a p p r o x i m a t e l y 7 N H C l a t 100°C. From t h e r e s u l t s p r e s e n t e d above, i t can be seen t h a t many compounds i n t e r f e r e w i t h t h e T N B S - a v a i l a b l e l y s i n e d e t e r m i n a t i o n . The method would r e q u i r e s e v e r a l c o r r e c t i o n s and m o d i f i c a t i o n s b e f o r e i t can y i e l d r e l i a b l e d a t a . C. The f l u o r o d i n i t r o b e n z e n e (FDNB) method. The FDNB d i f f e r e n c e method u t i l i z e s t h e r e a g e n t 1 - f l u o r o - 2 , 4 - d i n i t r o b e n z e n e (FDNB) t o b i n d the f r e e e-amino group'of l y s i n e . An e-DNP-lysine complex i s formed, which r e s i s t s a c i d h y d r o l y s i s . When t h e FDNB-reacted sample i s h y d r o l y z e d and a n a l y z e d f o r l y s i n e by t h e method o f Moore e t a l . ( 1 9 5 9 ) , the l y s i n e which was bound and n o t a v a i l a b l e f o r c o m b i n a t i o n w i t h FDNB, i s measured. T h i s i s c o n s i d e r e d t o be u n a v a i l a b l e l y s i n e . A v a i l a b l e l y s i n e , w h i c h was bound by FDNB, i s c a l c u l a t e d as the d i f f e r e n c e between t o t a l and u n a v a i l a b l e l y s i n e s . T a b l e 7 shows the l y s i n e found a f t e r a c i d h y d r o l y s i s , the u n a v a i l a b l e l y s i n e as d e t e r m i n e d by the FDNB p r o c e d u r e , and t h e a v a i l a b l e l y s i n e c o n t e n t o f t h e y p r o t e i n samples. The r e s u l t s are i n good agreement w i t h t h e r e p o r t e d v a l u e s . I n the case o f 3 - l a c t o g l o b u l i n , the a v a i l a b l e - l y s i n e c o n t e n t was found t o be 11.27%. P u r c e l l e t a l . (1976) r e p o r t e d a v a l u e o f 10.5% f o r a v a i l a b l e l y s i n e i n 3 - l a c t o g l o b u l i n , based on TABLE 7. A v a i l a b l e l y s i n e c o n t e n t o f p r o t e i n samples as d e t e r m i n e d by the f l u o r o d i n i -t r o b e n z e n e (FDNB) d i f f e r e n c e method. TOTAL LYSINE Repor-t e d UNAVAILABLE LYSINE AVAI-Repor-t e d P r o t e i n E x p e r i m e n t No. Experiment No. LABLE LYSINE (T - U) 1 2 3 4 AVE Value 1 2 3 AVE V a l u e G l u t e n 1.67 1. 69 1.73 1. 71 1. 70 1. 2r>~ 1.70 0.17 0.13 0 .15 1. 55 1.26? 1.54, 1.67 C a s e i n 7. 42 7. 83 7.59 7. 68 7.63 6.12:? 8.3l£ 8.7 0.22 0.25 0.22 0.23 7. 35 5 . 1 0 " 7.5 X £ 8.17 8 Lysozyme 5.28 5. 36 5.35 5.33 5.7 ^ 5.75 0.19 0.17 0.18 5.15 5.4 1 2 8 5.60 8 3 - l a c t o g . 13.60 12. 29 13.10 14. 41 13. 35 5 11.4 6 2.20 1.93 2.11 2.08 11.27 12 10.5 Egg 6. 92 6. 83 7.15 6. 46 6. 84 6.2^ 6.4 ; 0.25 0. 30 0.32 0.29 6.55 -The v a l u e s are g i v e n i n g l y s / 1 0 0 g p r o t e i n . 103. 1. Chromatographic procedure (Spackman e t a l . , 195 8) taken from F i n l e y and Friedman, 19 73. 2. T o t a l and u n a v a i l a b l e l y s i n e determined by the FDNB D i f f e r e n c e procedure, from Booth, 1971. 3. Chromatographic procedure (Spackman e t a l . , 195 8) , from H o l s i n g e r and P o s a t i , 1975. 4. T o t a l l y s i n e as determined by a c i d h y d r o l y s i s , from Stahmann and W o l d e g i o r g i s , 1975. 5. T o t a l l y s i n e by composition, from T r i s t r a m , 1953. 6. T o t a l l y s i n e from c o m p o s i t i o n a l data, from Jenness and Pat t o n , 1959. 7. T o t a l l y s i n e from c o m p o s i t i o n a l data, from O r r and Watt, 1957. 8. A v a i l a b l e l y s i n e determined by Carpenter's method, from Carpenter and Bjarnason, 1969 . 9. A v a i l a b l e l y s i n e determined by Carpenter's method, from Boctor and Harper, 196 8. 10. A v a i l a b l e l y s i n e determined by Carpenter's method, from Hussain, 1974. 11. A v a i l a b l e l y s i n e measured by the m o d i f i e d Carpenter method (Booth, 1971), from Stahmann and W o l d e g i o r g i s , 1975. 12. A v a i l a b l e l y s i n e c a l c u l a t e d from known amino a c i d sequence, from P u r c e l l e t a l . , 1976. 104. known amino a c i d sequence. P o r t e r (1948) have r e p o r t e d t h a t some p r o t e i n s such as 3 - l a c t o g l o b u l i n and serum g l o b u l i n s , i n c l u d e l y s i n e groups t h a t r e a c t w i t h FDNB o n l y a f t e r de-n a t u r a t i o n , w h i c h may be e f f e c t e d w i t h e t h a n o l , g u a n i d i n e o r m i l d a c i d . I t might be a d v i s a b l e t o t e s t t h e e f f e c t o f p r i o r d e n a t u r a t i o n on 3 - l a c t o g l o b u l i n , t o see whether a h i g h e r FDNB-r e a c t i v e l y s i n e r e s u l t i s o b t a i n e d . The o r i g i n a l FDNB p r o c e d u r e ( C a r p e n t e r , 1960) i s known t o y i e l d poor r e c o v e r i e s o f e-DNP-lysine and v a r i a b l e r e s u l t s , e s p e c i a l l y when a p p l i e d t o c e r e a l s and o i l s e e d meals. The poor performance o f the method i s a s s o c i a t e d w i t h t h e d e s t r u c t i o n o f e-DNP-lysine by c a r b o h y d r a t e s d u r i n g h y d r o l y s i s , and the f o r m a t i o n o f o t h e r y e l l o w p r o d u c t s w h i c h a re n o t e a s i l y s e p a r a t e d from e-DNP-lysine ( C a r p e n t e r e t a l . , 1957; Rao e t a l . , 1963). P a r t i a l d e s t r u c t i o n o f e-DNP-lysine does not cause d i f f i c u l t i e s when a v a i l a b l e l y s i n e i s d e t e r m i n e d by the FDNB d i f f e r e n c e p r o c e d u r e , s i n c e t h i s d e r i v a t i v e i s n o t measured w i t h t h i s t e c h n i q u e ; e-DNP-lysine i s mathema-t i c a l l y c a l c u l a t e d by d i f f e r e n c e . The d i f f e r e n c e t e c h n i q u e s u f f e r s from a major d i s a d v a n t a g e , t h a t i t i s e x t r e m e l y time consuming; 18 h r ( u n a v a i l a b l e l y s i n e ) and 24 h r ( t o t a l ) o f a c i d h y d r o l y s i s are r e q u i r e d f o r each d e t e r m i n a t i o n o f l y s i n e a v a i l a b i l i t y . Sub-sequent removal o f HCl from the sample and r e d u c t i o n o f t h e 105. sample to dryness on a rotary evaporator, i s , however, the most time consuming step. Hubbard and Finney (1976) have described a technique involving an Evapo-Mix evaporator, which greatly reduces the time and attention otherwise required. With t h i s system, 10 samples of 5.5 ml of solution were reduced to dryness i n about 35 minutes. Although time consuming, the FDNB difference technique i s the most accurate means presently available for the chemical determination of available l y s i n e . The values obtained w i l l include fr e e - l y s i n e and N-terminal l y s i n e . D. Pepsin-pancreatin digestion test . Although i n the f i n a l analysis, the n u t r i t i o n a l q u a l i t y of a protein must be assessed by feeding t r i a l s , the enzymatic methods for protein q u a l i t y evaluation are very useful for preliminary q u a l i t y evaluation. In v i t r o enzymatic digestion methods attempt to imitate the action of the mammalian digestive system. Compared to animal assays, the enzymatic methods have several advantages. They are less expensive and require less time than bioassays; they show less v a r i a t i o n than protein e f f i c i e n c y r a t i o assays with rats; a single assay can indicate the r e l a t i v e amounts of ess e n t i a l amino acids contained i n and released by the enzyme from the protein under assay. The enzymic methods have been used to monitor the adverse e f f e c t s of processing operations on proteins, that 106. may decrease the d i g e s t i b i l i t y of the p r o t e i n , reduce the a v a i l a b i l i t y o f l y s i n e or o x i d i s e s u l f u r amino a c i d s . Stahmann (1977) found t h a t peroxidase o r p o l yphenoloxidase i n the presence o f c h l o r o g e n i c a c i d and p r o t e i n , upon a c i d h y d r o l y s i s , r e v e a l e d no d e s t r u c t i o n w i t h i n c r e a s i n g concen-t r a t i o n s o f c h l o r o g e n i c a c i d and oxidase enzymes, but enzymatic h y d r o l y s i s r e v e a l e d a complete d e s t r u c t i o n of methionine. Even though the enzymic i n v i t r o d i g e s t i o n i s not complete, so t h a t the values o b t a i n e d are o n l y r e l a t i v e , the amount of the l i m i t i n g amino a c i d r e l e a s e d by enzymic h y d r o l y s i s i s a good i n d i c a t o r o f amino a c i d a v a i l a b i l i t y . L y s i n e t h a t has i t s e-amino group b l o c k e d has no n u t r i t i v e value s i n c e those groups are not s u s c e p t i b l e to enzymatic h y d r o l y s i s and the l y s i n e c o u l d not be f r e e d . The o r i g i n a l method of Akeson and Stahmann ( 1 9 4 6 ) used p i c r i c a c i d as a p r e c i p i t a n t of undigested p r o t e i n . However, t h i s procedure was found to be very i n c o n v e n i e n t s i n c e i t was necessary t o pass the t r e a t e d sample through a column i n o r d e r t o e l i m i n a t e the p i c r i c a c i d . In 1975, the author suggested the use of s u l f o s a l i c y l i c a c i d to p r e c i p i t a t e the u n d i g e s t e d p r o t e i n . T h i s i s an advantageous m o d i f i c a t i o n s i n c e t h i s reagent i s c o l o r l e s s , n i n h y d r i n n e g a t i v e and can be d i r e c t l y a p p l i e d to the columns f o r amino a c i d a n a l y s i s 107. (Hamilton, 1962). Menden and Gremer (1966) claimed that i t i s pre-ferable to digest the protein with a high concentration of enzyme for a short period, so that autohydrolysis of enzymes becomes n e g l i g i b l e . The pepsin-pancreatin digestion test of Stahmann and Woldegiorgis (1975) uses a digestion time of 27 hours, and an enzyme blank must be run along with each experiment. This blank value ranged between 0.5 85 and 1.42 6 yg of lysine per ml that was substracted from the sample value. Table 8 summarizes the results for available lysine as determined by the pepsin-pancreatin digestion t e s t . The resu l t s indicate the r e l a t i v e amount of lysine released by the enzymes under the conditions s p e c i f i e d for the test. A co r r e l a t i o n c o e f f i c i e n t of 0.995 was obtained between the enzymatic test and the available lysine determined by the FDNB o f f i c i a l method. This res u l t i s i n close agree-ment with the finding of Stahmann and Woldegiorgis (1975) who reported a co r r e l a t i o n c o e f f i c i e n t of 0.99 74 between the available lysine determined by the modified Carpenter method (Booth, 1971) and the lysine released by the digestive enzymes. Bujard et a l . (I967) also reported a high c o r r e l a t i o n f o r the available lysine as determined by the pepsin-pancreatin method of Mauron et a l . ( 1 9 5 5 ) . "by i n vivo feeding and by the 108. TABLE 8. Amount o f l y s i n e r e l e a s e d from p r o t e i n samples s u b j e c t e d t o p e p s i n - p a n c r e a t i n d i g e s t i o n . S amp l e E x p e r i m e n t No. 1 2 3 Ave v a l u e s C a s e i n 3. 463 4. 181 3. 994 3. 879 3. 8 1 B - l a c t o g l o b u l i n 5. 374 5. 690 6. 0111 5.692 1.5 N HCl G l u t e n 0. 542 0. 589 0. 559 0.563 Lysozyme 2. 200 2. 357 2. 365 2.313 Egg 2. 703 2. 762 2. 831 2. 765 2.74+0.13 1 2 . r The r e s u l t s are g i v e n i n g o f l y s i n e p e r 100 g p r o t e i n . 1. L y s i n e r e l e a s e d by the p e p s i n p a n c r e a t i n d i g e s t i o n t e s t . From: Stahmann and W o l d e g i o r g i s , 1975. 2. L y s i n e r e l e a s e d by t h e p e p s i n p a n c r e a t i n d i g e s t d y a l i s a t e method. From: Mauron, 1970. 109. Carpenter method, for a series of heat treated milk samples. Stahmann and Woldegiorgis (19 75) demonstrated that only the enzymatic method was able to show an increase i n available lysine following steaming of soybean meal. I t i s well known that raw soybeans contain a trypsin i n h i b i t o r , and the increase i n the l i b e r a t i o n of methionine, l y s i n e , arginine and h i s t i d i n e confirms that steaming destroys the i n h i b i t o r . I t i s apparent that the chemical methods for protein q u a l i t y may not reveal the a v a i l a b i l i t y of lysine when the proteins tested contain an i n h i b i t o r of one of the p r o t e o l y t i c enzymes that act during digestion. Sometimes, i t might be d i f f i c u l t to correlate chemical and b i o l o g i c a l a v a i l a b i l i t y since the l a t t e r could also involve protein d i g e s t i b i l i t y which may not be related to the r e a c t i v i t y of the e-amino group of ly s i n e . 110. GENERAL DISCUSSION A summary o f t h e r e s u l t s f o r a v a i l a b l e l y s i n e as de t e r m i n e d by the FDNB d i f f e r e n c e p r o c e d u r e , the DNBS and TNBS methods, and t h e p e p s i n p a n c r e a t i n d i g e s t i o n t e s t i s shown i n Ta b l e 9. F o r comparison o f the methods, the r e g r e s s i o n l i n e s and the c o r r e l a t i o n c o e f f i c i e n t s were e s t i m a t e d a c c o r d i n g t o the p r o c e d u r e d e s c r i b e d by Deming (Wakkers e t a l . , 1975). The " c l a s s i c a l p r o c e d u r e " t o e s t i m a t e the r e g r e s s i o n l i n e , assumes t h a t one o f the methods, used as the r e f e r e n c e , i s n o t s u b j e c t t o e r r o r . However, t h i s i s n o t always t r u e . Deming"s p r o c e d u r e has t h e advantage t h a t e s t i m a t e s are o b t a i n e d f o r the random e r r o s o f b o t h methods. Good agreement was o b t a i n e d between the FDNB o f f i c i a l method and t h e DNBS t e c h n i q u e , w i t h a c o r r e l a t i o n c o e f f i c i e n t o f 0.989 ( F i g u r e 12). When the TNBS method was compared t o the FDNB d i f f e r e n c e t e c h n i q u e , a c o r r e l a t i o n c o e f f i c i e n t o f 0.988 was found ( F i g u r e 13). The p e p s i n p a n c r e a t i n t e s t i n d i c a t e d the r e l a t i v e amount o f l y s i n e r e l e a s e d by the enzymes under the c o n d i t i o n s s p e c i f i e d f o r the t e s t . A c o r r e l a t i o n c o e f f i c i e n t o f 0.995 was found between the FDNB method and the enzymic t e s t ( F i g u r e 14). F i g u r e s 15, 16 and 17 show the r e g r e s s i o n l i n e s f o r comparison between the e n z y m a t i c t e s t and the DNBS method ( r = 0.998), the e n z y m a t i c t e s t and the TNBS method ( r = 0.987) 111. TABLE 9. L y s i n e a v a i l a b i l i t y d e t e r m i n e d by the FDNB o f f i c i a l p r o c e d u r e , the DNBS and t h e TNBS methods, and t h e e n z y m a t i c t e s t . S ample FDNB DNBS TNBS E n z y m a t i c T e s t G l u t e n 1.55 1. 75 1. 46 0.56 C a s e i n 7. 35 7. 52 7. 14 3.8 Lysozyme 5.15 5.43 5.73 2.31 ( 3 - l a c t o g l o b u l i n 11. 27 12. 26 13.93 5. 69 Whole egg 6.55 5.48 6. 71 2.77 The v a l u e s are g i v e n i n g p e r 100 g o f p r o t e i n . 112. 12. Comparison between the FDNB d i f f e r e n c e t e c h n i q u e and the DNBS method. The $ o f l y s i n e i s d e t e r -mined by g l y s i n e / 1 0 0 g p r o t e i n . FDNB (% lys ine) 113. URE 1 3 . Comparison between the FDNB d i f f e r e n c e technique and the TNBS method. The % of l y s i n e i s d e t e r -mined by g l y s i n e / 1 0 0 g p r o t e i n . FDNB (% l/sine ) 114. FIGURE 14. Comparison between the FDNB d i f f e r e n c e method and the enzymatic d i g e s t i o n t e s t . The % of l y s i n e i s determined by g l y s i n e / 1 0 0 g p r o t e i n . FDNB (% l y s i n e ) 115. FIGURE 1 5 . Comparison between the enzymatic d i g e s t i o n t e s t and the DNBS method. The % o f l y s i n e i s d e t e r -mined by g l y s i n e / 1 0 0 g p r o t e i n . E N Z . T E S T (% lysine) 116. FIGURE 1 6 . Comparison "between the enzymatic d i g e s t i o n t e s t and the TNBS method. The % o f l y s i n e i s d e t e r -mined by g l y s i n e / 1 0 0 g p r o t e i n . E N Z . T E S T ( % l y s i n e ) 117. FIGURE 17. Comparison between the TNBS and the DNBS methods. The % of l y s i n e i s determined by g l y s i n e / 1 0 0 g p r o t e i n . 15 10-c v> cn m z o r = 0 995 o A • • gluten lysozyme egg case in 0 - l ac tog lobul in 10 15 T N B S (%lys ine) 118. and the TNBS and DNBS method (r = 0.995), r e s p e c t i v e l y . The standard d e v i a t i o n o f the random e r r o r f o r each method i s shown i n Table 10. A m u l t i p l e range t e s t (Duncan's t e s t ) showed t h a t the TNBS method produced s i g n i f i c a n t l y lower r e p e a t a b i l i t y as compared to the o t h e r t h r e e methods. No s i g n i f i c a n t d i f f e r e n c e i n p r e c i s i o n was found among the FDNB o f f i c i a l technique, the DNBS method and the enzymatic t e s t . 119. TABLE 10. S t a n d a r d d e v i a t i o n o f the random e r r o r ( i n g l y s i n e / 100 g p r o t e i n ) i n the a v a i l a b l e l y s i n e methods, as e s t i m a t e d by Deming's p r o c e d u r e . .^ , Random e r r o r e s t i m a t e s i n method Methods compared FDNB DNBS TNBS E n z y m a t i c FDNB and DNBS 0.57 7 FDNB and TNBS 0.366 FDNB and Enz. 0.419 TNBS and DNBS Enz. and DNBS Enz. and TNBS 0.546 0.663 0.187 0.363 0.672 0.256 0.308 0.683 0.399 120. CONCLUSION From the r e s u l t s presented i n t h i s t h e s i s i t can be seen t h a t l y s i n e a v a i l a b i l i t y i n c a s e i n , a c i d s o l u b i l i z e d g l u t e n , egg, ( 3 - l a c t o g l o b u l i n and lysozyme, as determined by the o f f i c i a l FDNB d i f f e r e n c e technique gave r e s u l t s i n c l o s e agreement with the r e p o r t e d v a l u e s . Although the d i f f e r e n c e technique i s extremely time consuming, i t i s pr o b a b l y the most accurate means p r e s e n t l y a v a i l a b l e f o r d e t e r m i n a t i o n o f l y s i n e a v a i l a b i l i t y , I t i s not a f f e c t e d by the d e s t r u c t i o n o f €-DNP-lysine d u r i n g a c i d h y d r o l y s i s . The l y s i n e r e l e a s e d by the p e p s i n - p a n c r e a t i n d i g e s t i o n c o r r e l a t e d w e l l with the a v a i l a b l e l y s i n e as d e t e r -mined wi t h the FDNB chemical method. The DNBS and TNBS methods gave r e s u l t s i n c l o s e agreement with the r e s u l t s o b tained w i t h the FDNB o f f i c i a l t echnique. However, the data from the TNBS method showed a wide range of v a r i a b i l i t y and s i g n i f i c a n t l y lower r e p e a t i b i -l i t y . The use o f l y s i n e d e r i v a t i v e s to study the s p e c i -f i c i t y o f DNBS f o r the €-amino group of l y s i n e , showed t h a t DNBS r e a c t s with the -X-amino group o f l y s i n e , to a c e r t a i n e xtent. However, f o r p r o t e i n s and h i g h e r p o l y p e p t i d e s the c o n t r i b u t i o n o f the cK-amino group to the r e s u l t s becomes n e g l i g i b l e . 121. Only f o r s m a l l p e p t i d e s h a v i n g N - t e r m i n a l l y s i n e w i l l t he r e s u l t be s l i g h t l y h i g h e r t h a n t h e t r u e c o n t e n t o f a v a i l a b l e l y s i n e . The DNBS method was found t o be the s i m p l e s t and most r e l i a b l e method f o r d e t e r m i n a t i o n o f a v a i l a b l e l y s i n e , f o r the f o l l o w i n g r e a s o n s : 1) I t does n o t r e q u i r e a c i d h y d r o l y s i s o f the p r o t e i n . 2) I t i s s p e c i f i c f o r the e-amino group o f l y s i n e and p r a c t i c a l l y f r e e from i n t e r f e r e n c e s . 3) A l a r g e number o f samples can be a n a l y z e d s i m u l t a n e o u s l y i n a few h o u r s . 4) I t does not r e q u i r e e x p e n s i v e and l e n g t h y c h r o m a t o g r a p h i c amino a c i d a n a l y s i s . REFERENCES CITED AKESON, W.R. and M.A. Stahmann. 1964. A p e p s i n p a n c r e a t i n d i g e s t index o f p r o t e i n q u a l i t y e v a l u a t i o n . J . Nutr. 83» 257. BARAUD, J . 1957-Iodometric a n a l y s i s o f f r e e amino n i t r o g e n i n p r o t e i n by means o f t h e i r dithiocarbamate d e r i v a t i v e s . B u l l . 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N u t r i t i o n a l e v a l u a t i o n o f p r o t e i n foods. N a t l . Acad. S c i e n c e s - N a t l . Res. C o u n c i l , Washington, D.C. ( I n P r e s s ) . 146. APPENDIX 3% DNBS REAGENT D i s s o l v e 6 g DNBS and 6 g a c t i v a t e d c h a r c o a l i n d i s t i l l e d water and make the volume up to 100 ml. S t i r f o r 10 min and f i l t e r through Whatman # 1 . Measure the r e f r a c t i v e index o f the y e l l o w s o l u t i o n and d i l u t e i t to o b t a i n a 3% DNBS s o l u t i o n . R e f r a c t i v e index o f DNBS = 1 .3406 R e f r a c t i v e index o f d i s t i l l e d water = 1 .3340 D i f f e r e n c e = 0 . 0066 A d i f f e r e n c e o f 0.0046 corresponds to a 3% DNBS (RI = 1 .3386) s o l u t i o n , thus* 0 .0046 3% 0 .0066 x x = 4 . 3 % DNBS I f 4 .3% DNBS . 9 1 . 5 ml ( v o l . a f t e r f i l t e r e d ) 3.0% DNBS „ x x = 1 3 1 . 1 5 ml. T h e r e f o r e , d i l u t e the 4 .3% DNBS s o l u t i o n up to 1 3 1 . 1 5 ml by adding 3 9 - 6 5 ml d i s t i l l e d water. 

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