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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.Sc,  Universidad  A Thesis Submitted the  Javeriana,  i n Partial  197^  Fulfillment of  R e q u i r e m e n t s f o r t h e Degree o f MASTER OF SCIENCE in  The  F a c u l t y o f Graduate  (Department o f Food  We a c c e p t  this  the  THE  thesis  required  Studies  Science)  as conforming t o standard  UNIVERSITY OF B R I T I S H COLUMBIA FEBRUARY, 1 9 7 9  (c) M a r i l u z H o l g u i n ,  1979  In presenting  this thesis in partial fulfilment of the requirements for  an advanced degree at the University of B r i t i s h Columbia, I agree that the Library shall make i t f r e e l y available for reference  and  study.  I further agree that permission for extensive copying of this thesis for scholarly purposes may by his representatives.  be granted by the Head of my Department or It is understood that copying or publication  of this thesis for f i n a n c i a l gain shall not be allowed without my written permission.  Mariluz  _. . . ~ Department of  Food  Science  The University of B r i t i s h Columbia 2075 Wesbrook Place Vancouver, Canada V6T 1W5  Date  F e b r u a r y 2,  1979  Holguin  ii  ABSTRACT  Lysine since  i s o f prime n u t r i t i o n a l  i t i s t h e f i r s t l i m i t i n g amino a c i d  o f p l a n t o r i g i n and i s e a s i l y r e n d e r e d heat processing The t e r m  o r upon unfavourable  "available  which c o n t a i n f r e e chain.  lysine" refers  i n many f o o d s  unavailable  storage  upon  conditions.  t o forms o f l y s i n e  €-amino g r o u p s w i t h i n t h e p e p t i d e  Once t h e <= -amino g r o u p i s b l o c k e d ,  unavailable  significance  s i n c e i t c a n n o t be h y d r o l y z e d  lysine  becomes  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 |6-lactoglobulin,  acid  of lysines  dinitrobenzene  benzene s u l f o n i c  sulfonic  acid  acid  digestion test,  (DNBS) and t h e t r i n i t r o -  (TNBS) methods. The r e s u l t s  compared t o t h e f l u o r o d i n i t r o b e n z e n e a g r e e m e n t was o b t a i n e d and  lysozyme,  s o l u b i l i z e d g l u t e n and whole e g g  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 the  i n casein,  t h e DNBS t e c h n i q u e ,  (FDNB) method.  b e t w e e n t h e FDNB o f f i c i a l with  were  a correlation  Good  method  coefficient of  O.989. When t h e TNBS method was compared t o t h e FDNB d i f f e rence technique,  a correlation  coefficient of  O.988 was  found. The p e s i n p a n c r e a t i n  digestion test  indicated  the  r e l a t i v e amount o f l y s i n e  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  ficient  of  r e l e a s e d b y t h e enzymes u n d e r coef-  0.995 was f o u n d b e t w e e n t h e FDNB o f f i c i a l method  iii.  and  the enzymatic  test.  The s p e c i f i c i t y  o f DNBS f o r t h e €-amino  group  of lysine  was d e t e r m i n e d  u s i n g e < - and 6 - f o r m y l - l y s i n e s »  L-lysine,  L-lysyllysine,  L-lysylalanine  S-peptide.  DNBS was f o u n d  group b u t w i t h However,  a slight  i n t h e case  g r o u p s and N - t e r m i n a l  to react mainly  reactivity  o f proteins with lysine,  lysine, acid  and  h y d r o l y s i s o f the p r o t e i n s ;  amino a c i d  €-amino  negligible. t o be t h e s i m p l e s t  a) i t d o e s n o t r e q u i r e b) a l a r g e number o f  s i m u l t a n e o u s l y i n a few h o u r s ,  c) i t does n o t r e q u i r e e x p e n s i v e  tographic  several  group.  f o r determination of available  f o r the f o l l o w i n g reasons*  s a m p l e s c a n be a n a l y z e d  €-amino  the c o n t r i b u t i o n o f the  The DNBS method was f o u n d m o s t r e l i a b l e method  with  w i t h oO-amino  c(-amino g r o u p t o t h e r e s u l t s becomes  and  and r i b o n u c l e a s e -  analysis.  and l e n g t h y  chroma-  iv.  TABLE OF CONTENTS PAGE ABSTRACT  i .  ACKNOWLEDGEMENTS  i i i .  TABLE OF CONTENTS  iv.  L I S T OF FIGURES  v i .  L I S T OF TABLES  viii. 1  INTRODUCTION LITERATURE  SURVEY ON METHODS FOR  DETERMINATION 8  OF LYSINE- AVAILABILITY.  8  A. B i o a s s a y s B. M i c r o b i o l o g i c a l A s s a y s  12  C. C h e m i c a l Methods  16  1.  The f l u o r o d i n i t r o b e n z e n e  2.  The t r i n i t r o b e n z e n e method  method  1?  (FDNB)  sulfonic acid 28  (TNBS)  3.  Guanidination  35  4.  Acrylonitrile  36  5.  Methyl a c r y l a t e  36  6.  E t h y l v i n y l sulfone  37  7.  2-Chloro-3,5-dinitropyridine  37  8.  Sodium b o r o h y d r i d e  38  9. 10.  1  9  F  39  NMR  The s o d i u m d i n i t r o b e n z e n e method  sulfonate 42  (DNBS)  11.  Fluorescamine  48  12.  Dye-binding  48  13.  C h e m i c a l methods i n t h e d e t e r m i n a t i o n available  lysine i n materials  of  that  have u n d e r g o n e 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) 54  D. Enzymatic Methods  65  MATERIALS AND METHODS  65  A. M a t e r i a l s B. N i t r o g e n Determination  66  C. Amino A c i d A n a l y s i s D. L y s i n e Determination  "by the Dinitrobenzene  S u l f o n i c A c i d Method E. L y s i n e Determination  74  by the D i n i t r o f l u o r o 79  benzene Method G. Enzymatic D i g e s t i o n Test  85 89  RESULTS AND DISCUSSION A. The Dinitrobenzene  67  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 Method F. L y s i n e Determination  66  S u l f o n i c A c i d Method  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  89 96  C. The F l u o r o d i n i t r o b e n z e n e Method  101  D. 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  105  GENERAL DISCUSSION  110  CONCLUSION  120  REFERENCES CITED  122  APPENDIX  146  vi.  L I S T OF FIGURES F i g u r e No. 1(a) 1(b) 2 3  P a g e No. Structure of free lysine. B o d w e l l , 1976.  From: 2  L y s i n e as i t w o u l d e x i s t i n a p e p t i d e chain. From: B o d w e l l , 19 76. R e a c t i o n o f FDNB w i t h an amino F r o m : H a l l e t a l . , 1974.  acid.  R e a c t i o n o f TNBS w i t h an amino From: H a l l e t a l . , 1973.  acid.  3 21 29  19 4  F NMR s p e c t r u m o f t h e 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 dimethyl sulfoxide solution.  41  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. F r o m : Ney a n d Wirotama, 1970.  50  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 m e t h o d for determination of available lysine.  72  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 dinitrobenzene sulfonic acid.  73  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 t h e d e t e r m i n a t i o n o f available lysine.  77  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-trinitrobenzene sulfonic acid  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 tion of protein hydrolysates  82  11  The d i n i t r o f l u o r o b e n z e n e m e t h o d f o r determination of available lysine (difference technique). From: B l o m e t a l . , 1967.  84  5  6 7  8  9  vii.  L I S T OF FIGURES  (Continued)  F i g u r e No. 12  13  14  15  16  17  Page No. C o m p a r i s o n b e t w e e n t h e FDNB d i f f e r e n c e t e c h n i q u e a n d 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  C o m p a r i s o n b e t w e e n t h e 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 m e t h o d . 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  C o m p a r i s o n b e t w e e n t h e 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 test. 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  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 a n d t h e DNBS m e t h o d . The % o f l y s i n e i s d e t e r m i n e d b y g lysine/100 g protein.  115  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 a n d t h e TNBS m e t h o d . The % o f l y s i n e i s d e t e r m i n e d b y g lysine/100 g protein.  116  C o m p a r i s o n b e t w e e n t h e TNBS a n d t h e DNBS m e t h o d s . The % o f l y s i n e / 1 0 0 g protein.  117  viii.  LIST OF  TABLES  Table No. 1 2 3  Page Reagents used and d e r i v a t e s i n D i r e c t Methods.  formed  Reagents used and d e r i v a t e s i n I n d i r e c t Methods.  formed  18 19  P r o t e i n content of samples assayed for lysine a v a i l a b i l i t y .  90  A v a i l a b l e l y s i n e content of p r o t e i n samples as determined by the d i n i t r o benzene s u l f o n i c a c i d (DNBS) method.  91  5  Reaction of,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).  94  6  A v a i l a b l e l y s i n e content of p r o t e i n samples as determined by the t r i n i t r o benzene s u l f o n i c a c i d (TNBS) method.  97  4  7  8  9  10  A v a i l a b l e l y s i n e content of p r o t e i n samples as determined by the f l u o r o - d i n i t r o b e n z e n e (FDNB) method.  102  Amount of 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 to p e p s i n - p a n c r e a t i n digestion.  108  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 .  Ill  Standard d e v i a t i o n of the random e r r o r ( i n g lysine/100 g p r o t e i n ) i n the a v a i l able l y s i n e methods, as estimated with Deming's procedure.  119  No.  ix.  ACKNOWLEDGEMENTS  I wish t o express my g r a t i t u d e t o Dr. S. N a k a i , Chairman  o f my committee  ment i n the p r e p a r a t i o n  f o r h i s s u p e r v i s i o n and encourageof t h i s t h e s i s .  members o f my committee:  A l s o t o the other  Drs. Powrie, Vanderstoep and  Beames my s i n c e r e thanks. To V a l e r i e ( t e c h n i c i a n ) I am t r u l y g r a t e f u l f o r her c o - o p e r a t i o n  and a s s i s t a n c e .  I should  a l s o l i k e t o thank my husband  help and encouragement  for his  i n the p r e p a r i n g o f 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 n o t o n l y bility  on t h e amino a c i d  o f the p r o t e i n  availability. percentage  itself  Availability  synthesis  a s t h e amount o r  i n the food which  i s the only  G r o w t h may t h e r e f o r e  upon the p h y s i o l o g i c a l  P a n be d e f i n e d  i n the organism  when t h e s a i d amino a c i d diet.  hut also  o f a g i v e n amino a c i d  for protein  c o m p o s i t i o n and d i g e s t i -  i s utilized  ( g r o w t h and m a i n t e n a n c e ) limiting  be c o n s i d e r e d  f a c t o r o f the  t o 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 . Dietary I n many f o o d s , small  lysine  insufficiency often  i s limiting  amounts a r e i n c o r p o r a t e d  synthesis due  lysine  but also  to factors  sugars,  limits  n o t Only because  i n the p r o t e i n s  heat,  alkali,  relatively  during  because o f secondary chemical  such as l i g h t ,  growth.  bio-  changes  and r e d u c i n g  a s a r e s u l t o f w h i c h l y s i n e becomes n u t r i t i o n a l l y  unavailable. Lysine a  free  f o r m h a s a f r e e eC-amino g r o u p ,  €-amino g r o u p and a f r e e  proteins, acids  i n i t s free  carboxyl  95 to 100$ o f the l y s i n e  through peptide  linkages 1).  group.  i s linked  involving  I n most  to other  t h e oC-amino and  carboxyl  groups (Figure  to  react  w i t h o t h e r compounds s u c h a s s u g a r s o r amide  of  o t h e r amino  acids.  amino  O n l y t h e €-amino g r o u p  i s free groups  2.  FIGURE 1 .  (a) Structure  of free lysine.  From* B o d w e l l ,  1976.  €-amino g r o u p  —•  NH CH  2  2  1 1  CH  c  P  1  c  1  c  H~N\-  C - GOOH  t  H  ^  U-amino  1  group  3.  FIGURE !••  (t>)  L y s i n e as i t would e x i s t  i n a portion of  a peptide chain.  Bodwell, 1976.  NH  2  GH  2  From*  i  CH.  i  i  CH,  CH  2  CH  2  2  i  II  H H 0  Threonine  i  i  i  H i  N-C-G  N-C-G i  S  CH  H-C-OH i  GHo  II  i  N-C-C II  H H 0  H H 0  Lysine  Glycine  N-C-C i  i  II  H H 0  Methionine  4. Lysine group i s f r e 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 I f t h e €-amino g r o u p i s b l o c k e d  through a  c h e m i c a l bond, t h e segment o f t h e p r o t e i n n e a r t h e a f f e c t e d lysine  residue  may n o t be  I f the dipeptides,  a  p r o t e i n i s b r o k e n down t o amino a c i d s o r  the reacted  gastrointestinal  digested.  l y s i n e may n o t be a b s o r b e d f r o m t h e  tract.  Even i f absorbed,  the l y s i n e having  ' d e r i v a t i z e d * €-amino g r o u p may n o t be u t i l i z e d b u t r a t h e r  excreted  i n the u r i n e . Lysine  particular lyze  may a l s o be b u r i e d  i n a protein matrix of a  sequence o r c o n f o r m a t i o n which i s slow t o h y d r o -  or i s not hydrolyzed  a t a l l by animal proteases.  l y s i n e may o r may n o t a p p e a r a s c h e m i c a l l y  a v a i l a b l e by  c h e m i c a l methods and y e t be t o t a l l y u n a v a i l a b l e Proteolysis within  the i n t e s t i n a l  c a r r i e d out by the p a n c r e a t i c and  nutritionally.  lumen i s l a r g e l y  p r o t e o l y t i c enzymes.  Trypsin  c h y m o t r y p s i n a c c o u n t f o r 78% o f t h e t o t a l p r o t e o l y t i c  activity. residue The  Such  Trypsin  acts  on a p r o t e i n a t e i t h e r an a r g i n i n e  or a l y s i n e residue  a c t i o n o f other  w h i c h h a s a f r e e amino g r o u p .  proteases  i s only p a r t i a l l y i n h i b i t e d  when t h e €-amino g r o u p o f l y s i n e i s b l o c k e d . Under severe o r p r o l o n g e d h e a t i n g  c o n d i t i o n s , the  €-amino g r o u p o f l y s i n e c a n r e a c t w i t h t h e ) ( - c a r b o x y l g r o u p o f g l u t a m i c a c i d o r t h e (3-carboxyl g r o u p o f a s p a r t i c acid  on a n o t h e r p r o t e i n o r w i t h i n  t h e same p r o t e i n m o l e c u l e .  5.  As  a result,  resistant  a so  called  isopeptide  t o h y d r o l y s i s by  b e l i e v d t o be  gut  proteases.  mes  are  specific  The  a s p a r t y l - or g l u t a m y l - l y s i n e  However, M a u r o n ( 1 9 7 2 ) shown t h a t l y s i n e by  the  rat.  be  The  p o s s i b l y by  Booth (1973)  be  utilized  dipeptides  as  are p r o b a b l y  and  reduce  hindrance  the  o f the  i n the  alanine  of p r o t e i n digestion,  sites  cysteine  Dehydroalanine  of attack  by  thus formed r e a c t s w i t h (LAL).  nutritionally  available since  i n the  m e a l was  the  im  The  heating  dehydro-  €-amino g r o u p t o  form  I t appears t h a t l y s i n o a l a n i n e i s 38-65% o f the  form o f a l k a l i - t r e a t e d  recovered  with  i s f o r m e d by  under a l k a l i n e c o n d i t i o n s .  lysinoalanine  rats  kid-  enzymes.  dehydroalanine residues. or  as  might  Another r e a c t i o n of l y s i n e i s c r o s s - l i n k a g e  serine  of  absorbed  hydrolyzed  rate  have  sources  o f numerous s u c h c r o s s - l i n k a g e s  to a t l e a s t steric  linkages.  remains a f t e r p r o t e o l y s i s .  C a r p e n t e r and  intestinal wall  presence  expected  digestive  The  P r o t e o l y t i c enzy-  f o r t h e <£,£-peptide  these d e r i v a t i v e s can  such through the neys.  and  bond i s formed, w h i c h i s  the  feces  LAL  ingested  soybean c a s e i n or  ( d e G r o o t and  not by  soybean I969).  Slump,  A more common r e a s o n 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  reducing  sugars r e a c t with  Frampton (1967) all  carbohydrates.  react with  lysine.  showed t h a t s u c r o s e ,  Both reducing El-Nockrashy  non-  and  r a f f i n o s e and  l y s i n e to render i t u n a v a i l a b l e .  and  trehalose  Maillard  6.  reaction  i n w h i c h t h e €-amino g r o u p o f l y s i n e r e a c t s  aldehyde groups o f r e d u c i n g of l i p i d s ,  i s very well  sugars,  or with carbonyl  with groups  known t o d e c r e a s e l y s i n e a v a i l a b i -  lity. Thompson e t a l . ( 1 9 7 6 ) of  available  system. The  l y s i n e during  Available  first  have s t u d i e d  thermal processing  p h a s e 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  characterized reaction  on  acted  (FDNB).  order reaction  The t h i r d  utilized  glucose  r a p i d phase  kinetics.  p h a s e was lysine. occurred  W o l f and  a model system c o n s i s t i n g o f  glucose,and m i c r o c r y s t a l l i n e  predicting  activity  procedure  r e s u l t i n g i n the i n i t i a l  Thompson ( 1 9 7 7 )  found t h a t  unavailable.  b y 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  according to f i r s t  protein,  i n which  i n a v a i l a b l e l y s i n e when m e a s u r e d b y C a r p e n t e r ' s  fluorodinitrobenzene  The  phases.  s i g n i f i c a n t (98$ confiden-  Phase two showed a s t a t i s t i c a l l y increase  cellulose.  I t was  and t e m p e r a t u r e h a d t h e d o m i n a n t e f f e c t  the s p e c i f i c r e a c t i o n  a n d pH a l s o  influenced  rate  (k-j.).  the p r e d i c t i o n  though an i n t e r a c t i o n w i t h g l u c o s e .  Water o f k^ b u t t h e y  Jokinen et a l .  (1976)  developed an e q u a t i o n which p r e d i c t s  lysine  bility  as a f u n c t i o n  glucose,  and  sucrose  lysine  rate  o f a s o y model  l y s i n e l o s s went t h r o u g h t h r e e  30 t o 60% o f t h e a v a i l a b l e l y s i n e was made  ce)  the l o s s  o f pH, w a t e r a c t i v i t y ,  i n phase t h r e e .  i n fortified  availa* starch,  Thermal l o s s o f a v a i l a b l e  r i c e m e a l was k i n e t i c a l l y  assessed by  7.  T s a o e t a l . (1978). Because o f the extreme diet, and  and the a d d i t i o n a l  internal  cessing,  some t y p e s  s e v e r a l m e t h o d s h a v e "been p r o p o s e d  categorized  available  of lysine  considerations of protein  cross-linkages following  nutritionally  in vitro  importance  lysine.  i n four groupst  o f pro-  T h e s e methods c a n be  Biological  evaluation studies, and c h e m i -  cal  analyses.  ble  c h e m i c a l methods which a r e q u i c k and s u i t a b l e  tine  h a s b e e n made t o e s t a b l i s h  relia-  f o r rou-  assay. The  availability the  damage  f o r estimating  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 Much e f f o r t  i n the  object of this with  two o f t h e most p o p u l a r  dinitrobenzene sulfonic  benzene s u l f o n i c r e s u l t s with  s t u d y was t o d e t e r m i n e  acid  acid  methods,  (DNBS) and t h e t r i n i t r o -  (TNBS) t e c h n i q u e s ,  the f l u o r o  chemical  lysine  dinitrobenzene  and t o compare t h e  (FDNB). AOAC o f f i c i a l  method. Enzymatic to  correlate  example, mical  chemical  and b i o l o g i c a l  i t has been demonstrated  determinations  tibility  d i g e s t i o n assays  were done i n a n availabilities,  attempt 'i F o r  t h a t , i n some c a s e s ,  do n o t show t h e e x t e n t o f p r o t e i n  o r the presence  of trypsin  inhibitors.  chediges-  8.  METHODS FOR DETERMINATION OF LYSINE AVAILABILITY A.  BIOASSAYS The u l t i m a t e standards f o r the i n v i t r o methods  o f measuring amino a c i d a v a i l a b i l i t y animals.  are b i o a s s a y s w i t h  Animal assays are based on the o b s e r v a t i o n t h a t  the amount and q u a l i t y o f a p r o t e i n can r e s u l t i n a g a i n or l o s s o f body s u b s t a n c e .  Such g a i n or l o s s can be  identified  by a change i n body weight or by a change i n a body component which o f t e n i s n i t r o g e n c o n t e n t . The P r o t e i n E f f i c i e n c y R a t i o (PER), which i s o f f i c i a l AOAC p r o c e d u r e , and the Net P r o t e i n R a t i o are based on weight changes.  the  (NPR)  The methods based on body  n i t r o g e n change are the Net P r o t e i n U t i l i z a t i o n  (NPU) where  c a r c a s s n i t r o g e n i s measured, and the B i o l o g i c a l Value  (BV)  where n i t r o g e n balance i s measured. PER i s the s l o p e o f the l i n e r e l a t i n g weight g a i n of a rat  to p r o t e i n intake consumed under s t a n d a r d i z e d ex-  perimental  conditions.  Despite i t s long h i s t o r y ,  wide  usage, and o f f i c i a l s t a t u s , PER i s not a good assay procedure (Hegsted and Chang, 1965$ Hegsted and Samonds, 1978). the c r i t e r i a d e f i n e d f o r a v a l i d b i o a s s a y , i . e . , reproducibility, statistical validity,  Of  precision,  proportionality,  s i m p l i c i t y and low c o s t (Hegsted and Samonds, 1978) PER c o u l d be c o n s i d e r e d as meeting o n l y the c r i t e r i o n of s i m plicity.  Some improvement can be made by i n c l u s i o n o f a  9-  group o f animals period. Net  When m o d i f i e d  i n t h i s way  P r o t e i n R a t i o (NPR)  f i c a t i o n o f NPR by  consuming a n o n - p r o t e i n d i e t  called  for a  the procedure  ( B e n d e r and R e l a t i v e NPR  Doell,  similar  i s called  1957).  A modi-  (RelNPR) i s recommended  t h e 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  - National  Re-  s e a r c h 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 ( Y o u n g  and  1978)  Pellett, assay  cannot  as a u s e f u l p r o c e d u r e  be  performed.  i d e n t i c a l manner as NPR, tive  R e l a t i v e NPR  but  i n an  are expressed  rela-  or 100.  Bioassays tiple-point  with both  are c l a s s e d as e i t h e r t w o - p o i n t  assays.  c o n n e c t i n g the rats  two  W i t h two  (Hegsted  entirely valid,  and  or mul-  a straight  assumed.  l i -  Studies  N e f f , 1 9 7 0 j Hegsted, 1971)  and  have shown t h i s  not  assumption  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 ponse o b t a i n e d  p o i n t assays,  p o i n t s must be  humans (Young e t a l . , 1 9 7 7 ) t o be  the r e s u l t s  i s performed  t o t h e v a l u e o b t a i n e d w i t h an 8fo l a c t a l b u m i n d i e t  t a k e n as 1 . 0 0  ne  i f a multiple-point  (weight,  i n v o l v e measurements o f t h e  body water, o r body n i t r o g e n )  resin re-  l a t i o n t o p r o t e i n comsumed; a t s e v e r a l l e v e l s o f p r o t e i n c o n s u m p t i o n . These l e v e l s must be  selected with care, since only  f o r a s m a l l range can a s t r a i g h t l i n e v e l s more and more p r o t e i n w o u l d be energy,  while  a t v e r y low  be  assumed* a t h i g h l e -  d e a m i n a t e d and  l e v e l s d e v i a t i o n from  used  for  linearity  o c c u r b e c a u s e o f s u c h phenomena a s a d a p t i v e r e s p o n s e s ,  could  with  10.  consequently This  more e f f i c i e n t  is essentially  utilization  o f amino a c i d s .  t r u e when t h e l i m i t i n g  amino a c i d  is  lysine. Very s a y s and  good c o r r e l a t i o n s a r e  amino a c i d  Hegsted, 1 9 7 1 ) .  still  have a BV  amino a c i d  man,  Proteins completely  are  t o 40,  and  of proteins.  relationship  lacking  ifeo r e u t i l i z e  i n lysine  or t o t a l  response  including  amino a c i d s f o r t h e  F o r t h e human a b o u t 5 - 6  ving  i n an  i n t e r m e d i a t e manner.  While  such as the R e l a t i v e P r o t e i n Value 1 9 7 8 ) , c a n g i v e more r e a l i s t i c q u a l i t y p r o t e i n s , no animal  r i o d by  bioassay  can conserve  and  s l o w i n g down l y s i n e  Fortunately,  Not a l l  an absence o f  w i t h the o t h e r e s s e n t i a l  quality  have t h e s e v e r y low  procedures  estimates  i s exempt f r o m  f o r poor  this  problem. pe-  et a l . , 1972).  p r o t e i n s or r e a l  levels of essential  Pellett,  for a limited  o x i d a t i o n (Brooks  i n p r a c t i c e v e r y few  zero  ( Y o u n g and  recycle lysine  va-  amino a c i d s b e h a -  bioassay  (RPV)  syn-  g of p r o t e i n  s u l f u r amino a c i d s g i v e s t h e e x p e c t e d  f o r BV,  other  above z e r o . Young  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.  line  va-  (Bender,  h a v e shown t h a t a l l a n i m a l s ,  t h e amino a c i d s behave i n t h e same way;  An  biological  proteins lacking  c a n have v a l u e s s i g n i f i c a n t l y  have t h e a b i l i t y  thesis  The  a  amino a c i d b e l o w t h i s l e v e l  equal  Scrimshaw (1977)  and  1978).  (Pellett,  r i e s w i t h the l i m i t i n g  can  between b i o a s -  score f o r p r o t e i n s having  v a l u e g r e a t e r t h a n 40  I96I5  found  dietaries  amino a c i d s .  11.  The  content of available  comparing the performance  lysine  of animals  c a n he m e a s u r e d  receiving  a basal  supplemented w i t h the t e s t m a t e r i a l o r w i t h pure The  b a s a l p r o t e i n source has  tryptophan 1958;  ( W i l d e r and  ( P i o n and  Rerat, 1962)  s h o u l d be  a limiting factor  otherwise  they w i l l  of  or a mixture  (Bruggemann e t a l . , 1 9 6 9 ) .  ley  available  will  through  by  a growth response  changing  be  w h i c h have b e e n i  to a t e s t  level  deficient that  the  supplement can a l s o  be  relations  A. $ l y s i n e  used  as  C.  eaten: weight  e t a l . , 1963?  Guo  for calculation  gain/g food eaten,  gain/day.  calculated  consistently  i n the d i e t : weight  i n the d i e t : weight  i n lower  o f the d i e t  a  et a l . , 1967).  B. % l y s i n e  Carpenter  specifically  when added t o t h e d i e t ) ,  t h e amino a c i d b a l a n c e  t h e dose r e s p o n s e  resulted  animals,  to changes i n the  D i s c r e p a n c i e s have b e e n f o u n d  g lysine  of test  lysine  t h e 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 ,  whole ( M u e l e n a e r e  to  that  bar-  t h a t t h i s i s t h e o n l y amino a c i d  magnitude o f the response affected  sensitive  a b a s a l d i e t may  ( i n the sense  elicit  o f wheat and  It i s essential  i n the d i e t s  1 9 6 2 ) , wheat  lysine.  Although in lysine  n o t be  and  wheat (Howard e t a l . ,  M o r r i s o n e t a l . , , 1 9 6 3 - ; Mauron and M o t t u ,  gluten  diet  lysine.  been maize w i t h c y s t i n e  K r a y b i l l , 1947),  by  values  In general A (Calhoun  et a l . , 1971)'  according of  results  gain/day; and has  et a l . , i960; 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 e t a l . , 1969)  amino a c i d b a l a n c e  (Netke  pounds i n f l u e n c i n g  food consumption e t a l . (1963)  Carpenter (1965)  (Gupta  total  lysine  and S m i t h and S c o t t  values.  of  the c o r r e s -  v a l u e s obtained by chemical  analysis.  s o y a m e a l , 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 , obtained r e s u l t s  com-  e t a l . , 1958).  p r o t e i n t h a t were h i g h e r t h a n  With w e l l processed  tal  and b y o t h e r  obtained v a l u e s w i t h c h i c k s f o r the potency  undamaged f i s h ponding  a f f e c t e d by  w h e r e a s N e t k e and S c o t t  (1970)  e q u i v a l e n t t o o n l y 7 ^ t o 80% o f t h e t o -  Thus,  i t i s n o t p o s s i b l e t o make r e l i a b l e  com-  p a r i s o n s b e t w e e n m a t e r i a l s when t h e p o t e n c i e s a r e a s s e s s e d in different laboratories comparisons there  and, e v e n f o r w i t h i n - e x p e r i m e n t  i s a n e e d 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 to check the v a l i d i t y  remain the o n l y d i r e c t  means  o f claims f o r the n u t r i t i o n a l  rele-  vance o f v a l u e s o b t a i n e d by o t h e r  B.  procedures.  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 a c h i e v e d b y t h e i n t r o d u c t i o n b y F o r d o f m i c r o b i o l o g i c a l methods. rapid,  comparatively  Microbiological  cheap and p r o v i d e  (i960)  assays are  a means o f t e s t i n g  13.  l a r g e number o f s a m p l e s i n a s h o r t t i m e . T h e y c a n be for  1)  three main purposesi  availability total  Value  and  2)  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 ; expressed  (RNV), as a s s e s s e d b y  relative or  d e t e r m i n a t i o n o f amino  a f t e r enzyme h y d r o l y s i s ?  t i o n of p r o t e i n q u a l i t y ,  F o r d , 1965?  1970).  Kakowska-Lipinska,  determination and  the growth o f the  1969;  t h e T e t r a h y m e n a b i o a s s a y and  acid  3)  of  determina-  as R e l a t i v e N u t r i t i v e  to that with a standard p r o t e i n ,  whole e g g s ( B e n d e r ,  used  RNV  test  organism  usually casein Szklarska-Cyganska  i s a r e l a t i v e measure  n o t meant t o r e l a t e  of  to the r a t  RNV. The  microorganisms  are c u l t i v a t e d  t a i n i n g a l l amino a c i d s e x c e p t unknown sample. acids  If a strain  i s i n d i s p e n s a b l e , the  growth o f the c u l t u r e by  of  acid  lactic  formed.  acid  determine  by g r a p h i c  I f the  the  curve  t o be  detected  f o r which t h i s  the  o r by  turbidity  i n an  The  produced  determination of  e x t e n t o f growth, o r the  a g a i n s t the  amount o f amino  the amount acid  i s o b t a i n e d w h i c h makes i t p o s s i b l e  amino a c i d  content  o f t h e unknown sample  interpolation. The  requirement  t e s t microorganisms  used  f o r e x o g e n o u s amino a c i d ,  that of higher  animals.  con-  amino  c e l l s w i l l not m u l t i p l y .  i s measured by  i s plotted  added, a s t a n d a r d to  i s used  the growing microorganisms,  lactic  t h e one  on a medium  show a p a t t e r n o f broadly similar  to  14.  (1962)  Ford Streptococcus the  demonstrated the  zymogenes as  an  liminary partial  usefulness  i t has  considerable  when t e s t m a t e r i a l s  d i g e s t i o n with  papain,  are the  f o r a v a i l a b l e m e t h i o n i n e " show a g e n e r a l  lues  from animal assays ( M i l l e r  tely  this  o r g a n i s m does n o t  given values  R o c k l a n d and  Dunn ( 1 9 4 9 ) .  r e q u i r e s l y s i n e but an and  indispensable  i n d e x was  ty.  not  as  a n i m a l s . Viswana'tha  present  ammonia p r o d u c t i o n  N u t r i t i v e Value  No  the  and or  the  Tetra-  protein  quali-  over a  a means o f o b t a i n i n g  employed, b u t  i n Tetra-  also studied  i n c u b a t i o n were u s e d as  was  the  trypsin inhibitor  a method f o r d e t e r m i n i n g  (RNV).  not  soybean.  Rosen (1956)  O r g a n i s m c o u n t and  by  zymogenes  globular proteins  a f f e c t e d by  components o f raw  hymena b i o a s s a y  growth  s e r i n e which i s  proteinases  hymena were u n a b l e t o a t t a c k n a t i v e  and  va-  i t s a d d i t i o n to Tetrahymena c u l t u r e  medium. T h e y showed t h a t t h e  Fernell  to  demonstrated  showed a n e e d f o r p r e d i g e s t i o n o f  t h a t T e t r a h y m e n a was  pre-  obtain-  the  T e t r a h y m e n a u n l i k e S.  i t also requires  p r o t e i n sample p r i o r t o  hemaglutinin  a  lysine.  amino a c i d f o r h i g h e r  (1955)  Liener  pro-  Unfortuna-  Determination of p r o t e i n q u a l i t y using an  of  similarity  et a l . , 1965)•  require  o f T e t r a h y m e n a p y r i f o r m i s as  of  a s s a y o r g a n i s m f o r most  e s s e n t i a l amino a c i d s , s i n c e  t e o l y t i c ^powers and,  ed  has  a  p r e d i g e s t i o n o f the  four-day  Relative samples  p r o t e i n s were d e f a t t e d b e c a u s e  of  15. the  p o s s i b l e growth i n h i b i t i o n  Landers (1975)  i n t o the  who  food  pepsin  used a 24-hr  samples p r i o r  to  p o s i t i o n o f egg f o r the  p r o t e i n has  intact  The  an  the  high PER  cor-  of  the  microorganism  does n o t  that with  measure o f  too  way  that allows  against  laborious  cell  the counts.  Shorrock (1972)  the  found  o r g a n i s m s b r e a k up  turbidity  t o be  in  u s e d as  a  response. Guggenheim ( 1 9 6 7 )  have p o i n t e d  d i s t i n c t i o n b e t w e e n a v a i l a b l e and  a c i d s by m i c r o b i o l o g i c a l a s s a y , p a r t i c u l a r conditions  o f the  Much more r e s e a r c h Tetrahymena b i o a s s a y tivity  com-  heavy to remain i n suspension  t u r b i d i t y reading,  Szmelcman and the  are  controlled autoclaving  a characteristic  the  p r o d u c e a c i d , and  protozoa  ordinary  free  protein.  A l t h o u g h the an  simulating  o f 50 t o 60 as  RNV  u s u a l l y been measured by  the  incorpora-  i n media c o n t a i n i n g  r e s p o n s e has  that  their  protein.  amino a c i d s . A s o l u t i o n o f amino a c i d s  for  trypsin-bromelain  T e t r a h y m e n a g r o w t h and  T e t r a h y m e n a grows p o o r l y  100  predigestion  Tetrahymena growth media, r e p o r t e d  r e l a t i o n between the food  used a 3-hr  who  Frank e t a l . (1975)  p r e d i g e s t i o n o f the tion  acids  1973).  (Rolle,  and  from c e r t a i n f a t t y  to other  unavailable  i s a r b i t r a r y and  concerning  food  amino  r e l a t e s to  assay. the  applicability  t o a wide r a n g e o f f o o d s and  non-protein  out  ingredients  of  i t s sensi-  i s needed  before  16  the microorganism estimate  C.  food p r o t e i n  u s e d as  an e f f e c t i v e t o o l  to  quality.  CHEMICAL METHODS Since  (19^5) all  c a n be  a protein  application and  have  coworkers  nutritionally  a free  general  approach  available  A chemical  agent  stable  €-amino  derivatized (including c)  The  listed  Table  1,  a protean  the  as and  chemical  i s hydrolyzed  l y s i n e plus other free  is  follows« an  acid  €-amino  agent.  to y i e l d amino  acids  non-derivatized, unavailable lysine). lysine  i s determined  amount o f u n r e a c t e d  (direct  lysine  The  the  i n use  reagents  or proposed u s e d and  i n d i r e c t methods a r e l i s t e d  the in  2. In high  is  ( i n d i r e c t methods).  together with  formed.  lysine  summarized  d i r e c t methods c u r r e n t l y  i n Table  derivatives  and  protein  methods) a n d / o r t h e  The  in  estimate,  i s formed between f r e e  amount o f d e r i v a t i z e d  determined  lysine  group.  i s reacted with  'derivatized'  to  much o f t h e  c a n be  groups i n the p r o t e i n The  available  1957)t  for chemically estimating  lysine  derivative  technique  (1955a,  b e e n s i m p l e methods i n t e n d e d  d i r e c t l y o r i n d i r e c t l y , how  The  b)  of Sanger's  methods f o r e s t i m a t i n g t h e  p r e s e n t w h i c h has  a)  first  t o f o o d by C a r p e n t e r  chemical  either  the  carbohydrate  foods,  the  d i r e c t methods  are  17.  ( n o t 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)  fail  Use o f t h e  to yield  satisfactory  analytical  i n d i r e c t methods a p p e a r t o c i r c u m v e n t problems.  However,  relationship  of available  lysine  (1976),  i s n o t always  the by chemical satis-  85-95% a v a i l a b i l i t y  o b t a i n e d b y u s i n g c h e m i c a l methods, i s p r o b a b l y  an o v e r e s t i m a t e  f o r both  estimates o f available  r a t s and humans.  The c h e m i c a l  l y s i n e may be o v e r e s t i m a t e d  because:  I n t h e d i r e c t methods, t h e 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 available  b)  analytical  l e v e l s determined  F o r example, i n wheat p r o t e i n s ,  for lysine,  a)  these  as r e p o r t e d by Bodwell  methods and t h e b i o a s s a y s e s t i m a t e s , factory.  results.  In both  direct  tionally agents if be  estimates o f  lysine. a n d i n d i r e c t methods, some o f t h e n u t r i available  used  t o form  l y s i n e may r e a c t w i t h t h e c h e m i c a l acid  s t a b l e d e r i v a t i v e s and,  so, estimates o f the a v a i l a b l e  lysine  l e v e l would  exaggerated.  I n t h e f o l l o w i n g p a g e s , t h e c h e m i c a l methods 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 .  a d v a n t a g e s and d i s a d v a n t a g e s 1.  f o r each  When  listed  possible,  one w i l l  be g i v e n .  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 c h e m i c a l method  f o r the estimation 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 d e v e l o p e d h i s associates i n 1955work o f S a n g e r who u s e d  The p r o c e d u r e the reagent  b y C a r p e n t e r and  was b a s e d  l-fluoro-2  ,  on the  TABLE 1.  Reagents used  and d e r i v a t i v e s  formed  Method  Reagent  FDNB o r C a r p e n t e r  FDNB  DNP-lysine  (l-fluoro-2,4-dinitrobenzene)  (€,N-dinitrophenyl-lysine)  TNBS (<2,4,6-trinitrobenzene n i c acid)  TNP-lysine  TNBS  Guanidination 1  9  F  NMR  Used  i n d i r e c t methods  Derivative  Formed  sulfo(€TN-trinitrophenyl-lysine)  O-Methylisourea S-Ethyl-trifluorothioacetate  Homoarginine  Available  lysine  Acrylonitrile  €,N-trifluoroacetyl-lysine  Available  lysine  2TChloro-3 5-dinitropyridine  €,N-cyanoethyl-lysine  Available  lysine  Sodium  6,N-dinitropyridyl-lysine  DNBS  Available  lysine  Dye-binding  1  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  €-N,N-dimethyl-lysine DNP-lysine Fluorescamine  Dyed  complex  protein  co  TABLE 2.  R e a g e n t s u s e d and d e r i v a t i v e s f o r m e d i n i n d i r e c t  methods  Reagent Used  Derivative  FDNB  DNP-lysine  TNBS  TNP-lysine  T o t a l L y s i n e Minus Unavailable Lysine  Methyl a c r y l a t e  €,€,N,N-dicarboxyethyll y s i n e and €,N-monocarboxyethyl-lysine  T o t a l L y s i n e Minus Unavailab<be .Lys i n e  Ethyl vinyl  Method  Difference  or Silcock  T o t a l L y s i n e Minus Unavailable Lysine  sulfone  Formed  € ,N-ethylsulfonylethyllysine, or € ,€ , N , N t > b i s e t h y l s u l f o n y l ethyl-lysine  20.  ^-dinitrobenzene the d i f f e r e n t peptides.  acid,  N-terminal  Each  dinitrophenyl with  to determine  free  (DNP)  the  identities  amino g r o u p s  converted  column chromatography.  p o s i t i o n s Sanger obtained €-DNP-lysine l y s i n e ) w h i c h c o u l d a l s o be The ted  i n Figure  r e a c t i o n o f FDNB w i t h  is  and  e t h e r and  i s read d i r e c t l y .  methoxycarbonyl c h l o r i d e soluble  between the d i r e c t  by  DNP-  chromatography.  an amino a c i d  the  is  illustra-  Another p o r t i o n  aqueous p h a s e i s r e a d and  to an  The the  e'ther-  extinction difference  t h e r e a d i n g on t h e t r e a t e d . A  estimate  disadvantage  €-DNP-hydroxylysine,  dC-DNP-ornithine arid X - D N P - a r g i r i i n e , • as. l y s i n e . hand, t h e method d o e s n o t  aqueous  i s treated with  to c o n v e r t DNP-lysine  r e a d i n g and  hydrolysate  e x t i n c t i o n o f the  i s t a k e n as m e a s u r i n g € - D N P - l y s i n e .  lysine.  i n other  A p o r t i o n o f the  o f t h e method i s t h a t i t e s t i m a t e s  free  gave  ( a b b r e v i a t e d as  d e r i v a t i v e which i s then e x t r a c t e d .  o f the remaining  sample  by  o r i g i n a l method t h e p r o t e i n i s d i n i t r o -  then hydrolyzed.  extracted with  phase  acid.  lysine  present  had  2.  In Carpenter's phenylated  separated  a  digestion  identified  In a d d i t i o n N-terminal where l y s i n e was  with  u n i t Ithat  t o an <£-DNP-amino and  of  and  subsequent  amino a c i d  T h e s e y e l l o w compounds were s e p a r a t e d  and  i n proteins  that resisted  so t h a t e a c h N - t e r m i n a l  oC,€-di-DNP-lysine,  number  amino g r o u p became l a b e l l e d ,  group  o r i g i n a l l y r e a c t e d was  and  N-terminal  On  the  lysine  other or  FIGURE 2 .  of l-fluoro-2,4-dinitrobenzene  Reaction acid.  (From H a l l  (FDNB) w i t h a n amino  et a l . , 1973).  R i NH-CH-COOH NaHC0  R  3  + HCI  NH -CH-COOH 2  hydrolysis Amino  l-Fluoro - 2 , 4 - d i n i t r o b e n z e n e  acid  Dinitrophenylated amino acid  22.  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  carbohydrate, e s p e c i a l l y i n vegetable protein concentrates. Interference results €-DNP-lysine. DNP-lysine  The  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  e f f e c t i s due  to the d e s t r u c t i o n  by c a r b o h y d r a t e s d u r i n g a c i d The  i n 1955 was  procedure  hydrolysis.  d e s c r i b e d b y C a r p e n t e r and  m o d i f i e d by C a r p e n t e r ( i 9 6 0 )  of  Ellinger  t o remove  certain  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  of  feedstuffs. The m a j o r 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 app 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  difficult  to apply w i t h i n c r e a s i n g concentrations of carbohydrates. The  c o r r e c t i o n f a c t o r h a s b e e n a s s e s s e d b y 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 lysis  s t a g e and  t a k i n g i t through the r e s t of the  r e . W i t h a n i m a l m a t e r i a l s t h e r e c o v e r y was Accordingly, a l l values determined b y a f a c t o r 6f 1 0 0 / 9 2 = 1 . 0 9  found  s h o u l d be  hydro-  procedut o be  92%.  multiplied  to correct f o r l o s s e s during  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 t h e r e c o v e r y o f a d d e d €-DNPl y s i n e was  60-85%.  on t h e a s s u m p t i o n  The  use  of the c o r r e c t i o n f a c t o r  t h a t the €-DNP-lysine  that arises  depends from  t h e 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 t h e same e x t e n t as t h e added € - D N P - l y s i n e . assumption  i s invalid;  Booth (1971)  €-DNP-lysine  found t h a t t h i s  i n a p r o t e i n i s des-  troyed to a l e s s e r extent than €-DNP-lysine  t h a t has  been  23.  added as a f r e e compound.  Thus, the c o r r e c t i o n f a c t o r i n the  Carpenter method i s l e s s than f o r m e r l y supposed. that contain  no carbohydrates a f a c t o r  For m a t e r i a l s  o f 1 . 0 5 i s more  s u i t a b l e than Carpenter's o r i g i n a l 1.09, although v e r y s o l u b l e albumins need the l a t t e r .  For wheat and some o t h e r c e r e a l s  the r e s u l t s need to be c o r r e c t e d by m u l t i p l y i n g  by 1 . 2 .  Between these two groups, l i e beans, ground nut, and  maize,  r e q u i r i n g a f a c t o r of approximately 100/88 = 1.14. The presence of carbohydrates r e s u l t s i n v e r y low r e c o v e r i e s o f DNP-lysine, due to r e d u c t i o n o f n i t r o groups i n the d i n i t r o p h e n y l a t e d amino a c i d to amino groups, which g i v e der 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 Handwerck e t . a l . ( i 9 6 0 )  lysine.  DNP-  r e p o r t e d t h a t sugars are  l e s s d e s t r u c t i v e i f they have p r e v i o u s l y been i n c o n t a c t with FDNB.  S t a r c h can be e q u a l l y d e s t r u c t i v e and may  not be  i n a c t i v a t e d by p r e v i o u s c o n t a c t w i t h FDNB ( E r b e r s d o b l e r and Zucker, 1 9 6 4 ; El-Nockrashy, 1 9 6 5 ) .  Destruction  due to  carbohydrates i s l e s s e n e d 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 d i n i t r o p h e n o l , p i c r i c a c i d or FDNB i t s e l f (Blom e t a l . , 1 9 6 ? ; Matheson.1968; presumably lysine.  Ruderus and K i h l b e r g , 1 9 7 0 ; Booth, 1 9 7 D .  by competition with the n i t r o groups from T h i s p r o t e c t i o n may  DNP-  be achieved by adding these  compounds to the suspension b e f o r e r e f l u x i n g begins or by l e a v i n g the d i n i t r o p h e n o l and FDNB from the f i r s t stage o f  24.  the r e a c t i o n 1955a;  i n the r e a c t i o n  Carpenter, i 9 6 0 ) .  the q u a n t i t i e s protective cedure  left  f l a s k ( C a r p e n t e r and E l l i n g e r ,  Matheson (1968)  i n t h i s way  effect.  With  the d e s t r u c t i o n  has  shown  are s u f f i c i e n t  to give  sugars c a r r i e d through  of DNP-lysine  added  the  t h i s pro-  immediately  hydrolysis  i s no more t h a n 5 - 1 0 $ b u t  i t c a n be  (Matheson,  1968)  carbohydrates  or with s t r u c t u r a l  that  before  larger with  starch  (Booth,  197D • Because o f the t e c h n i c a l  difficulties  encountered  the complete  recovery o f €-DNP-lysine,  an  approach  b e e n t o measure a v a i l a b l e  lysine indirectly.  The  has  difference  between the t o t a l  lysine  a!nd,  o f the f r e e  DNFB, i s assumed t o be  €-amino g r o u p s  Difference  stable  o f the d e r i v a t i v e  difficulties,  to d e s t r u c t i o n  by  and  sample  by r e a c t i o n  the  partial  during acid hydrolysis  that  acid.  exchange chromatography equipment  lysine i t s e l f  with  sample.  known as  t e c h n i q u e o r S i l c o c k method, a r e t h a t  destruction not cause  o f the  theiav.aiibable l y s i n e o f the  main advantages o f t h i s t e c h n i q u e , a l s o  sample  the;residual  c o n t e n t measured a f t e r a c i d h y d r o l y s i s  a f t e r blockage  The  alternative  l y s i n e content of the  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 ,  in  does  i s extremely  However, a u t o m a t i c  ion-  i s n e c e s s a r y f o r ease  in  estimation of lysine concentration. Rao as a c h e c k  e t a l . (1963)  on t h e p r o c e d u r e  f i r s t used  the D i f f e r e n c e  they developed  technique  f o r determination  25-  of a v a i l a b l e l y s i n e (1967)  were t h e  i n o i l s e e d meal p r o t e i n s .  first  t o use  w o r k i n g r e p l a c e m e n t f o r the compared  the  methods o f  (1963)  with  lysine  i n fishmeal  the  S i l c o c k method hydrates.  the  Difference  i s not  p r o c e d u r e as  C a r p e n t e r FDNB method.  Carpenter  (i960)  S i l c o c k method f o r t h e and  Roach e t a l .  Rao  and  (1970)  interference  by  Compared t o C a r p e n t e r ' s p r o c e d u r e ,  the  Similar  resultsvere  reported  absorbance of  the  of dinitrophenylated lysine.  co-workers  the  carboSilcock  i n groundnut  Ostrowski et a l .  the  error  proteins  i n the  in color  ether soluble  i n t e n s i t y of  the  m e t h y l - c h l o r o f o r m a t e and e t h e r was  t a k e n as  phenol at  360 nm  the  in acid  acid  by  measured hydrolisates of  Carpenter  reacting  with methyl  available reduced  the  chloroformate  lysine derivative.  The  difference  hydrolysate before reaction after reaction  i s the  phenylation of proteins. advantage o f  (i960)  and  a measure o f a v a i l a b l e  Dinitrophenol  directly  i n t h e i r estimation  analysis  e-dinitrophenylhydrazone-lysine t o p r o d u c e an  (1957)  yellow ether-extracted  In a l a t e r m o d i f i c a t i o n  some o f  taking  by  available  r e l a t i n g to soybean meal. C a r p e n t e r and  the  of  showed t h a t  method gave l o w e r v a l u e s f o r a v a i l a b l e l y s i n e meal.  They et a l .  estimation  groundnut meal, subjectivto  and  a  difference and  extraction  with  lysine.  major by-product of  C o n k e r t o n and  with  the  dinitro-  Frampton ( 1 9 5 9 ) 1  i n absorbance of  hy  dinitro-  a l k a l i n e m e d i a , were a b l e  to  26.  correct  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  protein hydrolysate. in  the h y d r o l y s a t e  y e l l o w substances might  o f some d i n i t r o p h e n y l a t e d i960)  (Handwerck e t a l . , metric  Other  and  introduce errors  estimation of available  brown h u m i n p i g m e n t s t h a t l y s a t e s may  p r e s e n t i n the  a l s o be  lysine.  invariably  expected  are e l i m i n a t e d i n the p r o c e d u r e  proteins in a  colori-  In addition,  occur i n acid  to c o n t r i b u t e  Many o f t h e s o u r c e s o f e r r o r f o u n d  to the  error.  i n the e a r l i e r  d e s c r i b e d b y Rao  methods,  yellow derivatives  o f t h e r e a c t i o n b e t w e e n DNFB and  hydrolysis, is  as f r o m  through  developed  dinitrophenol  and  o f an i o n - e x c h a n g e  with a mixture  of methyl  ethyl  acid  column  ketone  other  the meal  t h e brown humin p r o d u c t s o f  t h e use  (1963)  et a l  i s s e p a r a t e d from  as w e l l  the  hydro-  where € - D N P - l y s i n e  proteins,  occur  that  and  aqueous HC'l. Blom e t a l . ( 1 9 6 7 )  a l s o made u s e  s e p a r a t i o n technique to p u r i f y The  of a  the r e s u l t i n g  a c i d h y d r o l y z e d FDNB-treated  sample was  €-DNP-lysine. passed  a n y l o n powder c o l u m n t o s e p a r a t e € - D N P - l y s i n e of  interfering  compounds.  three d e t e c t i o n procedures  Blom and  through  from  a number  his associates  outlined  f o r determination of  DNP-lysine:  a)  D i r e c t photometry  b)  Photometry o f the r e a c t i o n p r o d u c t 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 b a s e d group  o f the y e l l o w  chromatographic  a t the d r o p p i n g mercury  colour.  on r e d u c i n g the  electrode.  nitro  27.  In the o r i g i n a l ornithine  and l y s i n e  was p o o r ,  estimation of lysine. small by  shoulder  lengthening  due in  to maintain  a higher  resolved  pressure  t o improve  to determine t o t a l  used a m o d i f i e d  no a u t o m a t i c  and u n a v a i l a b l e  analyzer  and f r e e l y s i n e  i s no p r o b l e m f r o m p a r t i a l  more d i f f i c u l t  column  i s required  lysine. published  b y Couch i n  action.  advantages o f the D i f f e r e n c e technique  during hydrolysis.  short  t h e 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 .  DNFB D i f f e r e n c e t e c h n i q u e  lysine  o f the  sample.  1975 has been adopted as o f f i c i a l - A O A C f i r s t  N-terminal  i s high  o f t h e r e s i d u a l l y s i n e p e a k , and s o  o f the analyzed  using t h e i r technique,  The  area  Sometimes, however, t h e o r n i t h i n e l e v e l  available lysine  technical  rate.  and n o t c l e a r l y  c o u l d be a c o n s i d e r a b l e - e r r o r i n t h e e s t i m a t i o n  technique  would  o f i t , i t i s p o s s i b l e t o compute t h e t r u e  comparison to the area  there  that the  a s u f f i c i e n t l y , r a p i d flow  Ostrowski e t a l . (1970)  By  reported  p e a k c o u l d be t o t a l l y  t h e column, a l t h o u g h  to l y s i n e .  true  some e r r o r i n t h e  f r o m t h e l y s i n e peak, b u t i s c l e a r l y d i s t i n g u i s h -  as p a r t  there  (1967)  where t h e o r n i t h i n e p e a k i s s m a l l  separated able  thus causing  Williams  on the l y s i n e  t h e n be n e c e s s a r y In cases  S i l c o c k method t h e s e p a r a t i o n o f  are included,  The  are that and t h a t  r e d u c t i o n o f DNP-lysine  The d i s a d v a n t a g e s a r e t h a t l y s i n e i s  to estimate  than DNP-lysine unless  one h a s  28. automated ion-exchange  c h r o m a t o g r a p h y e q u i p m e n t , and t h a t  FDNB i s n o t w a t e r s o l u b l e and h a s a v e s i c a n t e f f e c t man  skin.  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 s i m p l e r t e s t based  of Carpenter  Kakade and L i e n e r  as t h a t  acid  (TNBS),  instead of  (FDNB), h a s b e e n s u g g e s t e d  by  (1969). The r e a c t i o n o f TNBS w i t h a n a m i -  i s illustrated The  o n t h e same p r i n c i p l e  sulfonic  l-fluoro-2,4-dinitrobenzene  acid  (TNBS)  (i960) i n w h i c h u s e i s made o f t h e r e a g e n t  2,4,6-trinitrobenzene  no  a c i d method  on hu-  original  i n Figure  3.  work w i t h t h i s compound on p u r e  a c i d s and p e p t i d e s was c a r r i e d  amino  o u t b y Okuyama and S a t a k e  (i960) a n d S a t a k e 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 b y p a p e r  chroma-  tography  with  and s p e c t r o s c o p y .  No r e a c t i o n was o b s e r v e d  the s i d e c h a i n s o f h i s t i d i n e , a f t e r treatment This  tyrosine,  t h r e o n i n e and s e r i n e  f o r t h r e e days a t e l e v a t e d  favourable s p e c i f i c i t y  o f TNBS p r o m p t e d  the m o d i f i c a t i o n o f cytochrome c (Takemori haemoglobin (Shinoda, and  Handler,  1964).  Gold'farb  et a l . ,  I962),  1965), and x a n t h i n e o x i d a s e ( G r e e n l e e  t o r e a c t w i t h TNBS ( K o t a k i e t a l . ,  (I966) d e s c r i b e d t h e r e a c t i o n o f TNBS w i t h  human serum a l b u m i n three  i t s use i n  The SH g r o u p s o f c y s t e i n e and m e r c a p t o -  e t h a n o l were o b s e r v e d  1964).  temperatures.  and a n a l y z e d  i t s course  c l a s s e s o f amino g r o u p s o f d i f f e r e n t  i n terms o f reactivities.  FIGURE 3.  Reaction of 2,4,6-trinitrobenzene amino a c i d .  (From H a l l  et a l . ,  sulfonic  acid  NH-CH-COOH  2  R  NaHC0  NHg-CH-COOH  ,4,6-Trinitrobenzene sulfonic  acid  an  1973)'  S0 OH  Amino  (TNBS) w i t h  3  +  HCL i  hydrolysis  acid  Trinitropheny lated amino  acid  30.  The  same r e a g e n t was  o f amino g r o u p s et  al.,  used  i n some n a t u r a l  the r e a c t i v i t i e s TNBS.  of a variety  Kinetic  spectrophotometer. they found t h a t  reactive found  s u g g e s t e d by  -NH-TNP g r o u p  quantitatively  a t 3^0 nm  species.  order reaction.  The  SH  group  pH  amino g r o u p  a t 340 nm  i>s  7 . 4 and  and  the was  groups. pH  and  has  t h a n t h e -NH-TNP  f o u n d t h a t b o t h amino g r o u p s  were 30 t i m e s more r e a c t i v e  a t room  of  lysine  temperature,  t o TNBS t h a n  groups. u s i n g TNBS, Habeeb ( 1 9 6 6 )  reaction of free  found  results  dependence  i s unstable at alkaline  t h e €-amino g r o u p s  sulfate,  recording  of N-acetylcysteine  However, a t pH  By  on a  t o TNBS t h a n most amino  -S-TNP g r o u p  also  The  r e a c t w i t h TNBS.  cC-amino  by m o n i t o r i n g  t h e r e a c t i o n o f t h e TNBS w i t h amino a c i d s  a much l o w e r e x t i n c t i o n c o e f f i c i e n t  the  Ray  o f amino a c i d s and p e p t i d e s  o n l y the unprotonated  I t was  examined  s t u d i e s were p e r f o r m e d  t o be more r e a c t i v e  group.  role  (Haynes  I n agreement w i t h G o l d f a r b ' s  i s a second  However, t h e  inhibitors  procedure  Radda ( 1 9 6 8 )  the e x t i n c t i o n o f the  showed t h a t  the f u n c t i o n a l  (1961).  Freedman and  peptides  trypsin  1967) w i t h a mathematical  and K o s h l a n d  towards  to e s t a b l i s h  amino g r o u p s  potassium  t h a t p r o t e i n s s u c h as b o v i n e have  able to study  i n p r o t e i n w i t h sodium  t h i o c y a n a t e and  and human g a m m a - g l o b u l i n  was  formaldehyde. serum a l b u m i n ,  the p r o p e r t y o f  the  dodecyl It  was  ovalbumin  binding  sodium d o d e c y l s u l f a t e through the e-amino group. group i n v o l v e d at t h i s b i n d i n g  The  amino  s i t e became u n r e a c t i v e  P a r t i a l r e a c t i o n of potassium thiocyanate  with the  to TNBS.  f r e e amino  groups i n bovine serum albumin, ovalbumin, human a - g l o b u l i n and  l a c t a l y s a t e was  demonstrated by r e a c t i o n w i t h TNBS.  was  a l s o found t h a t the  r e a c t i o n of bovine serum albumin  w i t h formaldehyde i n v o l v e s the formation l i n k s between the  amino group on one  guanyl, i n d o l e , and The  It  imidazole  of methylene c r o s s  hand and  the  groups on the other  method developed by Habeed s u f f e r s the  amide, hand.  disadvantage  that i t does not d i f f e r e n t i a t e between f r e e e-amino  and  N - t e r m i n a l amino groups of p r o t e i n s .  critical  i n the case of p e p t i d e s , as i n s u l i n , or p r o t e i n s or  T h i s c o u l d be  low m o l e c u l a r weight p r o t e i n s containing  s e v e r a l peptide  such  chains  subunits. Kakade and  Liener  (1969) developed a method to  deter-  mine s p e c i f i c a l l y the a v a i l a b l e l y s i n e content of p r o t e i n foodstuffs.  The  s p e c i f i c i t y of the technique f o r the  e-amino groups of p r o t e i n s r e s i d e s i n the  f a c t t h a t , subsequent  to a c i d h y d r o l y s i s of the TNP-protein, the ct-TNP amino a c i d s may  be e x t r a c t e d with e t h e r , whereas e-TNP-lysine remains  i n the  aqueous phase where i t i s determined speetrophoto-  metrically. 6 N HCI  A h y d r o l y s i s p e r i o d of 1 hr at 120°C w i t h  seems s u f f i c i e n t f o r q u a n t i t a t i v e recovery.  h y d r o l y s i s of the p r o t e i n i s not necessary s i n c e  Complete  small  32.  T N P - p e p t i d e s p o s s e s s e s s e n t i a l l y t h e same s o l u b i l i t y a n d spectrophotometric  p r o p e r t i e s as t h e f r e e TNP-amino  The amount o f e - T N P - l y s i n e curve  obtained  i s c a l c u l a t e d from a  acids.  standard  with v a r i o u s l e v e l s o f e-TNP-lysine  has been s u b j e c t e d  which  t o t h e same p r o c e d u r e a s 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 t h e available  l y s i n e values  d e t e r m i n e d w i t h TNBS a n d t h e v a l u e s  o b t a i n e d w i t h t h e DNFB m e t h o d 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) u s e d t h e TNBS m e t h o d o f Kakade and L i e n e r t o e v a l u a t e precipitated  caseins.  the a v a i l a b l e lysine of acid  a ^-,and 3 - c a s e i n y i e l d e d a v a i l a b l e g  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 available total  lysine.  However,  l y s i n e o f t h e K - f r a c t i o n was 15% h i g h e r  l y s i n e content.  An e x p l a n a t i o n  f o r these  than t h e  high  values  c o u l d be t h e p r e s e n c e o f D - g l u c o s a m i n e a n d D - g a l a c t o s a m i n e , and  probably  other uncharacterized  g l y c o p r o t e i n fragments.  Amino s u g a r s r e a c t w i t h TNBS t o f o r m c o l o r e d d e r i v a t i v e s w h i c h a r e n o t r e m o v e d by e t h e r e x t r a c t i o n . concluded  that although  exact values  Holsinger et a l .  of available lysine  c o u l d n o t be d e t e r m i n e d b y TNBS i n f o o d s 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 , t h e p r o c e d u r e c o u l d s t i l l application i n the determination  of r e l a t i v e values  i n a c t i v a t i o n during processing o f products composition.  find of lysine  of identical  P o s a t i e t a l . (1972) n o t i c e d  t h a t the presence o f  l a c t o s e i n cheese whey i n t e r f e r e d with 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 whey p r o t e i n when TNBS was of e-TNP-lysine was  dependent on the  the t e s t m a t e r i a l .  The  also influenced  used.  Loss  amount o f l a c t o s e i n  sample weight used f o r a n a l y s i s ,  the r e s u l t s .  The  l a r g e r the sample o f e i t h e  pure p r o t e i n or carbohydrate c o n t a i n i n g  m a t e r i a l , the  smalle  the amount of a v a i l a b l e l y s i n e found. H a l l et a l . (1973) proposed a method f o r the mination of a v a i l a b l e l y s i n e w i t h TNBS i n animal which d i f f e r s i n s e v e r a l aspects from t h a t Liener.  The  sample, was the  eliminated  by the use  used by Kakade and  at 60°C;  2-hr  They a l s o m o d i f i e d Liener  (0.1%  suggested, 0.5%  h y d r o l y s i s w i t h 11 N HCl  T  and small  o f agar suspensions o f  r e a c t i n g with the p r o t e i n f o r 75 min  the  reaction  TNBS s o l u t i o n f o r i n an  TNBS s o l u t i o n  at 40°C, f o l l o w e d  by  i n a b o i l i n g water, bath.  b o i l i n g water bath overcomes the s e r i o u s of the HCl  o f Kakade  h y d r o l y s i s p e r i o d with 6 N HC1  autoclave at 120°C) and  a 2-hr  proteins  of accurate weighing of a very  f i n e l y ground m a t e r i a l .  conditions 2-hr  necessity  deter-  corrosive  upon the m e t a l l i c i n t e r i o r of the  The  action  autoclave.  T h e i r 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 i n animal p r o t e i n concent r a t e s were i n c l o s e agreement w i t h those measured by  the  Carpenter procedure. H a l l et a l . (1973) i n agreement w i t h Ousterhout Wood (1970) proposed the use  and  of pure l y s i n e as a standard  i n s t e a d of e-TNP-lysine, s i n c e 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 6N H C l a n d i s e a s i l y and S a t a k e ,  formed  i s unstable i n hot  converted i n t o e-TNP-lysine  (Kotaki  1964).  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 a n d o r n i t h i n e , a n d f o u n d t h a t t h e s e compounds r e a c t t o g i v e T N P - p r o d u c t s 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  absorbance  values s i m i l a r  TNBS i s s e r i o u s l y  and have  t o e-TNP-lysine. a f f e c t e d by t h e presence  o f carbo-  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 coupled w i t h aldose g r o u p s , t h u s m a k i n g 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 b e c a u s e o f t h e a d s o r p t i o n o f the e-TNP-lysine and  on t o t h e c a r b o n p a r t i c l e s  t o t h e i n t e r f e r i n g absorbance  (from c h a r r i n g )  o f the browning  In 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 be  found i n samples c o n t a i n i n g carbohydrate  Furthermore, taurine  t h e amines agmatine,  spermine,  products.  l y s i n e can  compounds. s p e r m i d i n e and  f o u n d i n numerous p l a n t s p e c i e s ( S m i t h , 1 9 7 2 ) 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 samples are h i g h e r than t h e t o t a l  lysine  lysine  i n certain  content  (Hall eta l .  1975).-  H a l l e t a l . (1975) concluded for determining available  t h a t TNBS c a n be u s e d  lysine i n carbohydrate-rich  m a t e r i a l , p r o v i d e d t h a t t h e s a m p l e mass d o e s n o t e x c e e d 5 mg a n d t h a t t h e 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 m i n a t 30°C,  i n s t e a d o f 7 5 m i n a t 40°C.  Under these c o n d i t i o n s  the  'blank' absorbance,  i . e . the value of the  sample simply heated w i t h HCI  unreacted  i s very high i n r e l a t i o n t o  the absorbance of the a c t u a l e - t r i n i t r o p h e n y l a t e d Attempts t o reduce  product.  t h i s value t o an a n a l y t i c a l l y more  acceptable l e v e l have had l i m i t e d success. Eklund  (1976) s l i g h t l y m o d i f i e d the method o f Kakade  and L i e n e r (1969) by i n c r e a s i n g the sample s i z e as w e l l as by s u b j e c t i n g the TNP-proteins 90 min.  Eklund determined  rapeseed with  t o h y d r o l y s i s at 110°C  for  a v a i l a b l e l y s i n e i n c a s e i n and  'his procedure,  t h a t o f Kakade and L i e n e r  and the method of Rao e t a l . (1963).  Throughout, he  obtained  h i g h e r values w i t h h i s m o d i f i e d method, but the degree of v a r i a b i l i t y between repeated analyses of the same m a t e r i a l was  lower as compared t o the v a r i a b i l i t y o b t a i n e d with the  method of Kakade and L i e n e r . 3.  Guanidination Due  t o 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 a c i d h y d r o l y s i s (Carpenter, 1960), Mauron and B u j a r d (1963) suggested  the use o f O-methyl-isourea  t i o n of a v a i l a b l e l y s i n e .  f o r determina-  The g u a n i d a t i o n w i t h O-methyl-  i s o u r e a transforms the e-amino group of l y s i n e i n t o a guanidine d e r i v a t i v e , which on h y d r o l y s i s y i e l d s  homoarginine.  The h y d r o l y s a t e i s analyzed by column chromatography  and  homoarginine i s e l u t e d with the b a s i c amino a c i d s , where i t appears  after arginine.  No homoarginine i s formed when the  l y s i n e u n i t s are combined with sugar i n a M a i l l a r d  reaction  ( F i n o t and Mauron, 1972) .' A l t h o u g h m e t h y l - i s o u r e a 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 , p r o b a b l y , the l o n g r e a c t i o n time (3 days) needed has use o f the 4„  p r e c l u d e d any  extensive  approach.  Acrylonitrile 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 a g a i n  slowly,  w i t h the e-amino groups of l y s i n e t o form a c y a n o e t h y l a t e d d e r i v a t i v e t h a t i s s t a b l e under the o r d i n a r y 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 1966).  When the r e a g e n t was  conditions  (Riehm and  for  Scheraga,  used by P i s a n o e t a l . ( 1 9 6 8 )  and by H a r d i n g and Rogers ( 1 9 7 1 ) t o measure the  reactive  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 w i t h the v a l u e s f o r e-N- (X-glutairiyl) l y s i n e o b t a i n e d by enzymic d i g e s t i o n i n v i t r o . 5.  Methyl  aerylate M e t h y l a c r y l a t e i s an a l t e r n a t i v e t o  (Cavins and F r i e d m a n , 1 9 6 7 ) , and  i t s use  acrylonitrile  for n u t r i t i o n a l  s t u d i e s has been proposed by F i n l e y and Friedman The  m e t h y l a c r y l a t e method i s based on  (1973).  condensing  an a, 3 - u n s a t u r a t e d compound w i t 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 e x c e s s m e t h y l a c r y l a t e a t 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 s m a l l amount of e , N - m o n O c a r b o x y e t h y l l y s i n e .  The  reaction  t i m e c a n be c u t down t o 4 h r b y u s i n g a 7 5 % DMSO  (dipolar  aprotic  Available  Finley  6.  The  as  proteins.  sulfone  e t h y l v i n y l s u l f o n e method  t h e mono- a n d d i s u b s t i t u t e d  t i v e s o f l y s i n e and a l s o during acid hydrolysis sulfone treatment. difference  content  m e t h o d a n d TNBS a n d DNFB p r o c e d u r e s on  (Friedman and F i n l e y ,  may be u s e d t o m e a s u r e n u t r i t i o n a l l y  material  Buffer.  and F r i e d m a n r e p o r t e d good a g r e e m e n t b e t w e e n  o f pure  Ethylvinyl  197 5)  pH 9.1  alkylation.  methyl aerylate  a series  2 5 % (v/v)  l y s i n e i s m e a s u r e d by c o m p a r i n g l y s i n e  b e f o r e and a f t e r  the  solvent):  available  ethylsulfonylethyl  unavailable  lysine that  derivai s liberated  as l y s i n e r e m a i n i n g a f t e r  Available  lysine  ethylvinyl  l y s i n e i s measured as t h e  between t h e l y s i n e  content of the s t a r t i n g  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  treatment.  In p r i n c i p l e , e t h y l v i n y l sulfone can a l k y l a t e a l l functional  groups c o n t a i n i n g  for instance,  active  t h e e-amino g r o u p  hydrogens i n p r o t e i n s ;  o f l y s i n e , side  c h a i n s and  i m i d a z o l e g r o u p s 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 nucleophilic 7.  addition  reactions.  2-Chloro-3,5-dinitropyridine Selim  dinitrobenzene  (1965) e m p l o y e d S a n g e r ' s r e a g e n t  (FDNB) t o e s t i m a t e t h e l y s i n e c o n t e n t o f  protein'hydrolyzates after blocking the  free  1-fluoro-2,4-  amino a c i d s w i t h  copper.  t h e ot-amino g r o u p s o f  38.  A s i m i l a r p r i n c i p l e was u s e d b y T s a i e t a l . (1972) f o r the screening of l y s i n e content  i n maize seeds.  In  t h e i r method t h e 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 pronase o r a mixture yield The  with  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  f r e e amino a c i d s o r l o w m o l e c u l a r  weight  peptides.  a - c a r b o x y l a n d a-amino g r o u p s 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  l e a v i n g t h e e-amino g r o u p s o f l y s i n e  suspension),  free t o react.  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 then reaction with 2-chloro-3,5-dinitropyridine.  An  f o r m e d on  Extraction of  t h e 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 t h e 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 of e-DNPyr-lysine. determined  The a b s o r b a n c e o f t h i s s o l u t i o n i s t h e n  a t 400 nm.  Lysine content  as e s t i m a t e d  method i s u s a l l y h i g h e r t h a n  by t h i s  the estimate  colorimetric  of lysine  f r o m t h e amino a c i d a n a l y z e r , b u t i t i s i n g o o d agreement.  solution  The h i g h e r l y s i n e v a l u e s  found  content  general  i n the colorimetric  a s s a y s may be a c o n s e q u e n c e o f t h e r e l a t i v e l y m i n o r  contri-  b u t i o n made t o a b s o r b a n c e by t h e a r g i n i n e p r e s e n t . 8.  Sodium  borohydride  Thomas (1970) u s e d NaBH^ f o r t h e d e t e r m i n a t i o n o f t h e available  l y s i n e content o f cottonseed meal, a m a t e r i a l i n  w h i c h l y s i n e u n i t s may c o m b i n e w i t h t h e a l d e h y d e  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-  trolled. M a i l l a r d - t y p e compounds o f a l d e h y d e break  and l y s i n e  units  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  high recovery of lysine.  H o w e v e r , when t h e y a r e t r e a t e d  w i t h sodium borohydride they are reduced compounds.  to acid-stable  T h u s , when p r o t e i n s a r e t r e a t e d w i t h  h y d e 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 , lysine units  a r e formed  formalde-  e-N,N-dimethy1-  (Means a n d F e e n e y , 1 9 6 8 ) .  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  The  'total'  treatment  may, t h e r e f o r e , be a m e a s u r e o f t h o s e 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 Thomas  aldehydes.  (1970) a n d C o u c h a n d 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 FDNB-Difference The  technique  by t h e  and t h e s o d i u m b o r o h y d r i d e  method.  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  a d v a n t a g e o f t h e s o d i u m b o r o h y d r i d e m e t h o d , as  compared w i t h t h e o f f i c i a l D N F B - D i f f e r e n c e  technique, 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 m a k i n g 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 to 9.  obtain the available lysine 1 9  F  data.  NMR R a m i r e z e t a l . (1975) h a v e d e v e l o p e d  determine  a method t o  t h e e-amino g r o u p o f l y s i n e b y F NMR  F r e e e-amino g r o u p s o f l y s i n e  spectroscopy.  are t r i f l u o r o a c e t y l a t e d  with  40.  the reagent 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 sulfoxide solution  i n dimethyl  and t h e number o f s u c h g r o u p s  i s quan-  19 titatively  determined using standard  F NMR  technique.  D i m e t h y l s u l f o x i d e i s u s e d as t h e r e a c t i o n i n o r d e r t o improve  the homogeneity  medium  o f t h e s y s t e m , When t h e  m i x t u r e becomes homogeneous, a p o r t i o n i s a d d e d d i r e c t l y 19 an NMR  s a m p l e t u b e and t h e  A t y p i c a l spectrum The  spectrum i s obtained.  i s shown i n F i g u r e 4.  resonance a t lowest f i e l d  trifluoroacetic ester.  F NMR  The  (A) c o r r e s p o n d s  a c i d , a h y d r o l y s i s product of the  peak a t i n t e r m e d i a t e f i e l d  t h e t r i f l u o r o a c e t y l a t e d e-amino g r o u p s peak a t h i g h e s t f i e l d the t h i o l e s t e r The  to  to  thiol  (B) c o r r e s p o n d s  o f l y s i n e , and  to  the  ( C ) , t o the t r i f l u o r o a c e t y l group  of  (unreacted reagent).  relative integrated intensities  are  calculated  from the w e i g h t s o f the a p p r o p r i a t e l y c u t - o f f peaks.  The  molar r a t i o of l y s i n e to the o r i g i n a l t h i o l e s t e r i s given by t h e r a t i o o f t h e w e i g h t o f t h e i n t e r m e d i a t e p e a k t o t h e t o t a l weight of a l l peaks. The m o l a r o p t i m u m r a t i o o f t h i o l is  4:1.  reagent to  lysine  Higher concentrations of t h i o l reagent lead  d o u b l e 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 . c e n t r a t i o n s , n o t a l l t h e e-amino g r o u p s E x c e l l e n t a g r e e m e n t was  With lower  to con-  are a t t a c k e d .  found between the  t i o n a l d a t a f o r p u r e m i l k p r o t e i n s and t h e l y s i n e 19 determined with F NMR.  composicontent  1+1.  FIGURE 4 .  F  y  NMR  protein  spectrum of and  S-ethyl  dimethyl sulfoxide  the  r e a c t i o n products of  trifluorothioacetate in solution.  H  a  42.  F NMR s p e c t r o s c o p y f a s t and r e l a t i v e l y of v a r i a b i l i t y  o f f e r s a reasonably  simple procedure,  of results  without  accurate, the d i f f i c u l t y  f r o m v a r y i n g amounts o f p r o t e i n 19  present. and 10.  I t does r e q u i r e a c c e s s  fairly  concentrated  F NMR  spectrometer  protein solutions.  The s o d i u m d i n i t r o b e n z e n e The  to a  DNBS t e c h n i q u e  s u l f o n a t e method  for determination  (DNBS).  of available  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 s o f a r d e s c r i b e d 19 (2-chloro-3,5-dinitropyridine does n o t r e l y  and  F NMR a r e t h e e x c e p t i o n )  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  order t o determine 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 unhydrolyzed method  using  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  (Van S l y k e , 1 9 1 1 , 1912) a n d t h e Hofman r e a c t i o n a n d  i o d o m e t r i c back t i t r a t i o n method  (Baraud,  methods however were n o t s p e c i f i c  1957).  These  f o r t h e e-amino g r o u p o f  lysine. The (1959)/  w o r k 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  Hosoya  dinitrobenzene for determining  (1960) a n d Sugae  (196 0) s u g g e s t e d  that  s u l f o n a t e c o u l d be u s e d a s a s p e c i f i c  reagent  lysine i n proteins without hydrolysis.  E i s e n e t a l . (1953) d e m o n s t r a t e d t h a t DNBS r e a c t s w i t h o n l y amino g r o u p s .  specifical  L i (1956) r e a c t e d i n s u l i n  DNBS f o r 216 h r a t 3°C i n 0.1 M s o d i u m c a r b o n a t e  with  and found  t h a t t h e e-amino g r o u p 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 - t e r m i n a l g l y c i n e and p h e n y l a l a n i n e ,  r e s i d u e t y r o s i n e d i d not r e a c t with DNBS.  and the  When DNBS was  r e a c t e d with takaamylase-A a t pH 10.7, 37°C, only lysine residues  i n the enzyme molecule were  a f t e r 50 h r (Ikenaka, 1959). residues 1960).  dinitrophenylated  S i m i l a r l y , 3 out o f 25 l y s i n e  i n b a c t e r i a l amylase r e a c t e d The c a l c i u m  11 o f 22  a f t e r 74 h r (Sugae,  a c e t a t e , used t o minimize d e n a t u r a t i o n of  the enzyme, and the low pH used by these authors c o u l d many o f the l y s i n e r e s i d u e s u n r e a c t i v e  render  by s t e r i c and e l e c t r o -  static factors. Concon  (1971) confirmed the v a l i d i t y o f DNBS as a  reagent f o r l y s i n e determination  i n unhydrolyzed p r o t e i n s ,  by r e a c t i n g p u r i f i e d p r o t e i n s w i t h DNBS. l y s i n e residues  The number o f  p e r mole o f p r o t e i n was i n f u l l  with the r e p o r t e d  agreement  values.  Concon attempted t o spe'ed up the DNBS r e a c t i o n by i n c r e a s i n g the pH, temperature and r e a c t i o n time. became e v i d e n t the  w i l l react. cant the  that the d i f f e r e n c e i n r e a c t i o n r a t e between  a- and e-amino groups o f l y s i n e i s such t h a t  conditions  It  reaction  c o u l d be chosen i n which o n l y the e-amino group The optimum combination o f f a c t o r s f o r s i g n i f i -  r e a c t i o n o f the e-amino group w i t h DNBS, l a y w i t h i n following l i m i t s :  pH 9.0 - 13.0; temperature 30 - 60°C;  r e a c t i o n time 0.5 - 2.0 hr. groups w i t h DNBS, i n c r e a s e d  The r e a c t i v i t y o f the e-amino almost l i n e a r l y w i t h pH at 40°C.  No r e a c t i o n was observed a t pH 9.  The maximum r e a c t i v i t y  a p p e a r e d t o be b e t w e e n reactivity  pH 12 a n d 13.  A t pH above 12, t h e  o f t h e a-amino g r o u p was a p p a r e n t l y a b s e n t d u e t o  instability  o f t h e a~NH -DNP d e r i v a t i v e . 2  e-NB^-DNP  d e r i v a t i v e was s t a b l e a t pH 12 e v e n a f t e r 3 h r a t 40°C. was  There  no a d v a n t a g e t o be g a i n e d i n t e r m s o f s p e c i f i c i t y b y  increasing  t h e t e m p e r a t u r e t o 45°C a n d a t t h e same t i m e  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 workable  c o m b i n a t i o n o f pH 12.3, 40°C a n d 1 h r r e a c t i o n  time  (pH 10.5, 60°C, 1 h r f o r r i c e ) g a v e q u i t e s a t i s f a c t o r y results.  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 u t  with pure l y s i n e these conditions w i t h no d e t e c t a b l e The  gave  absorbance readings  c o n t r i b u t i o n f r o m t h e a-amino g r o u p s .  specificity  o f t h e DNBS u n d e r 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 b y p a p e r c h r o m a t o g r a p h y h y d r o l y z e d , DNBS-treated p r o t e i n s . detected  of the acid  O n l y e - N - D N P - l y s i n e was  ( C o n c o n , 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 t h e s p e c i f i c r e a c t i o n c o n s t a n t s f o r p u r e l y s i n e and c e r e a l p r o t e i n s .  rate  For grains  a n a l y z e d a t pH 12.3, 40°C, 1 h r t h e a v e r a g e v a l u e o f K was -3  2.5  x 10  For  rice,  -1  min  .  The same v a l u e was f o u n d f o r p u r e l y s i n e .  a n a l y z e d a t pH 10.5, 60°C, 1 h r t h e a v e r a g e v a l u e -3  o f K was 6.2 x 10 of  -1  min  l y s i n e under these  , w h i c h was a l s o e x a c t l y t h e v a l u e  conditions.  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  rice  i s d e t e r m i n e d a t pH 12.3 a n d 40°C.  this  i s due t o t h e h i g h a r g i n i n e  I t i sbelieved  content o f r i c e .  Under  that  high  alkaline conditions  and e l e v a t e d  may d e g r a d e t o o r n i t h i n e . O r n i t h i n e  temperature,  (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 ) . arginine with  a pK^ = 12.5 may r e a c t  DNBS a t pH 1 2 . 3 .  In addition,  significantly  with  L o w e r i n g t h e pH t o 10.5 a p p a r e n t l y  m i z e d t h e s e r e a c t i o n s , e v e n a t 60°C. necessitated  arginine  mini-  The d r o p i n pH  a change i n s u l f h y d r y l masking agent t o  mercuric chloride  (HgCl^) s i n c e p h e n y l m e r c u r i c  (PMC) p r e c i p i t a t e s b e l o w pH 12. lysine values, same c o n d i t i o n s  Other grains  chloride give  by as much as 3 0 - 4 0 % , when a n a l y z e d used f o r t h e r i c e samples.  lower under t h e  I ti s possible  t h a t because o f the high  arginine content,  rice  proteins  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 e n o u g h t o p e r m i t more  c o m p l e t e d i s s o c i a t i o n o f t h e e-amino g r o u p s e v e n t h o u g h t h e pH o f t h e s u r r o u n d i n g by  s o l u t i o n i s lower than t h a t  the Henderson-Hasselbach  permitted  equation.  A t pH 1 2 . 3 , 40°C, 1 h r , t h e DNBS r e a c t i o n i s incomplete;  only  a b o u t 14% o f t h e t o t a l  lysine i s reacted.  However, i t i s p o s s i b l e t o d e t e r m i n e d i r e c t l y 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 . i n d i c a t e that i n the presence o f excess follows f i r s t order The  k i n e t i c s as d e s c r i b e d  Lt  =  [(e  k t  -l)  by e q u a t i o n  lysine originally  / e  k t  ] L-  0  Kinetic  studies  DNBS, t h e r e a c t i o n  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  proportional t o the t o t a l  t h e amount o f  1.  time i s d i r e c t l y present. ( E q u a t i o n 1),  where L  ?0  = 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 ;  = the total  lysine o r i g i n a l l y present;  reaction rate constant; logarithm. time.  k - the s p e c i f i c  and e = t h e base o f t h e n a t u r a l  L-j. i s d i r e c t l y p r o p o r t i o n a l  to L  Q  a t any g i v e n  T h e r e f o r e , f r o m t h e a b s o r b a n c e due t o L ^ , L may be Q  determined d i r e c t l y . versus L  C Q  A p l o t o f t h e a b s o r b a n c e due t o L^-  conforms r e m a r k a b l y t o B e e r and Lambert's l a w .  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 d e p e n d e n t o n 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  time, every protein-bound  e-amino  g r o u p must be e q u a l l y  accessible  These  requirements are s a t i s f i e d lation  reaction  at high  and r e a c t i v e .  by c o n d u c t i n g t h e d i n i t r o p h e n y -  pH and i n h i g h  concentrations  of  urea. 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 g r o u p o f  lysine i n unhydrolyzed proteins. amino a c i d s may a l s o r e a c t w i t h cysteine  On t h e o t h e r h a n d , DNBS.  free  Tryptophan and  a r e among t h e m o s t r e a c t i v e .  Cysteine  i s seven  t i m e s more r e a c t i v e t h a n f r e e l y s i n e b e c a u s e t h e SH g r o u p also reacts with  DNBS.  The DNBS d e r i v a t i v e s o f f r e e  acids, except f o r cysteine, removed  by e t h e r  interference the  arginine  extraction.  i seliminated  and h i s t i d i n e ,  2  amino a c i d s  c a n be  A major p o r t i o n o f the cysteine  by e t h e r  extraction.  However,  s u l f h y d r y l g r o u p s h o u l d be m a s k e d b y m e r c u r a t i o n  PMC o r H g C l .  amino  The s l i g h t r e a c t i v i t i e s o f t h e o t h e r  a r e reduced by m e r c u r a t i o n .  Mercury  with basic  forms  47.  c o m p l e x e s 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  reactivities  o f a r g i n i n e and  q u i t e s m a l l when c o m p a r e d t o l y s i n e . be  histidine  Their interference  eliminated to a c e r t a i n extent with mercuric  ( b u t n o t PMC)  and by  use  use  of a s m a l l e r sample s i z e .  When approaches  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 e v e n w i t h of mercuric The  N-terminal  amino groups o f p e p t i d e s  For example, the  tryptophanyl-tryptophan proteins,  a t pH  12,  reactivity  of tryptophan  40°C, 1 h r r e a c t i o n t i m e , unreactive  t o e t h e r e x t r a c t i o n , and nm.  i s b e t w e e n 36 0 and  The 365  of the r e a c t i o n media  (PMC,  as In  N-terminal  t o DNBS.  the  the t e s t p r o t e i n i s optical density i s  Unfortunately,  the  e-DNP-lysine components  DNBS, p h o s p h a t e s ) and  c e r e a l pigments, absorb s t r o n g l y at these The  the  maximum w a v e l e n g t h o f  nm.  even  as much as 9 3%.  Once r e a c t e d w i t h DNBS r e a g e n t ,  m e a s u r e d a t 385  are  a-amino g r o u p s o f f r e e amino  i s r e d u c e d by  amino g r o u p i s v i r t u a l l y  subjected  the  chloride.  l e s s r e a c t i v e t o DNBS t h a n t h e acids.  can  chloride  the r a t i o of f r e e a r g i n i n e or h i s t i d i n e to l y s i n e 1:2,  are  DNBS m e t h o d a p p e a r s t o be  the  various  wavelengths.  r a p i d and  sensitive for  the  e-amino g r o u p s o f l y s i n e , w i t h t h e a d v a n t a g e t h a t 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 t h e p r o t e i n s l i k e most  the o t h e r  chemical  availability.  methods u s e d f o r d e t e r m i n a t i o n  of  of lysine  48.  11.  Fluorescamine P u r e e 1 1 e t a l . (1976) h a v e d e v e l o p e d a r a p i d m e t h o d  of the  determination of  proteins,  where p r o t e i n ,  f r e e amino g r o u p s i n i n t a c t p u r e hydrolysis  labeled  l y s i n e d e r i v a t i v e i s not  requires  only  with  two  steps:  and  extraction  necessary.  l a b e l i n g the  The  of  a  method  p r i m a r y amino g r o u p s  4 - p h e n y l - s p i r o - [ f u r a n - 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 labeled  f l u o r e s c a m i n e , and  g r o u p s by a b s o r p t i o n  375-390  measuring  s p e c t r o s c o p y i n the  the  range  nm. Fluorescamine, f i r s t synthesized  (1972), reacts groups to y i e l d  s p e c i f i c a l l y with a fluorescent  by W e i g e l e e t a l .  p r i m a r y and  and  secondary  a nonfluorescent  amino  product  respectively. The yields  r e s u l t of r e a c t i n g  fluorescamine with  a d i r e c t e s t i m a t e of the  g r o u p s and  hence, of the  unsubstituted  a-amino g r o u p s o f t h e  labeled,  i s a minor c o n t r i b u t i o n  12.  protein t o the  chain  is  results  proteins. also obtained.  Dye-binding. Dye-binding procedures are  w h i c h have been s u c c e s s f u l l y automatic systems of Three c l a s s e s quality evaluation: azo  e-amino  a v a i l a b l e l y s i n e content of  However, the and  proteins  dyes  r a p i d , i n e x p e n s i v e methods  a d a p t e d t o a u t o m a t i c and  semi-  analysis. o f d y e s t u f f s have been u s e d f o r phthalein  ( L a k i n , 1973).  Ney  d y e s , r e a c t i v e d y e s and  and  V7irotama (1970) h a v e  protein acid reported  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 the d e t e r m i n a t i o n  of available lysine i n milk  blue  R for  and c h e e s e .  The d y e , whose s t r u c t u r e i s shown i n F i g u r e 5 , when c o n verted t o the v i n y l will the  ( a c t i v e ) f o r m by h e a t i n g  i n strong  alkali,  form a c o v a l e n t bond w i t h t h e f r e e €-amino g r o u p o f lysine  residues  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 w e r e o b s e r v e d w i t h t h e h y d r o x y l s e r i n e and t y r o s i n e .  The a u t h o r s  c y s t e i n e c a n be d i s r e g a r d e d For determining  that reaction with  i n the case o f m i l k p r o t e i n s .  a v a i l a b l e l y s i n e the p r o t e i n i s  reacted w i t h remazol b r i l l i a n t p a s s e d o v e r S e p h a d e x G-25  claimed  groups o f  blue  coarse.  R, and t h e m i x t u r e i s The d y e d p r o t e i n i s  e l u t e d a f t e r 10 m i n u t e s 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 t h e 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 determined spectrophometrically. is  The amount o f b o u n d dye  c a l c u l a t e d by c o m p a r i n g t h e 280 nm e x t i n c t i o n o f t h e  eluate with that of d i f f e r e n t s o l u t i o n s of 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 . was at  This  f o u n d t o d e c r e a s e w i t h t i m e when m i l k was 95°C;  value maintained  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 b e c a u s e o f t h e i r c o n t e n t  of available lysine  fell.  FIGURE 5 i  Structure Ney  o f t h e r e a c t i v e dye  and W i r o t a m a ,  1970).  remazol b r i l l i a n t  b l u e R.  (From  51.  Although Pruss and Ney  (1972) demonstrated no  inter-  f e r e n c e from l a c t o s e and o t h e r sugars, the method s u f f e r s from the disadvantage  t h a t the r e a c t i o n i s dependent on  the q u a n t i t y o f p r o t e i n p r e s e n t i n the sample.  As  the  q u a n t i t y of p r o t e i n i n c r e a s e s , the amount o f dye bound  de-  creases. Most of the dye-binding r e s u l t s r e p o r t e d i n the l i t e r a t u r e are given i n terms of "dye-binding c a p a c i t y " or "DBC".  DBC  i s an almost p s e u d o - s c i e n t i f i c term  t h a t a g i v e n amount of p r o t e i n has amount of dye.  an a f f i n i t y  T h i s value depends upon the  which i m p l i e s f o r a given  experimental  c o n d i t i o n s , the s t r u c t u r e of the dye molecule, of the b u f f e r system and the p u r i t y of the dye. ture of the system and i t s pH are a l s o important  the  composition'  The  tempera-  factors.  T h e r e f o r e , unless these parameters are s p e c i f i e d , a s t a t e ment of dye-binding c a p a c i t y has no r e a l meaning and  may  even prove t o be m i s l e a d i n g ( L a k i n , 1973). Although q u a n t i t a t i v e 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 s d i f f i c u l t w i t h t h i s technique, dye-binding cedures  c o u l d be used t o monitor  pro-  losses of l y s i n e during  the p r o c e s s i n g o f food p r o t e i n s , as long as the p r o t e i n content of the m a t e r i a l remains constant. 13.  Chemical methods i n 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 i n m a t e r i a l s t h a t have undergone Mai H a r d reactions. The type o f damage which some o f the chemical p r o -  cedures  fail  to measure adequately i s t h a t o c c u r r i n g 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 s u g a r s have been i n  prolonged contact a t r e l a t i v e l y i n which  'early' Maillard  low temperatures  compounds, o f w h i c h  (e-N-deoxyfructosy1)-lysine  (FFL) i s a model  (37°C), a n d  a-N-formyl( F i n o t and  M a u r o n , 1972), w o u l d be e x p e c t e d . F i n o t and Mauron  (1972)  reacted deoxy-ketosyl  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 , 1 9 6 9 ) , and w i t h O - m e t h y l - i s o u r e a  and B u j a r d , 1 9 6 3 ) .  (Mauron  When C a r p e n t e r ' s m e t h o d was a p p l i e d t o  F F L i t was f o u n d t h a t 14-2 8% o f t h i s u n a v a i l a b l e f o r m 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 . iso.urea was  was t h e m o s t s p e c i f i c  completely non-specific  Guanidination using  0-methyl-  ( 0 % ) , w h e r e a s t h e TNBS m e t h o d  (82-89%).  P r e s u m a b l y , TNBS  r e a c t s w i t h t h e b a s i c s e c o n d a r y amine g r o u p s o f t h e e a r l y M a i l l a r d compound a n d 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 t h e TNP g r o u p s w e a k e n s t h e s u g a r l i n k a g e w h i c h s p l i t s  off to  give a high y i e l d of TNP-lysine. O - m e t h y l - i s o u r e a a p p e a r s t o be a h i g h l y  sensitive  i n d i c a t o r of a l l types of lysine binding, including the 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.  Although i t cannot  be recommended i n i t s p r e s e n t f o r m a s a r o u t i n e c o n t r o l measure because  o f the time taken  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  quality-  (3 d a y s )  f o r an  b e c a u s e 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  interest  as  a r e f e r e n c e method. When F F L was  (Finot total  and Mauron, 1972;  available  FDNB v/ould s t i l l epsilon  lysine.  react  amino g r o u p  with  was  complex b r e a k s  neither  isourea  c a n be  and  l y s i n e was This finding  down on  acid  that  (Mottu  damage  1  protein  available  sensitive  FDNB-lysine-  to y i e l d  C a r p e n t e r ' s FDNB, O - m e t h y l ( H u r r e l l and  available  lysine  With  'advanced  these procedures  l y s i n e , but  indicators  Carpenter, in  Maillard'  damage, where t h e r e i s a d e c r e a s e  digestibility,  estimate  The  the  'early  i n r o l l e r - d r i e d m i l k powders  and Mauron, 1967).  protein-protein  i n which  that  s i g n i f i c a n t extent.  b o r o h y d r i d e methods  as s e e n  1974) ,  showed  hydrolysis  t o any  g i v e a good e s t i m a t e o f the  MaiHard  unit  treatment  considerably  bound t o a s u g a r .  concluded  sodium  and C a r p e n t e r ,  a lysine  l y s i n e nor DNP-lysine It  1974)  Hurrell  l y s i n e minus F D N B - r e a c t i v e  greater than  sugar  subjected to FDNB-difference  they  o f damage t h a n  may  still  and  in overall over-  a r e , o f c o u r s e , more i s total-lysine  determination. Finot available  (1973) s u g g e s t e d  lysine  i n heat-damaged m i l k powders b a s e d  determination of t o t a l lysine after  acid  hydrolysis  ketosyl-lysine  a method f o r t h e e v a l u a t i o n  and  furosine  o f the p r o t e i n .  derivatives  compounds a r e o b t a i n e d :  on  the  c o n t e n t measured  When p u r e  e-N-deoxy-  a r e h y d r o l y z e d , t h r e e main  lysine,  furosine  and  pyridosine.  of  These compounds are always p r e s e n t the r e s u l t being  dependent only on the  a c i d used f o r h y d r o l y s i s  unavailable  concentration  the  The  i n the deoxy-ketosyl form, t h a t  l y s i n e , can be c a l c u l a t e d d i r e c t l y  acid hydrolysate  of  ( F i n o t and Mauron, 1972).  q u a n t i t y o f l y s i n e blocked is,  i n the same p r o p o r t i o n s ,  from an  of the p r o t e i n .  H u r r e l l and Carpenter  (1974) r e p o r t e d t h a t  autoclaving  p r o t e i n w i t h sucrose f o r 2 h r r e s u l t e d i n l e s s f u r o s i n e production  than d i d 1 h r .  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 r e a c t i o n do not break down t o f u r o s i n e on a c i d h y d r o l y s i s . Sulser  (1973) has  reported  similar results.  presence of f u r o s i n e i n the a c i d - h y d r o l y s a t e  While  the  of a t e s t  m a t e r i a l i n d i c a t e s t h a t some of i t s l y s i n e has been i n v o l v e d i n M a i l l a r d r e a c t i o n s , i t cannot be used as a q u a n t i t a t i v e i n d i c a t o r i n a l l instances of protein-sugar D.  damage.  ENZYMATIC METHODS Even though the  amino a c i d p r o f i l e i s important i n  e v a l u a t i n g the n u t r i t i v e q u a l i t y o f a p r o t e i n , the  digesti-  b i l i t y of t h a t p r o t e i n i s the primary determinant of a v a i l a b i l i t y o f i t s amino a c i d s . food p r o t e i n may  be o b t a i n e d  t h i s i s an expensive and Several  The  by using  the  d i g e s t i b i l i t y of a a r a t bioassay  but  time comsuming procedure.  i n vitro-enzymic  d i g e s t i o n methods f o r the  measurement of p r o t e i n a v a i l a b i l i t y have been developed. One  o f the e a r l i e s t procedures was  t h a t o f Melnick e t 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  with pancreatin. mixture  At i n t e r v a l s ,  w e r e w i t h d r a w n and  m e a s u r e d by  formol  also increased tic  the  a l i q u o t s of the  I t was  o f soy  and  a l s o found t h a t methionine i s  co-workers  (1946),  and  Riesen  (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 t h e t o t a l composition,  the  protein  o f the p r o t e i n t o enzyma-  ' e a r l i e r f r o m h e a t p r o c e s s e d s o y m e a l t h a n f r o m raw Melnick  was  found t h a t those  the n u t r i t i v e value  susceptibility  d i g e s t i o n . I t was  incubation  the degree of h y d r o l y s i s  titration.  f a c t o r s known t o i n c r e a s e  digested  released soy  meal.  et a l .  amino a c i d  r a t e o f r e l e a s e o f amino a c i d s f r o m 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  important  n u t r i t i o n a l q u a l i t y of a p r o t e i n . b y H o r n e t a l . (195 3) t o e v a l u a t e  This  factor in  the  c o n c e p t was  utilized  the n u t r i t i o n a l q u a l i t y  o f f o o d 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 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  individual  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 cottonseed  meal w h i c h had  of processing; be  been s u b j e c t e d  h o w e v e r , t h e r e was  u s e d t o compare p r o t e i n s E v a n s and  Butts  no  to various  degrees  i n d i c a t i o n that i t could  from d i f f e r e n t sources.  (1948) f o u n d t h a t e n z y m i c h y d r o l y s i s  o f s o y b e a n p r o t e i n b r o u g h t a b o u t 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 t h e unavailable  s e n s e t h a t t h e y had  f o r l i b e r a t i o n by  were e v i d e n t l y r e g e n e r a t e d i n the presence of sucrose, s u l f u r amino a c i d s had  by  of  been  rendered  d i g e s t i v e enzymes, though acid hydrolysis.  When  heating  l a r g e amounts o f t h e b a s i c  becQme u n a v a i l a b l e and  the  they  and  losses  w e r e much g r e a t e r t h a n i n t h e Sheffner  absence of  e t a l . (1956) by  combining the p a t t e r n  e s s e n t i a l amino a c i d s r e l e a s e d by with  the  (PDR)  i n v i t r o pepsin  amino a c i d p a t t e r n o f t h e  described  digestibility  digestion  Pepsin-Digest-Residue  D i v i s i o n o f t h e PDR  c o e f f i c i e n t of the  of  remainder of the p r o t e i n ,  an i n t e g r a t e d i n d e x , t h e  amino a c i d i n d e x .  sucrose.  i n d e x by  the  respective proteins,  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 of the p r o t e i n s Since  studied.  e n z y m a t i c h y d r o l y s i s may  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 dialysis  (Mauron e t a l . , 1955)  S a l t e r , 1966) they are  and  be  i n h i b i t e d by  released.  r a p i d l y absorbed,  gel f i l t r a t i o n  (Ford  digested  and  test protein  A l t h o u g h the method has  o c c a s i o n a l l y f o r other  f o o d p r o t e i n s , i t has  was  l o s s of l y s i n e a v a i l a b i l i t y  been u s e d  those achieved  to a Maillard  method  of  for  by t h i s e n z y m a t i c p r o c e d u r e  with Carpenter's chemical  This  action  A comparison of the values  l y s i n e obtained  milk.  lactose.  e-amino g r o u p i s r e s i s t a n t t o t h e  t h e d i g e s t i v e enzymes. available  i s due  l y s i n e side chain with  and  been employed  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 of heat p r o c e s s e d  l i n k a g e at the  as  d i a l y z e d , f i r s t with pepsin  then w i t h p a n c r e a t i n .  type r e a c t i o n of the  and  Mauron e t a l . (1955) e m p l o y e d an i n -  v i t r o d i g e s t i o n procedure i n which the  In m i l k , the  the  h a v e b e e n u s e d t o remove t h e a m i n o a c i d s  simultaneously  value  (1960)  with and  those from b i o l o g i c a l e v a l u a t i o n good conformity between the  three methods, on  m i l k samples (Bujard e t a l . , F o r d and  Salter  based on growth, showed  Mottu and  1967;  system, by  Mauron,  (1966) attempted the  removal of r e a c t i o n products from the  a series  of  I967).  continuous  in v i t r o digestion  causing the enzyme-substrate mixture t o pass  through a c a l i b r a t e d column o f Sephadex g e l G-25.  Each  d i g e s t was  residue  thus r e s o l v e d  and  three s o l u b l e  and  ' f r e e amino a c i d s ' .  i n t o four  fractions:  the  f r a c t i o n s , 'soluble p r o t e i n ' , Freeze-dried  cod  'peptide',  fillets  subjected  t o d i f f e r e n t heat treatments were used as t e s t  proteins.  Microbiological  the o r i g i n a l  test protein c r e a t i n and increasing  'free amino a c i d  component i n  the  samples by the  acids.  i n - v i t r o amino a c i d  from the data of Ford and  value i n the  amino  o r i g i n a l unheated meal  the s u l p h u r - c o n t a i n i n g amino  Mauron (197 0) c a l c u l a t e d  He  1  With  became i n c r e a s i n g l y d e f i c i e n t i n s e v e r a l  r e l a t i v e to t h e i r content i n the  availability  pan-  gave b r o a d l y s i m i l a r r e s u l t s .  temperature, the  n o t a b l y i n l y s i n e and  the  f r a c t i o n s and  a f t e r prolonged d i g e s t i o n w i t h p e p s i n , erepsin,  the d i g e s t s acids  assay of a l l the  S a l t e r , by  'free amino a c i d ' f r a c t i o n of the  corresponding value i n the  dividing heated  unheated sample.  found good agreement between h i s i n - v i t r o d i g e s t i o n  cedure a f t e r d i a l y s i s and  the  r e s u l t s o f F o r d and  pro-  Salter.  58.  A n o t h e r 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 the  low m o l e c u l a r weight s o l u b l e  by P r a h l by  and T a u f e l  filtration  (1966).  f r a c t i o n s was  experimented  These a u t h o r s r e p l a c e d  t h r o u g h a membrane u n d e r p r e s s u r e .  dialysis,  Amino  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, only nitrogen  acid  since  was m e a s u r e d .  A k e s o n and Stahmann pancreatin  from  digestion  (1964) u s e d p e p s i n f o l l o w e d  to estimate the d i g e s t i b i l i t y  p r o t e i n q u a l i t y of processed foods.  by  and  Upon d i g e s t i o n o f t h e  p r o t e i n , t h e 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 w e r e p r e c i p i t a ted with p i c r i c  a c i d and t h e a m i n o a c i d s w e r e 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 w h o l e e g g 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 pancreatin  i n d e x v a l u e s f o r 12 p r o t e i n s  values reported In  i n the l i t e r a t u r e ,  and t h e b i o l o g i c a l  from feeding  trials.  1 9 7 5 , Stahmann and W o l d e g i o r g i s s l i g h t l y  t h e p r o c e d u r e o f A k e s o n and Stahmann b y i n t r o d u c i n g salicylic  acid, instead of p i c r i c  of the undigested p r o t e i n acid i s ninhydrin be d i r e c t l y  negative  applied  pancreatin gives casein  (19 66)  sulfo-  Sulfosalicylic  a n d H a n s e n , 1969) and c a n  t o t h e c o l u m n s f o r amino a c i d  Menden and C r e m e r  modified  a c i d , as a p r e c i p i t a n t  and p e p t i d e s . (Perry  pepsin-  reported  alone o r i n combination w i t h  analysis.  that hydrolysis  acid  by  hydrolysis  a better i n d i c a t i o n of protein q u a l i t y of processed and meat p r o d u c t s t h a n a c i d h y d r o l y s i s  subjected  the p r o t e i n t o a short  alone.  d i g e s t i o n time with  They a  large  amount o f p a n c r e a t i n .  The authors argue t h a t the i n i t i a l  steps i n d i g e s t i o n are of importance  and t h a t i t i s p r e -  f e r a b l e to d i g e s t with a high c o n c e n t r a t i o n o f enzyme f o r a s h o r t e r p e r i o d , so as t o a v o i d a u t o - h y d r o l y s i s o f the enzymes  used. Buchanan  (1969) and Buchanan and Byers  c r i b e d an i n v i t r o system f o r measuring  protein  (1969) desdigestibility  on a v<heat l e a f p r o t e i n concentrate w i t h an enzymatic d i g e s t i o n u t i l i z i n g papain.  Examination o f the data shows a  f a i r degree of c o r r e l a t i o n between the i n v i v o f e e d i n g trials  and i n v i t r o f i g u r e s on a f r e e z e - d r i e d m a t e r i a l , but  a s e r i o u s d i f f e r e n c e i n the case o f the same m a t e r i a l moist heat treatment.  after  Buchanan was unable to o b t a i n a  c o r r e l a t i o n of p r o t e i n d i g e s t i b i l i t y by Akeson and Stahmann's procedure, and h i s r e s u l t s with papain showed poor  correla-  t i o n between i n v i v o and i n v i t r o data. Saunders e t a l . (1973) employed the p e p s i n - p a n c r e a t i n system d e s c r i b e d by Akeson and Stahmann papain method of Buchanan and Byers  (1964), and the  (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 e x c e l l e n t c o r r e l a t i o n between the values o b t a i n e d from the enzyme system used by Akeson and Stahmann and i n v i v o data  (0.874) , whereas very poor c o r r e l a t i o n was found between  the papain d i g e s t i o n and i n v i v o data. may have produced  The papain  system  low values compared t o the i n v i v o data  60.  b e c a u s e 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 level  i n alfalfa protein  lipid  concentrates.  S a u n d e r s 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, the i n v i t r o ( r = 0.914) w i t h trysin  i n vivo d i g e s t i b i l i t y .  well  A f t e r the  pepsin-  d i g e s t i o n , t h e 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  analyzed was  r e s u l t s of which c o r r e l a t e d  f o r nitrogen  c o n t e n t and t h e p r o t e i n  digestibility  c a l c u l a t e d as t h e d i f f e r e n c e b e t w e e n t h e n i t r o g e n  protein concentrate  and t h e n i t r o g e n  i n the  i n the undigested  fraction. R a y n e r and F o x (1976) h a v e c r i t i s i z e d t h e f a c t most o f t h e i n v i t r o maximum d i g e s t i b i l i t y determination an  absolute  standard is  therefore  e n z y m a t i c methods do n o t r e s u l t i n during  enzymolysis,  o f amino a c i d s  released  so t h a t t h e  from a p r o t e i n i s not  method o f m e a s u r i n g a v a i l a b i l i t y .  represented  that  A  reference  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  necessary.  R a y n e r and F o x (1976) u s e d t h e enzyme p r o n a s e t o digest autoclaved  rapeseed meals.  A f t e r incubation, the  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 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 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 in•vitro  available lysine with  The r e c o v e r y  o f amino a c i d s  Silcock available  from the pronase  e n z y m o l y s i s showed r e c o v e r i e s  a c i d and analyzer. when c o m p a r i n g lysine.  in-vitro  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 , methionine  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  interesting  the h i g h r e c o v e r y of t r y p t o p h a n .  r e s u l t was  acid.  An  A k e s o n 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  t h a t t r y p t o p h a n c a n be d e t e r m i n e d by t h i s The  a p p l i c a t i o n o f the pronase  and  indicated  procedure.  in vitro  procedure,  i  a l s o a l l o w s t o d i s c o v e r t h e p r e s e n c e o f ."trypsin i n h i b i t o r s . T r o p and B i r k inhibitors  (19 70)  inactivate  r e p o r t e d t h a t 90% o f n a t u r a l t r y p s i n pronase.  The m e t h o d d e v e l o p e d by D v o r a k  (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 d e t e r m i n e 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 .  Available  essential  amino a c i d s f r o m n a t i v e and h e a t - t r e a t e d b e e f s e r u m 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 microbiologically,  T h i s enzyme c o m b i n a t i o n was (1962).  prolidase.  by H i l l  and  I n t h i s system l e u c i n e aminopeptidase i s  papain h y d r o l y s a t e s , except those which nitrogen of p r o l i n e .  ability  and  p r e v i o u s l y used  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  prolidase.  determined  a f t e r t o t a l enzymic h y d r o l y s i s of the  p r o t e i n by p a p a i n , l e u c i n e a m i n o p e p t i d a s e  Schmidt  albumin,  remain i n t a c t i n c o n t a i n the imino  P r o l i n e p e p t i d e s a r e h y d r o l y z e d by  R e s u l t s o f t h i s s t u d y showed t h a t t h e  avail-  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  digestion,  corresponds  to the t h e o r e t i c a l amount i n the p r o t e i n .  Maga e t a l . (1973) p o i n t e d out t h a t the i n i t i a l r a t e s of  h y d r o l y s i s by t r y p s i n on some commonly used p r o t e i n  sources were good i n d i c a t o r s o f the p r o t e i n  digestibility.  A f t e r i n c u b a t i o n of the p r o t e i n with t r y p s i n , i n c u b a t i o n 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 p r o d u c t s , most of the  p r o t e o l y s i s o c c u r r e d d u r i n g the f i r s t remained  constant.  few minutes and  then  Steaming of the products r e s u l t e d i n  faster hydrolysis rates.  I t became apparent t h a t  c e r t a i n v e g e t a b l e p r o t e i n s may  although  have a h i g h n u t r i t i o n a l  b i o c h e m i c a l v a l u e s , t h e i r d i g e s t i v e a c c e p t a b i l i t y may  and be  q u i t e poor s i n c e they are not r a p i d l y h y d r o l y z e d i n the digestive  system.  Rhinehart  (1975) m o d i f i e d Maga's procedure  and  examined s e v e r a l enzyme systems which i n c l u d e d t r y p s i n , p e p s i n - t r y p s i n , t r y p s i n - c h y m o t r y p s i n , and  trypsin-  chymotrypsin-peptidase  r e s u l t s were  combinations.  The  encouraging, w i t h c o r r e l a t i o n c o e f f i c i e n t s of 0.79, 0.80  and 0.74,  0.72,  respectively.  These aforementioned  i n v i t r o methods have not been  w i d e l y accepted, e i t h e r because no corresponding i n v i v o data were p r e s e n t e d or because the procedures were complicated and time consuming, consequently/ d i f f i c u l t  for application in  routine in-plant quality  control.  H s u e t a l . (1977) d e v e l o p e d a m u l t i e n z y m e s y s t e m 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 m e t h o d was  b a s e d on t h e d i g e s t i o n o f an a q u e o u s p r o t e i n s u s p e n s i o n 37°C, pH 8, u s i n g a c o m b i n a t i o n and  peptidase.  the peptide  of trypsin,  chymotrypsin  As t h e p r o t e o l y t i c enzymes a t t a c k  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  and b r e a k  structure,  the  f r e e d c a r b o x y l groups t h a t formed,immediately  H ,  w h i c h , i n t u r n , l o w e r e d t h e pH o f t h e p r o t e i n  +  released suspension.  The pH d r o p 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 m e t e r . pH o f t h e p r o t e i n s u s p e n s i o n  at  The  a f t e r 10 m i n u t e s d i g e s t i o n w i t h  t h e m u l t i e n z y m e 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  of rats.  Hsu e t a l . were a b l e t o d e m o n s t r a t e t h a t t h e m u l t i enzyme  technique  s a l t s commonly  was u n a f f e c t e d b y f o o d  found i n foods,  lipids  and b u f f e r i n g  and was s e n s i t i v e e n o u g h t o  be a b l e t o d e t e c t t h e p r e s e n c e o f s o y b e a n t r y p s i n i n soy f l o u r s , trate.  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 c o n c e n -  The m e t h o d 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 heat processing  inhibitor  of s p e c i f i c  that occurred  during the  foods.  A multienzyme system c o u l d reduce the e f f e c t by  a s p e c i f i c enzyme i n h i b i t o r .  caused  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 under-predicting the d i g e s t i b i l i t y  alone,  could  of proteins  avoid  containing  trypsin inhibitor.  A s i n g l e enzyme system t h a t a t t a c k s at  a s p e c i f i c p e p t i d e bond may  give d i f f e r e n t r e s u l t s f o r  p r o t e i n s c o n t a i n i n g d i f f e r e n t c o n c e n t r a t i o n s o f the amino a c i d .  A multienzyme system reduces the  specific  limitations  t h a t are e v i d e n t f o r a s i n g l e enzyme system, and gives a b e t t e r approximation  of p r o t e i n 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 t h i s i n v i t r o method f o r p r e d i c t i n g apparent p r o t e i n d i g e s t i b i l i t y i s t h a t i t can completed w i t h i n one hour and w i t h a h i g h degree of sensitivity.  be  MATERIALS AND METHODS A.  Materials B - l a c t o g l o b u l i n was o b t a i n e d f r o m  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. l y z e d and l y o p h i l i z e d powder f r o m m i l k .  Nutritional I t was  crystal-  Salt-free  lysozyme  was p u r c h a s e d f r o m 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 H C I s o l u b i l i z e d g l u t e n was  a c c o r d i n g t o t h e m e t h o d o f Wu e t a l . ( 1 9 7 6 ) .  prepared  Casein  (pH 7.5). and d r i e d w h o l e e g g s o l i d s w e r e p r e p a r e d i n t h e laboratory.  Ribonuclease-S-peptide (bovine pancreas,  g r a d e X I I - P E , MW  2166.35), p a n c r e a t i n (porcine  g r a d e V I ) , and p e p s i n ( h o g s t o m a c h m u c o s a , 2x and  pancreas, crystallyzed  l y o p h i l i z e d powder) w e r e t h e p r o d u c t s o f S i g m a C h e m i c a l  Company, S a i n t L o u i s ,  Mo.  The a m i n o 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  dihydrochloride  L - l y s y l - L - a l a n i n e dihydrobromide  (MW  (MW  347.3)  and  379.1) w e r e f r o m S i g m a  C h e m i c a l Company. N-a-formyl-L-lysine  (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 t h e m e t h o d d e s c r i b e d by Hofmann e t a l . ( 1 9 6 0 ) . A c e t i c a n h y d r i d e was a d d e d t o a s o l u t i o n o f L - l y s i n e in  9 8% f o r m i c a c i d .  L-lysine  f o r m a t e was p r e p a r e d  formate  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 t h e f o r m a t e cycle.  The s o l v e n t s w e r e e v a p o r a t e d .  The r e s u l t i n g o i l  d i s s o l v e d i n e t h a n o l and t h e c r y s t a l s w e r e c o l l e c t e d and  was  recrystallized  from e t h a n o l .  a s s e s s e d on an amino a c i d  The  p u r i t y o f t h e compound  was  analyzer.  a-and e - c a r b o b e n z o x y - l y s i n e s w e r e o b t a i n e d f r o m Eastman O r g a n i c C h e m i c a l s , D a l l a s , Texas. HCI.  H 0 2  (MW;'. 411.:8)  was  £-TNP-L-lysine.  f r o m 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-fluoro-2,4-dinitrobenzene San  D i e g o , CA.  (FDNB)was from  2 ,4-dinitrobenzehe s u l f o n i c acid  2,4,6.-trinitrobenzene sulfonic acid trihydrate  (80% min.  B.  acid  (98%) was  (DNBS)  a n h y d r o u s by t i t r a t i o n ) w e r e o b t a i n e d 5-sulfosali-  f r o m A l d r i c h C h e m i c a l Company, I n c .  Nitrogen determination The  nitrogen content of $ - l a c t o g l o b u l 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 and e g g s was  lysozyme,  determined  by t h e 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 C o n c o n and (1973).  The  p r o t e i n c o n t e n t was  t h e n i t r o g e n c o n t e n by a f a c t o r o f 6.38 C.  and  (TNBS) i n t h e f o r m o f  f r o m E a s t m a n Kodak Company, R o c h e s t e r , N.Y. cylic.  Calbiochem,  for casein Amino a c i d The  system  and  c a l c u l a t e d by 6.25  (3-lactoglubin;  f o r egg  5.7  Soltess  multiplying and  lysozyme;  for gluten.  analysis.  digested protein  was  a n a l y z e d on a s i n g l e  (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)  attached  t o a P h o e n i x M o d e l M6 800 Amino A c i d A n a l y z e r ( P h o e n i x P r e c i s i o n I n s t r u m e n t Company).  column  D.  Lysine determination a c i d method. The  lysine  availability  determined according The  by t h e d i n i t r o b e n z e n e  o f v a r i o u s p r o t e i n s was  t o t h e m e t h o d d e v e l o p e d tfoy C o n c o n  2,4-dinitrobenzene  reagent  sulfonic  (1971).  s u l f o n i c a c i d i s u s e d t o com-  p l e x t h e f r e e e-amino g r o u p s o f l y s i n e . Reagents Reagent A 50 g o f u r e a in  a n d 7 g o f Na^PC^ ,.  I2H2O  36 m l o f h o t w a t e r w i t h a m a g n e t i c s t i r r i n g  were d i s s o l v e d bar,  then  100  mg o f p h e n y l m e r c u r i c  was  a l l o w e d t o c o o l a n d t h e v o l u m e 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  was  adjusted  DNBS  c h l o r i d e was a d d e d .  t o 1 2 . 3 0 - 12.35  Whatman N o . l  The s o l u t i o n  a n d t h e pH  w i t h I N NaOH.  reagent 6 g a c t i v a t e d c h a r c o a l was a d d e d t o 6 g o f DNBS  powder d i s p e r s e d i n 1 0 0 m l o f w a t e r . for  1 0 m i n u t e s and f i l t e r e d  refractive to was  through  I t was t h e n  stirred  Whatman No. 1 .  The  i n d e x was m e a s u r e d a n d t h e s o l u t i o n was  o b t a i n t h e f i n a l c o n c e n t r a t i o n o f 3% DNBS. kept frozen i n small v i a l s .  Ether  The  diluted reagent  (See a p p e n d i x 1 ) .  extractant Equal  distilled  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  w a t e r were m i x e d 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 time.  The a q u e o u s l a y e r was  68.  d i s c a r d e d and the w a t e r - s a t u r a t e d  e t h e r was used.  Other reagents and m a t e r i a l s 90% chips The  formic a c i d ;  0.07 5 N NaOH s o l u t i o n ;  (Hengar granules, Hengar Co., P h i l a d e l p h i a ,  boiling PA), 10 mesh.  c h i p s were soaked i n c o n c e n t r a t e d HCl f o r 30 minutes,  washed with water and d r i e d . Lysine  standard 2.5 mM  l y s i n e s o l u t i o n was prepared by  dissolving  45.66 mg o f l y s i n e monohydrochloride (MW = 146.20) i n d i s t i l l e d water and d i l u t i n g t o 100 ml. solution,  From t h i s  stock  f i v e l e v e l s o f l y s i n e standards were prepared as  f ollows: Tube No.  2.5 mM  lysine soln. (mis)  0.075 N NaOH (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 o f l y s i n e p e r 0.25 ml,  respectively.  They were used along w i t h each experiment f o r the c o n s t r u c t i o n o f the standard curve. i n small v i a l s .  The d i l u t i o n s were kept f r o z e n  Protein  solutions The  NaOH t o g i v e  t e s t p r o t e i n was d i s s o l v e d i n 25 m i s o f 0.075 N a final  concentration  r a n g e o f 30 - 100 ug o f  l y s i n e p e r 0.25 m l 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  s h a k e n on a v o r t e x  a t 2,500 x g f o r  20 m i n u t e s .  m i x e r and c e n t r i f u g e d  The s u p e r n a t a n t was c o l l e c t e d a n d u s e d f o r  analysis. Preparation  o f working protein  solutions  Protein  L i t e r a t u r e value (mg l y s i n e p e r 100 mg p r o t e i n )  Desired l e v e l (ug l y s i n e p e r 0.25 ml)  Gluten  1.6  40  250  Casein  7.6  76  100  Lysozyme  5.7  57  100  3-lactoglobulin  56.5  11. 3 6.2  Egg  Calculation  50  62  10. 3  RNase-S-peptide  mg p r o t e i n i n 25 m l 0.075N NaOH  100 50  51. 5  f o r gluten  100 mg g l u t e n 1.6 mg l y s i n e  40 yg l y s i n e 0.2 5 m l  = 250 mg g l u t e n  x  2  5  m  l  d i l u t e d t o 25 m l w i t h  ,  J a 0 H  0.075 N NaOH.  Procedure 0.5 m l o f r e a g e n t A was a d d e d t o a l l t u b e s w i t h o u t touching  the sides;  0.2 5 m l o f p r o t e i n s o l u t i o n was t h e n  pipetted  t o the surface  o f sample and sample b l a n k  tubes.  To t h e 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 m l o f 0.075 N  NaOH w e r e p i p e t t e d ;  to the standards,  0.25 m l o f t w o 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 surface o f reagent  A.  The s o l u t i o n was m i x e d t h o r o u g h l y b y  f i r m w r i s t motion,  a n d a l l o w e d t o s t a n d f o r a few m i n u t e s .  0.25 m l o f 3% DNBS was v e r y c a r e f u l l y a d d e d t o a l l t u b e s , except The  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 m l o f w a t e r .  DNBS was a d d e d , f i r s t t o r e a g e n t  standards  blanks  and then t o  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 t u b e s were immersed i n a w a t e r bath  a t 40°C a n d i n c u b a t e d The  for1 hr.  tubes were then  c o o l e d i n an i c e - w a t e r  2.0 m l HCL was added t o a l l t u b e s  except  bath.  f o r sample b l a n k s ,  t o w h i c h 1.75 m l HCL a n d 0.25 m l 3% DNBS w e r e a d d e d . E t h e r e x t r a c t i o n was c a r r i e d o u t t h r e e t i m e s 3.0,  2.5 a n d 2.5 m l o f w a t e r - s a t u r a t e d e t h e r .  with  A vortex  m i x e r was u s e d f o r e f f e c t i v e b l e n d i n g a n d e x t r a c t i o n .  Each  t u b e was s h a k e n f o r a f e w s e c o n d s , t o a v o i d t h e f o r m a t i o n o f a g e l o r emulsion  a t t h e e t h e r phase.  The e t h e r l a y e r was  carefully  removed w i t h a P a s t e u r p i p e t b y means o f a g e n t l e  suction.  A c l e a n P a s t e u r p i p e t was u s e d f o r e a c h  0.5 m l o f 90% f o r m i c a c i d a n d one H e n g a r b o i l i n g were added t o a l l t u b a s . i n a water bath  solution. granule  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  a t 5 5°c f o r n o more t h a n  15 m i n u t e s .  s o l u t i o n was c o o l e d d o w i t o room t e m p e r a t u r e a b s o r b a n c e o f samples,. s a m p l e b l a n k s  The  and t h e  and s t a n d a r d s  were  m e a s u r e d 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 reagent blank. the absorbance  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  r e a d i n g s o n t h e f o l l o w i n g d a y , when t h e  d r o p l e t s on t h e s i d e s o f t h e t u b e s h a d s e t down.  In this  c a s e 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  light. 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 6.  The amount o f e D N P - l y s i n e was  from t h e s t a n d a r d curve o b t a i n e d w i t h d i f f e r e n t  calculated  levels of  l y s i n e , as shown i n F i g u r e 7. The same p r o c e d u r e was c a r r i e d o u t on a- a n d e - f o r m y l - l y s i n e s , a- and e - c a r b o b e n z o x y S - p e p t i d e and t h e d i p e p t i d e s in  lysines,  lysyl-lysine  ribonuclease-  and l y s y 1 - a l a n i n e ,  an a t t e m p t t o d e t e r m i n e t h e s p e c i f i c i t y  o f t h e DNBS  method f o r t h e e p s i l o n - a m i n o group o f l y s i n e . Calculation Casein absorbance,  a v e . o f 3 d e t e r m i n a t i o n s = 0.300.  From t h e s t a n d a r d c u r v e , an a b s o r b a n c e  o f 0.300  corresponds  t o 0.450 y m o l e s o f l y s i n e / 3 m l . Available  lysine  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  follows: 0.45,0 y m o l e s l y s / 3 m l  1 mol X  0.0010 g c a s e i n / 3 m l  7=  10 y m o l  x  146.2 g 1 mol l y s  = 6.58 g o f l y s i n e / 1 0 0 g c a s e i n .  x 100 g c a s e i n  FIGURE 6.  The d i n i t r o b e n z e n e for determination  s u l f o n a t e (DNBS) method of available  lysine.  Protein  DNBS t r e a t m e n t  e-DNP-protein  Ether  Ether  layer  - D N P - f r e e amino  acids  extraction  Aqueous  layer  CE-DNP-lysine  Read a t 385  nm  73.  FIGURE 7.  Standard  curve  for available  n a t i o n f o r a sample t r e a t e d sulfonic  lysine  determi-  with dinitrobenzene  acid.  £ c  to  . 125 LYSINE  .250  .375  .500  (umoles  / 3 ml reaction  .625 mixture)  For  t h e o t h e r p r o t e i n s , t h e amount o f s a m p l e p e r 3 m l 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 ;  eggs and l y s o z y m e ;  0.0025 g f o r g l u t e n ,  0.0010 g f o r a n d 0.00135 g f o r  RNase-S-peptide. E.  Lysine determination a c i d method. The  proteins The  by t h e t r i n i t r o b e n z e n e  TNBS m e t h o d t o d e t e r m i n e a v a i l a b l e l y s i n e i n  was d e r i v e d  from the procedure o f Eklund  reagent 2,4,6-trinitrobenzene  (1976).  s u l f o n i c a c i d i s used  t o c o m p l e x t h e f r e e e-amino g r o u p s o f l y s i n e . the  sulfonic  e x p e r i m e n t s was r e d u c e d t o o n e t e n t h  The s c a l e o f  of the original  procedure. Reagents 34 mM TNBS s o l u t i o n : benzene s u l f o n i c a c i d  590.26 mg o f 2 , 4 , 6 - t r i n i t r o -  (MW 347.21) w e r e 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 t h e v o l u m e made up t o 50 m l . stored  i n a dark b o t t l e and kept  The s o l u t i o n was  frozen.  0.4 8 M s o d i u m b i c a r b o n a t e s o l u t i o n : 8.07  pH 8.5 a n d  g o f s o d i u m 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  w a t e r a n d d i l u t e d t o 200 m l . Water-saturated d i e t h y l ether: described Protein  was p r e p a r e d as  f o r t h e DNBS t e c h n i q u e . solutions 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 m l o f  0.47  M NaHCO,, pH 8.5 s o t h a t t h e p r o t e i n e x t r a c t  contained  6 mg p r o t e i n fresh  per ml.  The p r o t e i n  f o r each experiment,  protein  i n sodium  since  extracts  were  prepared  the s t a b i l i t y o f the  b i c a r b o n a t e i s unknown.  e-TNP-lysine standards' 62 mg o f e - T N P - l y s i n e - H C L . I ^ O with  0.4 8 M NaHCO^ s o l u t i o n .  graded  w e r e made up t o 50 m l  A series  o f tubes  containing  l e v e l s o f e - T N P - l y s i n e w e r e s e t up as f o l l o w s :  Tube No.  e-TNP-lysine/NaHC0  3  soln.  0.48 M N a H C 0  (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  3  T h e s e 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 y m o l e s o f e - T N P - l y s i n e p e r f i n a l  They w e r e u s e d f o r t h e c o n s t r u c t i o n  v o l u m e o f 25 m l .  of the standard curve.  Procedure 0.5 m l o f p r o t e i n tube and shaken  extract  was p l a c e d i n a  hydrolysis  i n a w a t e r b a t h a t 40°C f o r 10 m i n u t e s .  0.5 m l o f 34 mM TNBS s o l u t i o n was a d d e d t o t h e p r o tein extract.  To a v o i d  c r y s t a l l i z a t i o n of the supersaturated  TNBS s o l u t i o n i n t h e p i p e t , p r i o r t o use. sample b l a n k s .  t h e s o l u t i o n was warmed t o 40°C  I n s t e a d o f TNBS, 1.5 m l HCL was a d d e d t o t h e  76.  A f t e r an i n c u b a t i o n p e r i o d o f 2 hr a t 40 C, the tubes were immersed i n an i c e - w a t e r bath, and 1.5 ml of concent r a t e d HCL was added t o samples; to  0.5 ml o f TNBS was added  sample b l a n k s . The tubes were s e a l e d and the contents were h y d r o l y z e d  for  90 minutes i n a Reacti-Therm  h e a t i n g module a t 110°C.  The tubes were allowed t o c o o l a t room temperature  and the  h y d r o l y s a t e s were f i l t e r e d through Whatman 1 and d i l u t e d t o 25 ml with d i s t i l l e d water. 5 m l - a l i q u o t s were t r a n s f e r r e d t o stoppered t e s t tubes and e x t r a c t e d four, times w i t h 5 ml o f w a t e r - s a t u r a t e d ether.  R e s i d u a l e t h e r was evaporated  i n a shaking water-bath  by p l a c i n g the tubes  a t 55°C u n t i l no e t h e r s m e l l c o u l d be  detected. The  absorbance  of the aqueous p r o t e i n s o l u t i o n was  measured a t 346 nm i n a Beckman Spectrophotometer  DB a g a i n s t  the sample blank. The  flow diagram  o f the experimental procedure  can be  seen i n F i g u r e 8. The  amount o f e-TNP-lysine  (or i t s l y s i n e e q u i v a l e n t )  was c a l c u l a t e d from the standard curve obtained w i t h v a r i o u s l e v e l s o f e-TNP-lysine, measured at 346 nm, as shown i n F i g u r e 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 o f a v a i l a b l e l y s i n e .  Protein  TNBS  a- and  treatment  e-TNP-protein  Acid  a-TNP-amino  hydrolysis  acids  + e-TNP-lysine  Ether  Ether  layer  a-TNP-amino  acids  extraction  Aqueous  layer  e-TNP-lysine  Read a t 346 nm  78.  FIGURE 9.  Standard  curve  f o r available  f o r a sample t r e a t e d sulfonic  • 301  lysine  w i t h 2,4,6  determination  - trinitrobenzene  acid.  .602  .903  E-TNP-LYSINE.HCL.H 0 2  1.204  (umoles/25  1.505 ml)  79\  Calculation Gluten absorbance, From the e-TNP-lysine corresponds  ave. of 3 determinations = 0.10 0.  standard curve, an absorbance o f 0.10  t o 0.240 ymoles o f e-TNP-lysine.  A v a i l a b l e l y s i n e content i n g l u t e n i s c a l c u l a t e d 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 g l u t e n . F.  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 by the d i n i t r o f l u o r o benzene method. The  ' d i f f e r e n c e ' technique d e s c r i b e d by Couch  has been used  to determine  available lysine of proteins.  T h i s method has been adopted The  (1975)  as o f f i c i a l - f i r s t  reagent 1 - f l u o r o - 2 , 4 - d i n i t r o b e n z e n e  complex the e-amino groups o f l y s i n e .  a c t i o n , AOAC.  (DNFB) i s used to Available lysine 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 lysines. Reagents The reagent DNFB, s o l i d a t room temperature,  was  warmed up p r i o r t o use. 10% N a H C 0 3 s o l u t i o n :  2 g o f sodium bicarbonate was  d i s s o l v e d i n d e i o n i z e d water and the volume made up t o 20 ml. 6 N HCL:  495.87 ml o f c o n c e n t r a t e d h y d r o c h l o r i c a c i d  (12.1 N) were d i l u t e d t o 1 l i t e r with d e i o n i z e d water.  \  80.  Sodium c i t r a t e b u f f e r pH 2.2: 2^0  137.26 g Na c i t r a t e  was d i s s o l v e d i n water, and 26 ml HCI added.  40 g  B r i j - 3 5 was d i s s o l v e d i n water by h e a t i n g and added t o the s o l u t i o n , f o l l o w e d by 0.4 ml n - c a p r y l i c a c i d .  The s o l u t i o n  was d i l u t e d t o 4 l i t e r s and f i l t e r e d . Procedure 1.  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 with DNFB (Unavailable l y s i n e ) . 0.1 g o f p r o t e i n was p l a c e d i n a dry 2 - l i t e r  f l a s k , w i t h a few g l a s s beads. sample a t the bottom.  boiling  Care was taken t o d e p o s i t the  10 ml f r e s h l y prepared  10% NaHCO^  s o l u t i o n was added, care being taken t h a t no meal adhered t o the s i d e s o f the f l a s k .  The contents were thoroughly mixed  by g e n t l e s w i r l i n g and the suspension was allowed t o stand f o r 10 minutes. A s o l u t i o n o f 0.4 ml DNFB i n 10 ml e t h a n o l was then added and mixed by g e n t l e s w i r l i n g . was r i n s e d w i t h 3-5 ml e t h a n o l .  The s i d e of the f l a s k  The f l a s k was stoppered and  the contents were shaken under subdued l i g h t f o r 3 h r on a mechanical  shaker. 2 ml of 6 NrHCl was added.  The mixture was c o n c e n t r a -  t e d t o o i l y dryness i n a vacuum r o t a r y evaporator a t 40°C,  81.  and the r e s i d u e was e x t r a c t e d w i t h four-5 0 ml p o r t i o n s o f anhydrous e t h e r .  In each case, the e t h e r was mixed  thoroughly w i t h the s e m i s o l i d mass and the two phases were allowed t o separate completely b e f o r e the e t h e r was decanted.  F i n a l t r a c e s o f e t h e r were removed by a e r a t i o n  i n the r o t a r y  evaporator/without  vacuum.  125 ml of 6 N HCI was added and the mixture  boiled  under r e f l u x f o r 18 h r , m a i n t a i n i n g a constant stream o f p r e p u r i f i e d n i t r o g e n through a c a p i l l a r y tube which about 2.5 cm above the s u r f a c e o f the s o l u t i o n The mixture was allowed t o c o o l down t o room  reached  (Figure 10). temperature  and the condensor was washed down w i t h 6 N HCI. I f time p e r m i t t e d a t the c o o l i n g s t a g e , the f l a s k was kept o v e r n i g h t i n the r e f r i g e r a t o r , s i n c e i t helped t o p r e c i p i t a t e the excess DNFB and reduce extraction  (Carpenter e t a l . , 1957).  The mixture  was then evaporated t o a s t i c k y paste i n  a vacuum r o t a r y evaporator at 50°C. adding  the subsequent work o f  The paste was washed by  100 ml o f d e i o n i z e d water and d r y i n g back t o a paste  on the vacuum r o t a r y evaporator a t 5 0°C. repeated 5 times.  T h i s procedure was  The l a s t e v a p o r a t i o n was c a r r i e d on u n t i l  the sample was completely d r y . 100 ml o f pH 2.2 sodium c i t r a t e b u f f e r was added. The  f l a s k was stoppered, shaken f o r 5 minutes and f i l t e r e d  through Whatman 43 i n t o a storage b o t t l e t h a t was kept i n  82.  FIGURE 1 0 .  Refluxing of protein  system used  i n the  hydrolysates-  preparation  83.  a c o l d room a t 5°C; p r o t e i n was 2.  1.0  ml  filtrate  c o n t a i n i n g 0.72-0.88  u s e d f o r amino a c i d a n a l y s i s .  P r e p a r a t i o n of p r o t e i n h y d r o l y s a t e without (Total lysine) 0.025 g o f p r o t e i n s a m p l e was  r o u n d - b o t t o m f l a s k w i t h 4-5 HCI  was  a d d e d and  mantle, u n t i l The  mg  100  placed  g l a s s beads.  200  DNFB  in a 2  ml o f 6 N  the m i x t u r e  a l l o w e d t o b o i l on  m l HCI  evaporated.  mixture  was  had  hydrolyzed  liter  a heating  u n d e r r e f l u x f o r 24  hr,  as d e s c r i b e d u n d e r p r e p a r a t i o n o f s a m p l e w i t h DNFB. The  h y d r o l y s a t e was  vacuum r o t a r y e v a p o r a t o r w i t h 100 dryness  ml-portions  ml  a t 50°C.  43.  1.0  ml  washed 5  o f "sodium c i t r a t e b u f f e r , pH  filtrate  to  2.2  was Whatman  protein  was  analyzer.  flow diagram of the experimental  shown i n F i g u r e  times  f i l t e r e d through  c o n t a i n i n g 0.18-0.22 mg  u s e d f o r a n a l y s i s on an amino a c i d The  I t was  in a  evaporation.  a d d e d , t h e c o n t e n t s w e r e s h a k e n and No.  to s t i c k y paste  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  d u r i n g the l a s t A f t e r 100  evaporated  procedure i s  11.  Calculation , The  p e a k s on t h e c h r o m a t o g r a m s w e r e 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 the peak  (absorbance r e a d i n g  at  t h e maximum p e a k h e i g h t , m i n u s a b s o r b a n c e r e a d i n g a t t h e  base-  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 of a v a i l a b l e l y s i n e (difference  technique).  From: B l o m e t a l . , (1967) .  Protein  Acid  hydrolysis  Column' chromatography  DNFB t r e a t m e n t  Aqueous  layer  Acid  Total  hydrolysis  lysine  e-DNP-lysine  Unreacted  lysine  Evaporate t o dryness Citrate  buffer  Column c h r o m a t o g r a p h y Unreacted  T o t a l l y s i n e - Unreacted  lysine = Available lysine.  lysine  85.  line  o f t h e p e a k ) fay t h e w i d t h  count).  The  area  constant  f o r the given  at half  u n d e r t h e p e a k was amino a c i d  the height  then  (dot  d i v i d e d by the  so t h a t , micromoles  or  m i c r o g r a m s o f amino a c i d  p e r volume a p p l i e d t o t h e  analyzer,  vcere o b t a i n e d .  very  a  This  gram on t h e Monroe The v a l u e s amino a c i d s was  1.  done by u s i n g  pro-  calculator. were  adjusted  so t h a t  t h e sum  of a l l the  lysine  content  i n c a s e i n was c a l c u l a t e d  follows: Total  lysine  14.035  yg l y s i n e / m l  1403.5  yg lysine/100  5.614 2.  easily  100%.  Available, as  was  mg  Unavailable  lysine/100  (volume  applied)  m l / 2 5 mg mg  =  casein  =  casein  =  casein.  lysine  2.632 y g l y s i n e / m l = 2 6 3 . 2 y g l y s i n e / 1 0 0 m l / 1 0 0 mg 0 . 2 6 3 2 mg 3.  G.  lysine/100  mg  casein.  Available lysine =  Total lysine  Available  5 . 3 5 0 8 mg  lysine  =  Enzymatic digestion The d i g e s t i b i l i t y  pepsin-pancreatin  - Unavailable  lysine/100  mg  casein.  test o f p r o t e i n s was  determined by the  d i g e s t i o n method o f Akeson and  (1964) , as m o d i f i e d  lysine  by Stahmann  and W o l d e g i o r g i s  Stahmann (1975).  86.  Reagents Pepsin 100  ml v o l u m e t r i c  solution: flask  was p l a c e d i n a  and b r o u g h t t o v o l u m e w i t h 0.1 N H C I .  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  Pancreatin in  10 mg o f p e p s i n  a 50 m l v o l u m e t r i c  flask  placed  and b r o u g h t t o v o l u m e w i t h  pl-l 8.0 p h o s p h a t e b u f f e r . pH 8.0 s o d i u m p h o s p h a t e b u f f e r K HP0 .3H 0 2  4  2  (0.1M) :  a n d 0.523 g o f a n h y d r o u s K H P 0 2  i n w a t e r and d i l u t e d t o one 14%  sulfosalicylie  21.92 g o f  were d i s s o l v e d  4  liter. acid solution:  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 m l . No. 1 i n t o a d a r k  The s o l u t i o n was f i l t e r e d  through  Whatman  bottle.  C i t r a t e s a m p l e d i l u t e r pH 2.2 50 ppm m e r t h i o l a t e  (See DNFB  reagents).  solution.  Procedure 50 mg o f p r o t e i n s a m p l e was i n c u b a t e d of pepsin  s o l u t i o n i n a shaking water bath  w i t h 7.5 m l  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 m l o f 0.2 N NaOH, 3.75 m l of p a n c r e a t i n was  added.  s o l u t i o n a n d 0.25 m l o f 50 ppm 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  a d d i t i o n a l 2 4 hrs i n a shaking water bath To p r e c i p i t a t e t h e u n d i g e s t e d 5.0 m l o f d i g e s t i o n m i x t u r e  .was  merthiolate f o r an  a t 37°C.  p r o t e i n and p e p t i d e s ,  m i x e d w i t h 5.0 m l o f 14%  87.  sulfosalicylic  a c i d s o l u t i o n , t o b r i n g the f i n a l c o n c e n t r a -  tion of s u l f o s a l i c y l i c for  30 minutes  a c i d t o 4.7%.  The f l a s k s were shaken  and the pH was a d j u s t e d to 2.2 by adding drops  o f 6 N NaOH. The d i g e s t s were then t r a n s f e r r e d t o a 15 ml graduated tube and brought t o volume w i t h pH 2.2 c i t r a t e The s o l u t i o n was c e n t r i f u g e d minutes,  buffer.  a t 1,000 x g f o r 30  f i l t e r e d through Whatman No.42 and s t o r e d i n the  f r e e z e r a t -2 0°C f o r amino a c i d  analysis.  The same procedure was c a r r i e d out f o r an enzyme blank t h a t was prepared by i n c u b a t i o n  with the p r o t e i n  sample  omitted. Calculation: 15.2 5 ml o f d i g e s t i o n mixture tained  50 mg p r o t e i n ;  ( f i n a l volume) con-  thus, 5 ml o f d i g e s t i o n  c o n t a i n e d 16.39 mg p r o t e i n .  mixture  5.00 ml o f d i g e s t i o n  mixture  mixed w i t h s u l f o s a l i c y l i c a c i d and made up t o 15 ml with citrate  b u f f e r , contained 16.39 mg p r o t e i n ;  0.5 ml used f o r  amino a c i d a n a l y s i s , contained 0.5 46 mg p r o t e i n . The  areas under the peaks o f the e f f l u e n t curve were  c a l c u l 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 the peak by the  width a t h a l f the h e i g h t , and d i v i d i n g by the c o n s t a n t f o r the given amino a c i d .  Micromoles, or micrograms of amino  a c i d p e r volume a p p l i e d , v/ere o b t a i n e d .  88.  I f c a s e i n h a d 13.899 y g l y s i n e / 0 . 5 m l : 13.899 y g l y s i n e / 0 5 m l 0.546 mg p r o t e m / 0 . 5 m l  1 mg 1000 y g  2.54 4 mg l y s i n e / 1 0 0 mg  casein.  ^  89.  RESULTS AND DISCUSSION The  p r o t e i n content o f the samples,  as d e t e r m i n e d  by t h e r a p i d m i c r o - K j e l d a h l m e t h o d o f C o n c o n a n d 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) m e t h o d  The m e t h o d d e s c r i b e d b y C o n c o n reagent sodium  dinitrobenzenesulfonate  phosphate-phenylmercuric e-amino g r o u p s  (1975a) e m p l o y s t h e  (DNBS) i n a u r e a -  c h l o r i d e s o l u t i o n , t o complex t h e  of lysine.  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 a r e 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 t h e n m e a s u r e d a t 385 nm a n d t h e amount o f available lysine i s calculated T a b l e 4 shows t h e a v a i l a b l e  from a s t a n d a r d c u r v e .  lysine content of protein  samples  as d e t e r m i n e d b y t h e DNBS m e t h o d . From t h e s a m p l e s  s u b j e c t e d t o t h e DNBS p r o c e d u r e ,  o n l y 1.5 N FICL g l u t e n a n d w h o l e e g g s o l i d s h a d a h i g h b l a n k value.  This i s often encountered  i n undefatted materials  or i n those c e r e a l g r a i n s w i t h h i g h l y pigmented A c c o r d i n g t o Concon  layers.  ( 1 9 7 5 b ) , f o r t h e DNBS m e t h o d , l i p i d s  n o t be r e m o v e d e x c e p t i n h e a v i l y p i g m e n t e d b a r l e y , o a t s , r y e , and wheat. and e g g s a m p l e s  outer  need  varieties of  However, s u b j e c t i n g t h e g l u t e n  t o f a t e x t r a c t i o n might have produced  lower  blank values. The casein  sample b l a n k s f o r B - l a c t o g l o b u l i n ,  and RNase-S-peptide  gave l o w e r a b s o r b a n c e  lysozyme, readings  TABLE 3.  P r o t e i n content o f samples assayed f o r l y s i n e availability  Sample  g protein/100 g sample  1.5 N HCl Gluten  35.20  3-lactoglobulin  63.43  Lysozyme  89.20  Casein  72.30  Whole Egg ( f r e e z e - d r i e d )  37.26  91. TABLE 4.  Available  lysine  content  of protein  determined by the dinitrobenzene (DNBS) method. ( V a l u e s  sulfonic  i ng lysine/100 g  E x p e r i m e n t No.  Protein  2  3  1.801  1.668  1.712  1.818  1.668  1.838  1.702  1.715 1.764  Average  1.819  1.679  1.730  Casein  7.413 7.408 7.496  7.575 7.504  7.550  Gluten  Average  7.439  Lysozyme  5.463  Average /3 - l a c t o g .  Average Egg  Average  7.501 7.618 7.556  7.515  5.545 5.414 5.474  5.384  5.437  12.354 12.428  11.837  12.723  11.837  12.797  12.283  11.543  12.355  11.739  12.503 12.674  5.490  5.368  5.610  5.523 5.346  5-340  5.622  5.327  5.722  5.453  5.345  5.651  5.483  10.670  10.618  10.644  lysine  content,  e t a l . , 1976.  values  1.748  5.507 5.402  RNase-S-  protein).  Ave  5.327 5.396 5.430  nfintide  from P u r c e l l  7.575 7.551  acid  Reported  1  "Available  samples as  5.402 5.432  12.256  b a s e d o n known amino a c i d  5.4J  10.5  10.2J  sequence,  92.  than the reagent blank. ignored;  In t h i s case, the n e g a t i v e value  o b v i o u s l y , no absorbance  sample without d i n i t r o p h e n y l a t i o n . the reagent blank may  i s contributed  was  by the  The h i g h e r absorbance  of  come from the r e a c t i o n of DNBS w i t h  i m p u r i t i e s i n the reagents or simply contaminations  (Concon,  1971). The time o f d i n i t r o p h e n y l a t i o n g r e a t l y a f f e c t s the p r e c i s i o n of the DNBS r e s u l t s s i n c e the absorbance  o f the  s o l u t i o n d u r i n g c o l o r development i n c r e a s e s w i t h time. change of about 0.006 absorbance  A  u n i t s per minute p e r ml  w i t h a l y s i n e c o n c e n t r a t i o n of 0.4  moles per ml at pH  and 40°C, can be expected  1971).  (Concon,  In t h i s  12  thesis,  about 8 minutes e l a p s e d between a d d i t i o n o f DNBS t o the  first  tube and a d d i t i o n of the reagent t o the l a s t tube, b e f o r e i n c u b a t i o n at 40°C.  Thus, an absorbance  or l e s s , s i n c e a t room temperature  e r r o r of 0.048 u n i t s  very l i t t l e d i n i t r o p h e n y -  l a t i o n o c c u r s , can be expected i n the  results.  Studying the r e a c t i v i t y o f TNBS w i t h amino a c i d s and p e p t i d e s , Freedman and Radda (1968) showed t h a t , a t room temperature, pH 7.4,  the e-amino groups of l y s i n e s were 30  times more r e a c t i v e t o TNBS than were the a-amino groups. When the n e i g h b o r i n g c a r b o x y l group was.  amidated  i n a p e p t i d e bond, the a-amino groups became even reactive.  or joined less  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  o r p e p t i d e bond formation o f the c a r b o x y l r a d i c a l ,  accounted  f o r the decreased r e a c t i v i t y of TNBS.  suggested  I t was  then  t h a t DNBS, a reagent l e s s e l e c t r o p h i l i c than TNBS, might more s p e c i f i c f o r the e-amino group.  The  absence  be  of one  extra  ortho n i t r o group i n DNBS i s expected t o d i m i n i s h the c o r r e s ponding e l e c t r o p h i l i c i t y of the s u l f o n a t e r a d i c a l . other e l e c t r o s t a t i c f a c t o r s may  a l s o i n f l u e n c e the  of both DNBS and primary amino groups.  Steric  and  reactivity  T h e r e f o r e , under the  r e a c t i o n c o n d i t i o n s used, a v i r t u a l disappearance of the absorbance,  due t o a-amino groups may  result.  However, u s i n g  model l y s i n e d e r i v a t i v e s w i t h d i f f e r e n t numbers of a- and e-amino groups, i t has been found t h a t DNBS a l s o r e a c t s w i t h the a-amino groups to a c e r t a i n e x t e n t . r e s u l t s of t h i s experiment.  Table 5 i n d i c a t e s the  e - f o r m y l - l y s i n e which has  one  free a-amino group, when r e a c t e d w i t h DNBS, shows a 385  nm  absorbance  one  v a l u e of 0.091, but a - f o r m y l - l y s i n e t h a t has  f r e e e-amino group,  shows an absorbance  v a l u e of 0.2 89.  expected values of l y s i n e , l y s y l - l y s i n e ,  lysyl-alanine  The and  r i b o n u c l e a s e - S - p e p t i d e , have been c a l c u l a t e d assuming DNBS specificity  f o r the e-amino group.  The d i f f e r e n c e between  the experimental and the expected v a l u e s , shows the e x t e n t t o which the a-amino group r e a c t s w i t h DNBS. 0.095 f o r l y s i n e ,  The d i f f e r e n c e i s  0.069 f o r the d i p e p t i d e l y s y l - l y s i n e ,  only 0.026 f o r RNase-S-peptide.  A d i f f e r e n c e of 0.001  and  was  TABLE 5.  R e a c t i o n o f a m i n o 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  Amino a c i d o r peptide  (1)  Number o f e-NH  2  a--NH  (DNBS)  Experimental  E x p e c t e d (2)  Difference  2  e-formyl-lysine  0  1  0.091  0.000  0.091  a-formyl-lysine  1  0  0.289  0.289  0.000  Lysine  1  1  0.384  0.289  0.095  Lysyl-lysine  2  1  0.647  0.578  0.069  Alanine  0  1  0.000  0.000  0.000  Lysyl-alanine  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  lysy1-alanine,  a-amino g r o u p o f a l a n i n e  i n d i c a t i n g that the  i s n o t m e a s u r e d by t h e DNBS  technique. From t h e above f i g u r e s i t c a n be s e e n t h a t l y s i n e i s a t the N-terminal p o s i t i o n i n a p r o t e i n , s u l t w i l l 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 available the  l y s i n e , since  when  the r e -  content of  t h e a-amino g r o u p i s c o n t r i b u t i n g t o  results. R N a s e - S - p e p t i d e i s a 2 0 - r e s i d u e component w h i c h h a s  two  l y s i n e u n i t s , one o f them l o c a t e d  position the  at the N-terminal  ( R i c h a r d s and V i t h a y a t h i l , 1 9 5 9 ) .  peptide,  r a t i o o f e- t o a-amino g r o u p s i s 2 t o 1 , a n d t h u s , t h e  contribution t i v e l y high  o f t h e a-amino g r o u p t o t h e r e s u l t i s (0.026).  peptide, that  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  same f i g u r e was c a l c u l a t e d  b a s e d on known a m i n o a c i d In  compara-  I f t h e e x p e c t e d v a l u e f o r RNase-S-  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 , The  In t h i s  by P u r c e l l e t a l .  (1976),  sequence.  t h i s work, t h e a v a i l a b l e  S-peptide c a l c u l a t e d  the r e s u l t i s 10.2.  l y s i n e c o n t e n t o f RNase-  f r o m an a b s o r b a n c e v a l u e o f 0 . 6 0 4 , was  f o u n d t o be 1 0 . 6 4 g p e r 1 0 0 g o f p r o t e i n .  Purcell et al.  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, s u c h 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 t h e e- as w e l l  as t h e a-amino g r o u p s o f p r o t e i n s ,  obtained a value o f 15.3 g l y s i n e p e r 100 g o f RNase-S-peptide. In p r o t e i n s ,terminal  and h i g h e r p o l y p e p t i d e s t h a t  l y s i n e , t h e r a t i o w o u l d be t h a t  of several  h a v e Ne-amino  g r o u p s t o one a-amino g r o u p .  In this  case,  the c o n t r i b u t i o n  o f t h e a-amino g r o u p t o t h e r e s u l t s , becomes n e g l i g i b l e .  Thus,  the  will  lysine availability  give r e l i a b l e data  a s d e t e r m i n e d b y t h e DNBS m e t h o d ,  for proteins containing a substantial  number o f e-amino g r o u p s p e r c h a i n . a- a n d e - c a r b o b e n z o x y - l y s i n e s DNBS s p e c i f i c i t y  study.  were a l s o used f o r t h e  T h e s e d e r i v a t i v e s d i d n o t show any  a b s o r b a n c e v a l u e when r e a c t e d w i t h DNBS. be  explained  B.  This behavior  i n terms o f a s t e r i c b e n z y - b e n z y l  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 m e t h o d The  reagent 2,4,6-trinitrobenzene  could  hindrance.  (TNBS) sulfonic  a c i d was  u s e d t o c o m p l e x w i t h t h e f r e e e-amino g r o u p o f l y s i n e , a n d the  resulting  metrically  e - T N P - l y s i n e was d e t e r m i n e d  a f t e r 90. m i n 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  fair  spectrophoto-  agreement w i t h those  reported  f o ravailable l y s i n e , i n i n t h e l i t e r a t u r e , as  shown i n T a b l e 6. TNBS h a s b e e n f o u n d t o r e a c t w i t h v a r i o u s f e r i n g compounds; contradictory. Posati  thus, the reported values  inter-  a r e sometimes  H o l s i n g e r e t a l . (1970) a n d H o l s i n g e r a n d  (1975) r e p o r t e d  a n o m a l o u s l y h i g h r e s u l t s when t h e TNBS  p r o c e d u r e o f Kakade and L i e n e r The  a v a i l a b l e l y s i n e content  the  total  lysine content,  (1969) was a p p l i e d t o c a s e i n s .  f o r k - c a s e i n was 1 5 % h i g h e r  and whole c a s e i n v a l u e s  10-15% h i g h . They a s c r i b e d t h i s  than  were  f i n d i n g t o the presence o f  d - g l u c o s a m i n e 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  97.  TABLE 6 .  Available  l y s i n e c o n t e n t 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 (TNBS) method. ( V a l u e s  Experiment -Protein  Gluten Average Lysozyme Average  /3-lactoglob. Average  Available 1.  Reported  values  1.388 1.428 1.408  1.370 1.376  1.585 I.607 1.596  1.24  5-734 5.858 5.796  5.584  5.793 5.777 5-785  5.742 5.781  6.900  6.703 8.54-0.21^ 8.235 9.655 8.061  1.373 5.603 5.59^ 6.913 6.837 6.874  12.991  14.024 14.228 14.126  6.134  7*028  6.192  7.253 7.141  6.163  Average  No.  3  12.980 13.002  Egg  i n g lysine/100 g protein).  2  7.607  Average  acid  1  7-59^ 7.620  Casein  sulfonic  6.957 6.929 14.564  1  9.79  3  14.759 14.662 6.797 6.857 6.827  l y s i n e as d e t e r m i n e d b y t h e TNBS method1  From F i n l e y and F r i e d m a n ,  1973.  2 . From Kakade a n d L i e n e r ,  I969.  3.  From H o l s i n g e r e t a l . ,  1970.  4.  From E k l u n d ,  5.  From H a l l e t a l . ,  1976. 1973.  11.652  98.  uncharacterized glycoprotein  fragments  i n the casein  micelle.  Amino s u g a r s r e a c t w i t h TNBS t o f o r m c o l o r e d d e r i v a t i v e s  which  a r e n o t removed by e t h e r e x t r a t i o n . oi - a n d 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 , g a v e a v a i l a b l e e i t h e r equal to o r l e s s than the t o t a l  lysine contents, lysine present.  On t h e o t h e r h a n d , l o w TNBS v a l u e s h a v e b e e n r e p o r ted  by P o s a t i  lactose. in  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  containing  These a u t h o r s found t h a t t h e p r e s e n c e o f l a c t o s e  l e v e l s approaching the l y s i n e : l a c t o s e  r a t i o i n dehydrated  whey p o w d e r s , 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-TNPlysine  derivative. H a l l e t a l . (1975) a n d P o s a t i e t a l . (1972) h a v e  s t r e s s e d the importance  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. b o v i n e serum a l b u m i n gave about  A s a m p l e o f 0.05 mg o f  89% a v a i l a b l e l y s i n e , w h e r e a s  a s a m p l e s i z e o f 0.7 mg g a v e o n l y 7 5 % a v a i l a b l e (Posati e t a l . ,  1972).  On t h e o t h e r h a n d , E k l u n d  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 e m p l o y i n g There  lysine (1976)  a 30 mg  sample.  i s a l s o some c o n f l i c t i n t h e l i t e r a t u r e r e -  garding the r e a c t i v i t y  of free  l y s i n e towards  r e a c t s w i t h TNBS t o f o r m i n i t i a l l y K o t a k i and S a t a k e hydrochloric acid;  TNBS.  Lysine  a , e - d i T N P - l y s i n e , which  (1964) showed t o be u n s t a b l e i n h o t 6 N 81%  e - T N P - l y s i n e a n d 17% f r e e  lysine  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 , 1 9 6 0 ) .  Ousterhout  a n d Wood  (1970)  found,  h o w e v e r , 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-  lysine. free  H a l l and a s s o c i a t e s  (197 3) found t h a t r e c o v e r i e s o f  l y s i n e and e-TNP-lysine added as i n t e r n a l standards t o  p r o t e i n suspensions which were then s u b j e c t e d t o the TNBS r e a c t i o n and a c i d h y d r o l y s i s were v a r i a b l e .  Furthermore,  s i n c e a, e - d i T N P - l y s i n e i s e t h e r s o l u b l e , i n p r o t e i n s where l y s i n e i s at the N - t e r m i n a l p o s i t i o n , t h i s d e r i v a t i v e o r s m a l l p e p t i d e s c o n t a i n i n g i t , would be e x t r a c t e d i n t o the e t h e r phase and would escape  detection.  H a l l e t a l . (1975) found v a r i a b l e r e s u l t s f o r available lysine i n carbohydrate-rich materials.  According  to them,browning and c h a r r i n g produced d u r i n g a c i d h y d r o l y s i s accounted f o r the low r e s u l t s f o r a v a i l a b l e  lysine.  e-TNP-lysine was adsorbed on t o the carbon p a r t i c l e s  (from  charring) which were then removed d u r i n g the e t h e r e x t r a c t i o n process.  When the sample s i z e was decreased, H a l l e t a l .  found t h a t t h e • a v a i l a b l e l y s i n e measured by TNBS r e a c t i o n exceeded  the value f o r the t o t a l l y s i n e .  In m a t e r i a l s r i c h  i n s t a r c h e s , the p u r i f i e d s t a r c h e s themselves TNBS to g i v e an apparent l y s i n e content.  reacted with  From these  findings,  H a l l e t a l . recommended t h a t the TNBS r e a c t i o n be c a r r i e d out at 30°C f o r 30 minutes,  f o r determining a v a i l a b l e  lysine,  i n feeds and c e r e a l s r i c h i n carbohydrates. Not only carbohydrates and sugars i n t e r f e r e i n the 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 w i t h TNBS, but n a t u r a l l y o c c u r r i n g amines such as agmatine,  spermine,  spermidine and  t a u r i n e , r e a c t with TNBS and can be measured as a v a i l a b l e  100.  lysine. if  Cadaverine,  hydroxylysine, ornithine  p r e s e n t i n samples o f t i s s u e s  decomposition,  t h a t have u n d e r g o n e some  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 .  T h e r e i s some d i s a g r e e m e n t used t o draw t h e s t a n d a r d c u r v e and  L i e n e r (1969) and E k l u n d  standard.  and p u t r e s c i n e ,  i n regard t o the standard  f o r t h e TNBS m e t h o d .  (1976) u s e d e - T N P - l y s i n e  Graded l e v e l s o f e-TNP-lysine  t h e same p r o c e d u r e  Kakade  as t h e p r o t e i n  as a  were s u b j e c t e d t o  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 t h e U.K., H a l l e t a l . (1973) p o s e d t h e use o f l y s i n e as a s t a n d a r d . the product  pro-  They d e m o n s t r a t e d  that  f r o m 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 a n d  ether extraction  h a d v i r t u a l l y t h e same a d s o r b a n c e 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-TNP-lysine.  However,  when l y s i n e was r e a c t e d w i t h TNBS a n d s u b j e c t e d t o a c i d hydrolysis of  a n d e t h e r e x t r a c t i o n , we f o u n d  1.04 x 10  4  (moles/liter)  -1  cm  -1  .  a molar  e-TNP-lysine,  absorptivity subjected 4  t o t h e same p r o c e d u r e ,  h a d a m o l a r a b s o r p t i v i t y o f 0.89 x 10  (moles/liter)  B u t , when e - T N P - l y s i n e  directly  ^ cm  was  read  a t 346 nm, t h e m o l a r a b s o r p t i v i t y was f o u n d t o be  1.46 x 10^  (moles/liter)  - 1  cm  - 1  which  i s i n agreement w i t h  t h e v a l u e r e p o r t e d b y Kakade and L i e n e r (1969). In t h e p r e s e n t work, T N B S - a v a i l a b l e determined  f r o m a s t a n d a r d c u r v e w h i c h was b u i l t by r e a d i n g  d i r e c t l y t h e 346 nm a b s o r b a n c e o f g r a d e d lysine.  l y s i n e was  l e v e l s o f e-TNP-  A c c o r d i n g t o H a l l e t a l . (1973) a b o u t 1 0 0 % o f  e-TNP-lysine  c a n be r e c o v e r e d f r o m e i t h e r  d i r e c t l y i n 1 N. H C I ;  b) e - T N P - l y s i n e  a)  e-TNP-lysine  i n 1 N HCI a f t e r  ether  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 in  after 2 hr  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 a b o v e , i t c a n be s e e n  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 determination.  The m e t h o d w o u l d r e q u i r e s e v e r a l  and  modifications before  C.  The f l u o r o d i n i t r o b e n z e n e The  i t can y i e l d  group'of l y s i n e .  (FDNB) t o b i n d  the reagent  the free  e-amino  An e - D N P - l y s i n e c o m p l e x i s f o r m e d , w h i c h  acid hydrolysis.  hydrolyzed  data.  (FDNB) m e t h o d .  FDNB d i f f e r e n c e m e t h o d u t i l i z e s  1-fluoro-2,4-dinitrobenzene  resists  reliable  corrections  and a n a l y z e d  When t h e F D N B - r e a c t e d s a m p l e i s f o r l y s i n e b y t h e m e t h o d o f Moore  e t a l . ( 1 9 5 9 ) , t h e l y s i n e w h i c h was b o u n d a n d 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 m e a s u r e d . t o be u n a v a i l a b l e by  lysine.  This  i s considered  A v a i l a b l e l y s i n e , w h i c h was b o u n d  FDNB, i s c a l c u l a t e d as t h e d i f f e r e n c e b e t w e e n t o t a l a n d  unavailable  lysines.  T a b l e 7 shows t h e l y s i n e f o u n d a f t e r a c i d h y d r o l y s i s , the  unavailable  l y s i n e a s d e t e r m i n e d by t h e FDNB p r o c e d u r e ,  and  the a v a i l a b l e l y s i n e content of theyprotein  r e s u l t s a r e i n good agreement w i t h t h e r e p o r t e d the  values.  The In  c a s e o f 3 - l a c t o g l o b u l i n , 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 was  f o u n d t o be 1 1 . 2 7 % . of  samples.  10.5%  P u r c e l l e t a l . (1976)  reported  a  value  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 , b a s e d on  TABLE 7.  Available  l y s i n e c o n t e n t o f p r o t e i n s a m p l e s as d e t e r m i n e d  trobenzene  (FDNB) d i f f e r e n c e  TOTAL LYSINE Experiment  Protein 1  2  ted  3  4  AVE  Value  Gluten  1.67  1. 69  1.73  1. 71  1. 70  1. 2r>~ 1.70  Casein  7. 42  7. 83  7.59  7. 68  7.63  6.12:? 8.3l£ 8.7  Lysozyme 3-lactog. Egg  5.28  5. 36  13.60 12. 29 13.10  6. 92  6. 83  7.15  5.7 ^ 5.75  UNAVAILABLE Experiment 1  2  0.17  0.13  0.22  0.25  LYSINE  LABLE  No. 3  AVE 0 .15  0.22  AVAI-  0.23  LYSINE (T - U) 1. 55  7. 35  0.17  5 14. 41 13. 35 1 1 . 4  2.20  1.93  2.11  2.08  11.27  6.2^ 6.4  0.25  0. 30  0.32  0.29  6.55  6  6. 46  6. 84  The v a l u e s a r e g i v e n i n g l y s / 1 0 0 g p r o t e i n .  ;  Reported Value 1.26? 1.54, 1.67 5.10" 7.5 £ 8.17 X  8  0.19  5.33  5.35  dini-  method.  Repor-  No.  by t h e f l u o r o  0.18  5.15  5.4 5.60 10.5  -  1 2  12  8 8  103.  1.  Chromatographic procedure f r o m F i n l e y and F r i e d m a n ,  (Spackman e t a l . , 195 8) 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 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 p r o c e d u r e , f r o m B o o t h , 1971.  3.  C h r o m a t o g r a p h i c p r o c e d u r e (Spackman e t a l . , 195 8) , f r o m 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 d e t e r m i n e d by a c i d h y d r o l y s i s , Stahmann and W o l d e g i o r g i s , 1975.  5.  Total  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 d a t a , from Jenness P a t t o n , 1959.  7.  Total lysine 1957.  8.  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 C a r p e n t e r ' s method, C a r p e n t e r and B j a r n a s o n , 1969 .  from  9.  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 C a r p e n t e r ' s method, B o c t o r and H a r p e r , 196 8.  from  l y s i n e by c o m p o s i t i o n , f r o m T r i s t r a m ,  taken  from  1953. and  f r o m c o m p o s i t i o n a l d a t a , f r o m O r r and W a t t ,  10. A v a i l a b l e l y s i n e H u s s a i n , 1974.  d e t e r m i n e d by C a r p e n t e r ' s method,  from  11. A v a i l a b l e l y s i n e m e a s u r e d by t h e m o d i f i e d C a r p e n t e r method ( B o o t h , 1 9 7 1 ) , f r o m 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 sequence,  l y s i n e c a l c u l a t e d f r o m known amino f r o m P u r c e l l e t a l . , 1976.  acid  104.  known amino a c i d  sequence.  Porter  (1948)  some p r o t e i n s s u c h as 3 - l a c t o g l o b u l i n  have r e p o r t e d  and serum  that  globulins,  i n c l u d e l y s i n e g r o u p s t h a t r e a c t w i t h FDNB o n l y a f t e r d e 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 mild acid.  I t m i g h t 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 s e e w h e t h e r reactive  lysine The  a h i g h e r FDNB-  result i sobtained.  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 p o o r r e c o v e r i e s o f e - D N P - l y s i n e a n d 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 a n d o i l s e e d The  meals.  poor performance o f t h e 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 - 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  hydrolysis,  and t h e 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 r e n o t e a s i l y s e p a r a t e d from e-DNP-lysine Rao e t a l . ,  1963).  ( C a r p e n t e r e t a l . , 1957;  P a r t i a l d e s t r u c t i o n o f e-DNP-lysine  n o t c a u s e d i f f i c u l t i e s when a v a i l a b l e  does  l y s i n e i s determined  by t h e 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 tically  this  technique;  e - D N P - l y s i n e i s mathema-  c a l c u l a t e d by d i f f e r e n c e . The  difference technique suffers  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; (unavailable required  lysine)  and 24 h r ( t o t a l )  18 h r  of acid hydrolysis are  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  availability.  Sub-  sequent r e m o v a l o f H C l from t h e sample and r e d u c t i o n o f t h e  105.  sample to dryness on a r o t a r y e v a p o r a t o r , i s , however, the most time consuming s t e p .  Hubbard and Finney  (1976) have  d e s c r i b e d a technique i n v o l v i n g an Evapo-Mix e v a p o r a t o r , which g r e a t l y reduces the time and a t t e n t i o n required.  With t h i s system,  otherwise  10 samples o f 5.5 ml o f s o l u t i o n  were reduced t o dryness i n about  35 minutes.  Although time consuming, the FDNB d i f f e r e n c e technique i s the most a c c u r a t e means p r e s e n t l y a v a i l a b l e f o r the chemical 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 . obtained w i l l D.  The values  i n c l u d e f r e e - l y s i n e and N-terminal l y s i n e .  Pepsin-pancreatin digestion  test.  Although i n the f i n a l a n a l y s i s , the 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 must be assessed by f e e d i n g t r i a l s , the enzymatic methods 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 are very useful f o r preliminary quality evaluation.  In v i t r o  enzymatic  d i g e s t i o n methods attempt t o i m i t a t e the a c t i o n o f the mammalian d i g e s t i v e system.  Compared t o animal assays, the  enzymatic methods have s e v e r a l advantages.  They are l e s s  expensive and r e q u i r e l e s s time than b i o a s s a y s ;  they show  l e s s v a r i a t i o n than p r o t e i n e f f i c i e n c y r a t i o assays w i t h rats;  a s i n g l e assay can i n d i c a t e the r e l a t i v e amounts o f  e s s e n t i a l amino a c i d s c o n t a i n e d i n and r e l e a s e d by the enzyme from the p r o t e i n under assay. The enzymic methods have been used t o monitor the adverse e f f e c t s o f p r o c e s s i n g o p e r a t i o n s on p r o t e i n s , t h a t  106.  may  decrease  the  availability Stahmann in  the  of  digestibility l y s i n e or  (1977) f o u n d  presence of  of  oxidise  protein,  reduce  s u l f u r amino  acids.  that peroxidase or  chlorogenic  hydrolysis,  revealed  no  trations of  chlorogenic  enzymatic h y d r o l y s i s  the  a c i d and  destruction a c i d and  revealed  the  polyphenoloxidase p r o t e i n , upon  with increasing  o x i d a s e enzymes,  acid  concen-  but  a complete d e s t r u c t i o n  of  methionine. Even complete,  so  amount o f  the  hydrolysis Lysine value  that  the  enzymic i n v i t r o  values obtained  limiting  has  since  The  the  are  not  lysine could  a c i d as  f o u n d t o be  n e c e s s a r y t o pass the  column i n o r d e r t o e l i m i n a t e author the  suggested  the  use  undigested protein.  since  t h i s reagent  directly  applied  the  no n u t r i t i v e  to  enzymatic  freed. Stahmann  very  treated  picric  i s an  t o the  protein.  sample t h r o u g h In  1975,  a  the  a c i d to p r e c i p i t a t e  advantageous  i s colorless, ninhydrin  (1946)  inconvenient  acid.  of s u l f o s a l i c y l i c This  the  availability.  susceptible be  not  enzymic  amino a c i d  not  is  relative,  a p r e c i p i t a n t of undigested  However, t h i s p r o c e d u r e was i t was  by  o r i g i n a l method o f Akeson and  used p i c r i c  since  only  i t s e-amino g r o u p b l o c k e d has  those groups and  are  digestion  amino a c i d r e l e a s e d  i s a good i n d i c a t o r o f  that  hydrolysis  though the  modification  negative  columns f o r amino a c i d  and  analysis  can  be  107.  (Hamilton,  1962).  Menden and Gremer  (1966) claimed t h a t i t i s p r e -  f e r a b l e t o d i g e s t the p r o t e i n w i t h a high c o n c e n t r a t i o n o f enzyme f o r a s h o r t p e r i o d , so t h a t a u t o h y d r o l y s i s o f enzymes becomes n e g l i g i b l e .  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  of Stahmann and Woldegiorgis 27 hours,  (1975) uses a d i g e s t i o n time o f  and an enzyme blank must be run along w i t h each  experiment.  T h i s blank value ranged between 0.5 85 and  1.42 6 yg o f l y s i n e p e r ml t h a t was s u b s t r a c t e d from the sample v a l u e . Table as determined  8 summarizes the r e s u l t s f o r a v a i l a b l e  lysine  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  r e s u l t s i n d i c a t e 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 o b t a i n e d between the enzymatic  t e s t and the a v a i l a b l e l y s i n e  by the FDNB o f f i c i a l method.  determined  This result i s i n close  ment w i t h the f i n d i n g o f Stahmann and Woldegiorgis  agree-  (1975) who  r e p o r t e d 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 0.99 74 between the a v a i l a b l e l y s i n e determined  by the m o d i f i e d Carpenter method  (Booth, 1971) and the l y s i n e r e l e a s e d by the d i g e s t i v e enzymes. Bujard e t a l . (I967) a l s o r e p o r t e d a h i g h c o r r e l a t i o n f o r the a v a i l a b l e l y s i n e as determined of Mauron e t a l . ( 1 9 5 5 ) .  by the p e p s i n - p a n c r e a t i n method  "by i n v i v o f e e d i n g 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  subjected to pepsin-pancreatin digestion.  Experiment S amp l e  No.  1  2  3  Ave  values  Casein  3. 463  4. 181  3. 994  3. 879  3. 8  B-lactoglobulin  5. 374  5. 690  6. 0111  5.692  1.5 N H C l  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  Gluten  1  2.74+0.13 2.r  The r e s u l t s a r e 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 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 . From: Stahmann a n d 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 method. F r o m : M a u r o n , 1970.  pancreatin digest dyalisate  1  109.  Carpenter method, f o r a s e r i e s o f heat t r e a t e d milk Stahmann and Woldegiorgis  (19 75)  samples.  demonstrated t h a t  only the enzymatic method was able t o show an 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 f o l l o w i n g steaming o f soybean meal. w e l l known t h a t raw soybeans c o n t a i n  It is  a t r y p s i n i n h i b i t o r , and  the i n c r e a s e i n the l i b e r a t i o n o f methionine, l y s i n e , a r g i n i n e and h i s t i d i n e confirms t h a t steaming destroys  the i n h i b i t o r .  I t i s apparent t h a t the chemical methods f o r p r o t e i n q u a l i t y may not r e v e a l the a v a i l a b i l i t y o f l y s i n e when the p r o t e i n s t e s t e d c o n t a i n  an i n h i b i t o r o f one o f the  p r o t e o l y t i c enzymes that a c t d u r i n g  digestion.  Sometimes, i t might be d i f f i c u l t  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 y s i n c e the l a t t e r  could  a l s o i n v o l v e p r o t e i n d i g e s t i b i l i t y which may n o t be r e l a t e d t o the r e a c t i v i t y o f the e-amino group o f l y 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 d e t e r m i n e d b y t h e FDNB d i f f e r e n c e  p r o c e d u r e , t h e DNBS a n d  TNBS m e t h o d s , a n d 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 shown i n T a b l e 9. regression  l y s i n e as  test i s  F o r comparison o f t h e methods, t h e  l i n e s and t h e 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 t h e 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 . , 1 9 7 5 ) .  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  regression  the  reference, i s not subject to error.  always t r u e .  l i n e , assumes t h a t  one o f t h e methods, used as However, t h i s i s n o t  Deming"s p r o c e d u r e h a s t h e a d v a n t a g e t h a t  e s t i m a t e s a r e o b t a i n e d f o r t h e random e r r o s o f b o t h m e t h o d s . Good a g r e e m e n t was o b t a i n e d b e t w e e n t h e FDNB m e t h o d a n d 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 of the  0.989  (Figure  12).  FDNB d i f f e r e n c e  0.988  was f o u n d  coefficient  When t h e TNBS m e t h o d was c o m p a r e d t o  technique, a correlation  coefficient of  (Figure 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 b y t h e enzymes u n d e r t h e specified  official  f o r the t e s t .  A correlation  conditions  c o e f f i c i e n t o f 0.995  was f o u n d b e t w e e n t h e FDNB m e t h o d a n d t h e e n z y m i c t e s t (Figure 14). F i g u r e s 1 5 , 16 a n d 17 show t h e r e g r e s s i o n  lines f o r  c o m p a r i s o n b e t w e e n t h e e n z y m a t i c t e s t a n d t h e DNBS m e t h o d ( r = 0 . 9 9 8 ) , t h e e n z y m a t i c t e s t a n d t h e TNBS m e t h o d  ( r = 0.987)  111.  TABLE 9.  Lysine a v a i l a b i l i t y procedure, enzymatic  d e t e r m i n e d by t h e FDNB  official  t h e DNBS a n d t h e TNBS m e t h o d s , a n d t h e test.  Enzymatic  S ample  FDNB  DNBS  TNBS  Gluten  1.55  1. 75  1. 46  0.56  Casein  7. 35  7. 52  7. 14  3.8  Lysozyme  5.15  5.43  5.73  2.31  11. 27  12. 26  13.93  5. 69  6.55  5.48  6. 71  (3-lactoglobulin Whole e g g  The v a l u e s a r e g i v e n i n g p e r 100 g o f p r o t e i n .  2.77  Test  112.  12.  C o m p a r i s o n b e t w e e n t h e FDNB d i f f e r e n c e and t h e DNBS m e t h o d .  The $ o f l y s i n e  mined b y g l y s i n e / 1 0 0 g p r o t e i n .  FDNB  (%  lysine)  technique i s deter-  113.  URE 1 3 .  C o m p a r i s o n b e t w e e n t h e FDNB d i f f e r e n c e and  t h e TNBS method.  mined by g l y s i n e / 1 0 0  FDNB  technique  The % o f l y s i n e i s d e t e r g protein.  (% l / s i n e )  114.  FIGURE 14.  Comparison and  b e t w e e n t h e FDNB d i f f e r e n c e  the enzymatic  lysine  digestion  i s determined  FDNB  test.  The % o f  by g l y s i n e / 1 0 0 g  (% l y s i n e )  method  protein.  115.  FIGURE 1 5 .  Comparison between the enzymatic d i g e s t i o n and t h e DNBS method.  The % o f l y s i n e  mined by g l y s i n e / 1 0 0 g  ENZ.  TEST  (%  protein.  lysine)  test  i s deter-  116.  FIGURE 16.  C o m p a r i s o n "between t h e e n z y m a t i c and  t h e TNBS method.  mined by g l y s i n e / 1 0 0  ENZ.  digestion  The % o f l y s i n e g  TEST (%  protein.  lysine)  test  i s deter-  117.  FIGURE 17.  C o m p a r i s o n b e t w e e n t h e TNBS and t h e DNBS methods. The % o f l y s i n e  i s determined by g l y s i n e / 1 0 0  protein.  15  r  = 0  995  10c v>  cn m z o  o  gluten lysozyme  A  egg  •  casein  •  0-lactoglobulin  10 TNBS  (%lysine)  15  g  118.  and  t h e TNBS and DNBS method  (r = 0.995),  The s t a n d a r d d e v i a t i o n method i s shown i n T a b l e 10. test)  showed t h a t  repeatability significant official  respectively.  o f t h e random e r r o r  A multiple  range  t h e TNBS method p r o d u c e d  test  f o r each (Duncan's  s i g n i f i c a n t l y lower  as compared t o t h e o t h e r t h r e e methods.  difference  No  i n p r e c i s i o n was f o u n d among t h e FDNB  t e c h n i q u e , t h e DNBS method and t h e e n z y m a t i c  test.  119.  TABLE 10.  Standard deviation 100  g protein)  o f t h e random e r r o r  (in g lysine/  i n t h e a v a i l a b l e l y s i n e m e t h o d s , as  e s t i m a t e d b y Deming's p r o c e d u r e .  ^. , Methods  Random e r r o r e s t i m a t e s i n method  compared  FDNB  DNBS  FDNB and DNBS  0.57 7  0.546  FDNB a n d TNBS  0.366  FDNB a n d E n z .  0.419  TNBS  Enzymatic  0.663 0.187  TNBS a n d DNBS  0.363  Enz.  a n d DNBS  0.256  Enz.  a n d TNBS  0.672 0.308 0.683  0.399  120.  CONCLUSION From t h e r e s u l t s p r e s e n t e d seen t h a t gluten, the  lysine availability  i n casein,  FDNB d i f f e r e n c e  agreement w i t h technique  the reported  values.  most a c c u r a t e means p r e s e n t l y  digestion  Although the d i f f e r e n c e  available  acid  i t i s probably the f o r determination of  hydrolysis.  lysine released  correlated  i n close  I t i s n o t a f f e c t e d by the d e s t r u c t i o n  of €-DNP-lysine during The  as d e t e r m i n e d by  t e c h n i q u e gave r e s u l t s  i s e x t r e m e l y time consuming,  availability,  i t c a n be  acid solubilized  egg, ( 3 - l a c t o g l o b u l i n and lysozyme,  official  lysine  i n this thesis  well  by the p e p s i n - p a n c r e a t i n  with the a v a i l a b l e  lysine  as  deter-  m i n e d w i t h t h e FDNB c h e m i c a l method. The  DNBS and TNBS methods gave r e s u l t s i n c l o s e  agreement w i t h  t h e r e s u l t s o b t a i n e d w i t h t h e FDNB  official  t e c h n i q u e . However, t h e d a t a f r o m t h e TNBS method showed a wide r a n g e o f 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 l o w e r r e p e a t i b i -  lity. The ficity  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 -  o f DNBS f o r t h e €-amino g r o u p o f l y s i n e , showed  DNBS r e a c t s  w i t h t h e -X-amino g r o u p o f l y s i n e , t o a c e r t a i n  e x t e n t . However, f o r p r o t e i n s contribution negligible.  that  and h i g h e r p o l y p e p t i d e s t h e  o f t h e cK-amino g r o u p t o t h e r e s u l t s becomes  121.  Only f o r s m a l l peptides having N-terminal  l y s i n e w i l l the  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  of available  lysine. 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Therefore, by a d d i n g  d i l u t e t h e 4 . 3 % DNBS s o l u t i o n up t o 1 3 1 . 1 5 m l 3 9 - 6 5 ml d i s t i l l e d  water.  

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