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Covalent binding of methionine and tryptophan to soy protein Voutsinas, Leandros Panagis 1978

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COVALENT BINDING OF METHIONINE AND TRYPTOPHAN TO SOY PROTEIN  fey LEANDROS PANAGIS VOUTSINAS B.Sc., U n i v e r s i t y o f T h e s s a l o n i k i , 1969 A THESIS SUBMITTED IN PARTIAL FULFILLMENT THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES (Dept. o f Food Science)  " We accept t h i s t h e s i s as conforming to the r e q u i r e d standard  THE UNIVERSITY OF BRITISH COLUMBIA October,  @  1978  Leandros Panagis V o u t s i n a s ,  1978  In p r e s e n t i n g t h i s  thesis  in p a r t i a l  f u l f i l m e n t o f the requirements f o r  an advanced degree at the U n i v e r s i t y of B r i t i s h Columbia, the I  Library shall  make i t  freely available  f u r t h e r agree t h a t p e r m i s s i o n  for  r e f e r e n c e and  f o r e x t e n s i v e copying o f  this  that  study. thesis  s c h o l a r l y purposes may be granted by the Head of my Department or  by h i s of  for  I agree  this  representatives. thesis  It  is understood that copying or p u b l i c a t i o n  f o r f i n a n c i a l gain s h a l l  not be allowed without my  written permission.  Leandros P. Voutsinas  Department of The  Food Science  University of B r i t i s h  2075 Wesbrook P l a c e Vancouver, Canada V6T 1W5  Date  October 23, 1978  Columbia  ii.  ABSTRACT  One common method of improving the n u t r i t i o n a l quality of certain food proteins i s through f o r t i f i c a t i o n with necessary of l i m i t i n g essential  amino acids.  method, however, i s not the best.  amounts  This simple and convenient Several disadvantages are asso-  ciated with the addition of free amino acids to food proteins, such as changes i n flavor and color, losses of added amino acids during food processing or cooking, differences  i n s t a b i l i t y and metabolism  between free amino acids and amino acids i n proteins.  Covalent  attachment of the l i m i t i n g amino acids, however, should eliminate these problems, and moreover, could improve the n u t r i t i o n a l and functional properties of food proteins.  In this study,  therefore,  an attempt to improve the n u t r i t i o n a l value of the soy protein was made by using the carbodiimide condensation reaction to covalently bind methionine and tryptophan to soy protein.  In order to confine  as much as possible the binding, of amino acid to the protein a-carboxyl groups the soy protein i s o l a t e (SPI) was p a r t i a l l y hydrolyzed with pepsin to increase the number of a-carboxyls i n soy protein.  Various  conditions of the carbodiimide reaction were analysed by a f r a c t i o n a l f a c t o r i a l design i n an attempt to determine the factors affecting amino acid binding to soy protein hydrolysate (SPH). investigated,  Of the  the  factors  pH, SPH concentration, carbodiimide concentration,  a c t i v a t i o n time and reaction time were found to s i g n i f i c a n t l y the methionine binding e f f i c i e n c y ,  affect  whereas pH, SPH concentration,  carbodiimide concentration, amino acid concentration and reaction temperature were found to s i g n i f i c a n t l y influence the tryptophan binding efficiency  to SPH.  To determine the best l e v e l for each of the selected  factors an optimization of the carbodiimide reaction conditions was conducted by carrying out another f a c t o r i a l experiment.  Thus, under  the best condition found, the methionine and tryptophan contents of methionine - and tryptophan-bound SPH samples were increased and 18.0-fold,  7.7-fold  respectively.  An jin v i t r o pepsin-pancreatin digestion the bound amino acids were readily  test demonstrated that  released.  In order to improve the low y i e l d of the f i n a l product, another analysis of the carbodiimide reaction conditions was carried out.  Since  the y i e l d could not be markedly improved by this f a c t o r i a l design i n which peptic SPH was used, SPI without preliminary hydrolysis was used as the starting material.  A product with 95-99% y i e l d was obtained  and i t s methionine or tryptophan content was increased 6.3-fold 11.3-fold,  respectively.  or  High d i g e s t i b i l i t y was s t i l l maintained for  these products. Gel f i l t r a t i o n chromatography demonstrated that the carbodiimide reaction caused an increase fractions.  i n the molecular weights of soy protein  Furthermore, gel f i l t r a t i o n chromatography revealed  there was no selective amino acid binding among the different fractions during the carbodiimide reaction.  that.,  soy protein  iv.  TABLE OF CONTENTS PAGE INTRODUCTION  1  LITERATURE REVIEW  5  A. The chemistry of carbodiimides B. Physical and chemical properties of soybean proteins a. S o l u b i l i t y b. Molecular size d i s t r i b u t i o n c. Association-Dissociation reactions d. Subunit structure e. Amino acid composition MATERIALS AND METHODS A. B. C. D. E. F. G. H. I. J.  Materials Preparation of soy protein hydrolysate S t a t i s t i c a l analysis Covalent binding of methionine and tryptophan to soy protein hydrolysate Methionine determination Tryptophan determination Nitrogen determination Amino acid analysis Enzymatic digestion test Gel f i l t r a t i o n chromatography 1. Preparation and operation 2. S o l u b i l i z a t i o n of samples  RESULTS AND DISCUSSION A. B. C. D.  Preparation of soy protein hydrolysate S t a t i s t i c a l analysis Amino acid analysis Enzymatic digestion test  E. Gel f i l t r a t i o n chromatography  5 14 14 16 17 17 19 20 20 21 21 21 22 23 23 23 24 24 24 25 26 26 27 61 64 67  GENERAL DISCUSSION  80  CONCLUSION  83  LITERATURE CITED  85  APPENDIX  95  V.  LIST OF TABLES  TABLE  PAGE  1  F a c t o r s and t h e i r a s s i g n e d l e v e l s i n v e s t i g a t e d f o r t h e i r p o s s i b l e i n f l u e n c e on amino a c i d b i n d i n g to SPH.  28  2  A n a l y s i s of v a r i a n c e of the 2 - l e v e l f r a c t i o n a l f a c t o r i a l experiment f o r methionine b i n d i n g to SPH.  29  3  A n a l y s i s of v a r i a n c e of the 2 - l e v e l f r a c t i o n a l f a c t o r i a l experiment f o r t r y p t o p h a n b i n d i n g to SPH.  30  4  Assigned l e v e l s f o r f a c t o r s a c i d b i n d i n g to SPH.  40  5  A n a l y s i s of v a r i a n c e of the 3 - l e v e l f a c t o r i a l experiment f o r methionine b i n d i n g to SPH.  41  6  A n a l y s i s of v a r i a n c e of the 3 - l e v e l f a c t o r i a l experiment f o r tryptophan b i n d i n g to SPH.  42  7  F a c t o r s a f f e c t i n g amino a c i d b i n d i n g and t h e i r best l e v e l s .  56  8  A s s i g n e d l e v e l s f o r f a c t o r s i n v e s t i g a t e d by a 3 - l e v e l f a c t o r i a l experiment f o r t h e i r p o s s i b l e i n f l u e n c e on f i n a l p r o d u c t ' s y i e l d .  57  9  A n a l y s i s o f v a r i a n c e of the 3 - l e v e l f a c t o r i a l experiment f o r s e l e c t i o n of the f a c t o r s i n f l u e n c i n g the methionine-bound SPH y i e l d s and o p t i m i z a t i o n .  58  10  A n a l y s i s of v a r i a n c e of the 3 - l e v e l f a c t o r i a l experiment f o r s e l e c t i o n of the f a c t o r s i n f l u e n c i n g the tryptophan-bound SPH y i e l d s and o p t i m i z a t i o n .  59  11  F a c t o r s a f f e c t i n g f i n a l p r o d u c t ' s y i e l d and t h e i r best l e v e l s .  60  12  Amino a c i d c o m p o s i t i o n (g/16gN) of SPI and amino a c i d - b o u n d soy p r o t e i n samples w i t h the h i g h e s t coupling y i e l d s .  62  i n f l u e n c i n g the amino  Liberated amino acid (g/16gN) from SPI and amino acid-bound soy protein samples with the highest coupling yields by pepsinpancreatin digestion iji v i t r o . Proteins used i n c a l i b r a t i o n of Sephadex G-200 column eluted with 0.1M sodium phosphate buffer, pH 7.0, and containing 0.1% SDS.  vii.  LIST OF FIGURES  FIGURE  PAGE  1  A mechanism proposed by Khorana (1955) f o r the c a r b o d i i m i d e - m e d i a t e d p e p t i d e s y n t h e s i s .  6  2  E f f e c t of pH on the r a t e of methionine and t r y p t o p h a n b i n d i n g to SPH.  32  3  E f f e c t of SPH c o n c e n t r a t i o n on the r a t e of methionine and t r y p t o p h a n b i n d i n g to SPH.  33  4  E f f e c t of EDC c o n c e n t r a t i o n on the r a t e of methionine and t r y p t o p h a n b i n d i n g to SPH.  34  5  E f f e c t of a c t i v a t i o n time on the r a t e of methionine b i n d i n g to SPH.  35  6  E f f e c t of r e a c t i o n time on the r a t e of methionine b i n d i n g to SPH.  36  7  E f f e c t of amino a c i d (tryptophan) c o n c e n t r a t i o n on the r a t e of t r y p t o p h a n b i n d i n g to SPH.  37  8  E f f e c t of r e a c t i o n temperature on the r a t e of t r y p t o p h a n b i n d i n g to SPH.  38  9  E f f e c t of pH on the r a t e of methionine and t r y p t o p h a n b i n d i n g to SPH.  43  10  E f f e c t of SPH c o n c e n t r a t i o n on the r a t e of m e t h i o n i n e and tryptophan b i n d i n g to SPH.  46  11  E f f e c t of EDC c o n c e n t r a t i o n on the r a t e of m e t h i o n i n e and t r y p t o p h a n b i n d i n g to SPH.  47  12  E f f e c t of amino a c i d (tryptophan) c o n c e n t r a t i o n on the r a t e of tryptophan b i n d i n g to SPH.  48  13  E f f e c t of a c t i v a t i o n time on the r a t e of methionine b i n d i n g to SPH.  50  14  E f f e c t of r e a c t i o n time on the r a t e of methionine b i n d i n g to SPH.  52  15  E f f e c t of r e a c t i o n temperature on the r a t e of t r y p t o p h a n b i n d i n g to SPH.  54  C a l i b r a t i o n curve of Sephadex G-200. Gel f i l t r a t i o n pattern of SPH s o l u b i l i z e d with SDS on Sephadex G-200. Gel f i l t r a t i o n pattern of tryptophan-bound SPH s o l u b i l i z e d with SDS on Sephadex G-200. Gel f i l t r a t i o n patterns of SPH and tryptophan-bound SPH on Sephadex G-200. Gel f i l t r a t i o n pattern of SPH s o l u b i l i z e d with NaOH (at pH 12.0) on Sephadex G-200. Gel f i l t r a t i o n pattern of methionine-bound SPH s o l u b i l i z e d with NaOH (at pH 12.0) on Sephadex G-200. Gel f i l t r a t i o n patterns of SPH and methionine-bound SPH on Sephadex G-200. Gel f i l t r a t i o n pattern (A280nm) ° ^ tryptophan-bound SPH s o l u b i l i z e d with NaOH (at pH 12.0) on Sephadex G-200. Gel f i l t r a t i o n pattern (A2]_0nm) ° ^ tryptophan-bound SPH s o l u b i l i z e d with NaOH (at pH 12.0) on Sephadex G-200.  ACKNOWLEDGEMENTS  The author wishes t o express h i s s i n c e r e a p p r e c i a t i o n t o h i s s u p e r v i s o r , Dr. S. Nakai, P r o f e s s o r , Department of Food S c i e n c e , f o r h i s constant a d v i s e , h e l p and encouragement throughout t h e course of t h i s study, and i n t h e p r e p a r a t i o n of t h e t h e s i s . He  i s a l s o t h a n k f u l t o t h e members of h i s graduate  Dr. W. D. Powrie, Head, Department of Food  committee:  Science  Dr. J . F. R i c h a r d s , P r o f e s s o r , Department of Food  Science  Dr. J . Vanderstoep, A s s i s t a n t P r o f e s s o r , Department of Food  Science  for their interest  i n t h i s r e s e a r c h and f o r t h e review o f t h i s  thesis. Financial  aid, gratefully  acknowledged, was p r o v i d e d by Canada  Department of A g r i c u l t u r e through  support  of the p r o j e c t .  1.  INTRODUCTION  The e f f i c i e n t use of a p r o t e i n by man or animal r e q u i r e s it  contains  the e s s e n t i a l amino a c i d s as w e l l as n i t r o g e n i n  and p r o p o r t i o n s needed by the organism to meet i t s as w e l l as f o r g e n e r a l p h y s i o l o g i c a l however, t h a t w i t h the p o s s i b l e  functions.  needs f o r  It  that  amounts specific  i s w e l l known,  e x c e p t i o n of o n l y a v e r y few, most  p r o t e i n s do not c o n t a i n the e s s e n t i a l amino a c i d s p r o p o r t i o n s needed by man or a n i m a l .  Therefore,  i n the amounts  and  their u t i l i z a t i o n  tends to be i n e f f i c i e n t , p a r t i c u l a r l y f o r p r o t e i n foods d e r i v e d from the v e g e t a b l e kingdom  (Bressani,  of a p r o t e i n source i s u s u a l l y  1975).  The amino a c i d d e f i c i e n c i e s  overcome ( b a l a n c e d ) ,  as has been w e l l  documented, by the a d d i t i o n of the a p p r o p r i a t e amounts of the d e f i c i e n t amino a c i d s ; compounds  these can be i n the form of e i t h e r c r y s t a l l i n e  ( p r o t e i n f o r t i f i c a t i o n or complementation)  or as  (synthetic) constituents  of p r o t e i n s which are r i c h sources of the d e f i c i e n t amino a c i d s supplementation).  A d d i t i o n of s y n t h e t i c amino a c i d s r e s u l t s  (protein  i n increased  p r o t e i n q u a l i t y due to the improvement of the e s s e n t i a l amino a c i d p a t t e r n and due o n l y to t h a t , involved  (Bressani,  amino a c i d s  1974).  s i n c e t h e r e are no o t h e r  On the o t h e r hand, a d d i t i o n of the d e f i c i e n t  as p r o t e i n can make changes  i n a l l t h r e e major  a f f e c t i n g p r o t e i n u t i l i z a t i o n , namely, p r o t e i n q u a l i t y , and the t o t a l energy v a l u e  (Pellet,  animal feeds  (Waddell,  1958).  parameters  protein quantity,  1974).  F o r t i f i c a t i o n w i t h amino a c i d s lating  parameters  is  the normal p r a c t i c e i n formu-  S t u d i e s by Howe et a l .  (1965A,  1965B, 1967) have demonstrated the improvement i n q u a l i t y which can be o b t a i n e d by f o r t i f y i n g foods w i t h amino a c i d s .  Bread supplemented w i t h  2.  lysine is a far superior source of protein than ordinary bread (Altschul, 1974).  It has been estimated, moreover, that 10,000-  20,000 tons of 1-lysine w i l l by 1980, be added to cereals intended for human food (Chem. Engng News, 1973).  The rapid p r o l i f e r a t i o n of  meat analogs from soy and other vegetable protein sources w i l l i n crease the use of methionine i n foods based on meat and.milk model (Horan, 1974B).  Bressani (1975), reported experimental results from  human subjects indicating that addition of the l i m i t i n g amino acids represents not only a better protein n u t r i t i o n for the i n d i v i d u a l , but a significant  economic saving as w e l l , due to the  efficiency with which protein is u t i l i z e d .  increased  Although the interest i n  amino acid f o r t i f i c a t i o n of human foods i s certainly on the  increase,  the u t i l i z a t i o n of this option, at the present time, is not  sufficiently  used to make a significant  contribution to human protein supply.  f i c a t i o n with amino acids, however,  Forti-  is expected to become an essential  and regular adjunct of the human food system (Altschul, 1974).  In this  way, the technology w i l l contribute more f l e x i b i l i t y to counteract  the  pressures of greater populations and higher expectations. The f o r t i f i c a t i o n of food proteins with essential amino acids i s an important, simple and convenient method for improving their nutritional quality.  However, i t is not necessarily  of improving their n u t r i t i o n a l q u a l i t y .  the best method  This i s due to several defects  associated with the addition of free amino acids to food proteins. F i r s t l y , the added amino acids can easily be l o s t during the subsequent food processing,  cooking or through washing or discarding of the  (Bressani, 1975; Fujimaki _e_t a l . , 1977).  juices  Secondly, i t may easily occur  that the free amino acids suffer degradation or reaction with food ingredients to affect al.,  (e.g.  Strecker degradation or amino-carbonyl reaction)  the food q u a l i t i e s such as flavor and color (Yamashita et  1970).  T h i r d l y , considering the methionine f o r t i f i c a t i o n of  foods, i t s addition i s severely r e s t r i c t e d , because of the unattractive flavor which methionine, i n general, imparts to foods  (Klaui, 1974).  Furthermore, Beigler (1969) reported that methionine gives off an unpleasant odor upon high heating, such as s t e r i l i z a t i o n .  This finding  was recently confirmed by Hippe and Warthesen (1978) who reported that methionine f o r t i f i c a t i o n i n products that receive high heat treatment is not feasible from a flavor standpoint because of the formation of methional and dimethyl d i s u l f i d e .  A generally accepted reaction scheme  for the production of flavor compounds from methionine i s Strecker degradation (Ballance, 1961).  Methional, a product of this r e a c t i o n , has  been reported to have a flavor threshold i n the ppb range (Patton and Josephson, 1957).  Fourthly, a difference i n s t a b i l i t y between free  methionine and methionine i n peptides has been reported by A r a i (1974) , suggesting the existence of different s t a b i l i t y c h a r a c t e r i s t i c s between free amino acids and amino acids covalently bound to proteins.  Fifthly,  the metabolism of amino acids ingested as proteins d i f f e r s somewhat from that of free amino acids.  More rapid i n t e s t i n a l transport of  amino acids from small peptides than from the equivalent amount of free amino acid or amino acid mixture has been shown to occur i n man and several other mammalian species by many techniques including tolerance t e s t s , the use of tied loops of intestine or i n t e s t i n a l perfusion (Craft et a l . , 1968; Matthews et a l . , 1968; A d i b i , 1971),  4.  and measurements of uptake or transmural transport by small in v i t r o (Cheng et a l . , 1971; Rubins and Auricchio, 1971).  intestine Thus,  differences have been reported in i n t e s t i n a l transport between o l i g o peptide methionine and free methionine (Lis e_t a l . , 1972) as well as between oligopeptide lysine and free lysine (Burston et a l , , 1972). One possible method for removing or lessening the above defects is to supplement the desirable amino acids i n a form covalently bound with proteins or peptides i n foods. The high n u t r i t i v e value of the soybean protein has been recognised for many years and has been responsible for the enormous growth of i t s use in the animal feed industry (Horan, 1974A).  Insofar  as the food industry is concerned, soy proteins u n t i l recently were selected or used primarily for their functional effects i n imparting desirable properties to fabricated foods.  Now, hoever, they are being  incorporated for their n u t r i t i o n a l value (Wolf, 1972A).  Soy protein  offers the only expanding commercial source of protein able to meet the growing demand for a n u t r i t i o n a l l y balanced, high protein food suitable for both human and animal feeding (Coppock, 1974).  Soy  protein i s a good source of a l l the essential amino acids except methionine and tryptophan (Wolf, 1972A). The s p e c i f i c objectives,  therefore, of this work were:  (1)  Optimization of the carbodiimide condensation reaction used as a means for covalently binding methionine and tryptophan to soy protein; (2) evaluation of the n u t r i t i o n a l quality of the chemically modified soy protein obtained under the best condition; and (3) determination of the effect,  i f any, of the carbodiimide condensation reaction on the molecular  weight d i s t r i b u t i o n pattern of the soy protein.  5.  LITERATURE REVIEW  A.  The chemistry of carbodiimides In 1955 Sheehan and Hess introduced a new method for the formation  of peptide bonds using N,N'-dialkylated carbodiimides, preferably N , N ' dicyclohexylcarbodiimide.  This reagent became, i n a short time,  most important coupling reagent.  the  The carbodiimide method can be called  the most popular method of peptide synthesis.  The main reason for i t  has probably been the high r e a c t i v i t y of carbodiimides, since  this  assures that the desired coupling reactions proceed smoothly, usually to completion of the acylation within a short time (Bodanszky and Ondetti, 1966).  In addition to t h i s ,  the s i m p l i c i t y and convenience  of this technique as well as the commercial a v a i l a b i l i t y of the reagent are also an explanation for this unprecedented popularity. is s l i g h t 1975)  (Anderson and Callahan, 1958)  to n e g l i g i b l e  and this fact offers an additional advantage.  Racemization  (Rebek and F e i t l e r , The carbodiimide  condensation reaction can be expresssed i n the following equation:  R-COOH+H N-R' + R"-N=C=N-R" + R-CONH-R' + R"-NH-CO-NH-R"  [1]  2  The reaction mechanism was f i r s t discussed by Khorana (1953, 1955) and was more recently thoroughly reinvestigated by Detar and his coworkers (1966A,B,C).  Khorana (1953) suggested that the reaction proceeds through  the activation of the carboxyl group (I),  i n i t i a t e d by the addition of  i t s proton to the double bond of the carbodiimide(II), followed by the attack of the carboxylate anion to form the unstable reactive i n t e r mediate ( I I I ) , the O-acylisourea ( F i g . l ) .  This O-acylisourea(III),  in  turn, can undergo either an intramolecular rearrangement, v i a an oxygen to nitrogen (CKN) acyl transfer, to an N-acylurea(V) or conversion to two  r o II  +  R-C-OH + N-R'  II  [I]  R-C-0  o II  N-R'  C  I  II  N-R'  II  R-C-O-C  I  ,  NH-R:'  H-N-R'  [II]  [III]  N-R'  II  „  NH-R' RCOO R-C-O-C  'I  l-C-O-C I NH-R*  NH-R'  [III]  NH-R' C=0  R-C=0 \ 0 / R-C=0  +  NH-R' [Va]  [IV]  [VI]  (a) H„N-R" NH-R' H N-R" ^ R-CO-NH-R" + C=0  (b) ] H N-R" 2  X  0 II  R-C-N-R'  I  0=C  I  NH-R' [V]  7T  *  [VII]  I  NH-R'  R-CO-NH-R" [VII] +  [Vb] M  R-C-OH  —  [I]  Fig.  1.  A mechanism proposed by Khorana (1955) for the carbodiimidemediated peptide synthesis.  7.  activated forms (IV and VI) of the o r i g i n a l carboxyl component carboxylic acids, peptides or proteins). mentioned p o s s i b i l i t i e s ,  (e.g.  In addition to the above  Franzblau et_ a l .  (1963) suggested that i n  certain cases, the 0-acylisourea could c y c l i z e .  The f i r s t a l t e r n a t i v e ,  that i s the formation of N-acylurea, has a chance to occur when there are no further protons available (Schroder and Lubke, 1965) or when no other nucleophile is present or the reaction of those present i s slow or hindered (Kopple, 1966).  If excess carboxyl component i s the only a v a i l -  able reactant a second addition of proton takes place and the 0->-N acyl migration w i l l be prevented. S t a b i l i z a t i o n occurs by way of formation of the symmetrical anhydride (VI) and disubstituted urea (Va).  Although the  intramolecular rearrangement of O-acylisourea i s probably the most common route to the formation of N-acylureas, an alternative mechanism for their production, namely, by the subsequent acylation of the i n i t i a l l y formed disubstituted urea (Va) by the acid anhydride (VI), was discovered i n experiments carried out by Smith et^ a l .  (1958).  It has a chance to occur  when carbodiimide and carboxyl component are kept for a long time at room temperature and no other reactants are present.  This p o s s i b i l i t y , however,  has been subsequently excluded by De Tar and S i l v e r s t e i n (1966A). The synthesis of peptide derivatives  (VII), according to Khorana  (1955), may proceed through one or more of the following three routes: (a)  (IV)+NH2-R" -* (VII), since O-acylisourea has been found to be sensitive  to bases and reacts readily with nucleophiles  (Khorana, 1952); (b) acid  anhydride (VI)+NH -R" -> (VII); and (c) N-acylurea (V)+NH -R" + (VII). 2  2  Support for the above mechanism has been provided by Doleschall and Lempert (1963), who isolated a c y c l i c O-acylisourea and examined i t s reactions with  8.  different  nucleophiles.  The N-acylureas of the type (V) appear to be quite stable and show l i t t l e tendency to undergo condensation with an amino acid ester under mild conditions and thus conversion into desired peptide d e r i vative (VII), (Khorana, 1953).  It has been subsequently suggested,  however, that N-acylureas are not activated compounds and therefore are unreactive toward amines and do not form peptide bonds.  The N-acylurea  (path c) has, thus, been excluded as an acylating agent (Diehl, 1957; De Tar and S i l v e r s t e i n , 1966A, Klausner and Bodansky, 1972). Although the O-acylisourea is highly unstable and has not been isolated except i n one very special case (Doleschall and Lempert, 1963), i t s occurrence i s inferred from k i n e t i c data and from the appearance i n the reaction mixture of both the urea of the carbodiimide (as a by-product of peptide synthesis) and the N-acylurea resulting from the rearrangement of the proposed O-acylisourea (Steinman et^ a l . , 1966) .  Recently a mechani-  s t i c study of the carbodiimide-mediated peptide synthesis has been reported by Rebek and F e i t l e r (1973), who have determined the reaction  intermediates.  Their evidence indicated that the i n i t i a l acylating agent i n the carbodiimide reaction was distinguishable from the carboxylic acid anhydride under conditions normally encountered during peptide synthesis i n solution. Their results ind icated a maximum of 55% (5°C) to 60% (30°C) as the extent of anhydride's p a r t i c i p a t i o n during peptide synthesis i n solution with N,N'-dicyclohexylcarbodiimide.  Delayed addition of the amine component  was found to decrease the importance of O-acylisourea (path a) and increase the extent of anhydride's p a r t i c i p a t i o n (path b) i n peptide synthesis. One advantage of N,N'-dicyclohexylcarbodiimide claimed by Sheehan  9.  and Hess (1955) was the apparent lack of racemization i n the formation of o p t i c a l l y active peptide derivatives.  However, the fact that coupling  reactions promoted by carbodiimides may sometimes be attended by appreciable racemization was indicated by the finding of Hofmann e_t a l . (1958) and was promptly reaffirmed by Anderson and Callahan (1958), who have demonstrated an influence of solvent and temperature on the racemization. Low temperatures (DL-form).  (-5°C) reduce s i g n i f i c a n t l y the amount of racemic mixture  Recently Rebek and F e i t l e r (1975), however, working on the  peptide synthesis with carbodiimide, reported that racemization was i n the range of 0.01 to 0.1%.  From the racemization point of view, symmetrical  anhydrides may be undesirable intermediates,  since racemization might be  expected from the intermediate anhydride on the basis of the ready azlactone formation (Bodanszky and Ondetti, 1966).  Intermediary azlactone  formation is the most l i k e l y course that racemization takes when occurring during a condensation reaction (Schroder and Lubke, 1965). used dicyclohexylcarbodiimide reagent,  With the widely  racemization increases with temper-  ature and is more pronounced i n polar solvents such as dimethylformamide than i n nonpolar, e . g . , efficient  C ^ C ^ (Bodanszky et_ a l . , 1976).  The most  method to reduce racemization i n coupling with carbodiimide is  to use additives such as N-hydroxysuccinimide or 1-hydroxybenzotriazole.. Unfortunately the use of additives cannot be taken as a guarantee  against  racemization (Bodanszky e_t a l . , 1976) . The undesired formation of. N-acylurea is frequently observed (Khorana, 1955; M e r r i f i e l d and Woolley, 1956; Shankman and Schvo, 1958; and Sheehan e_t al^., 1956).  It is dependent upon a number of factors.  Low temperatures  reduce i t s formation (Helferich and Boshagen, 1959; Schneider, 1960); therefore the reaction i s normally carried out at a temperature of 0°C  10.  or lower (du Vigneaud ^t_ a l . , 1957; Katsoyannis and Suzuki, Sheehan and Yang, 1958).  However,in a few cases the reaction  carried out at higher temperatures Previero et_ al.,  1973).  1962;  (Theodoropoulos and Folsch, 1958;  The formation of N-acylurea is suppressed i n  methylene chloride ( M e r r i f i e l d , 1958; M e r r i f i e l d and Woolley, Sheehan et a l . , 1956) et a l . , 1956).  is  or a c e t o n i t r i l e  1958;  (Benoiton and Rydon, 1960;  Sheehan  Khorana (1955) pointed out that the r e l a t i v e proportions  of N-acylureas formed during the coupling reaction w i l l depend largely upon the p a r t i c u l a r peptide derivative used and the solvent employed and that their formation even in r e l a t i v e l y small amounts may complicate i s o l a t i o n of the desired products i n a satisfactory y i e l d . too, that,  since the rearrangement of O-acylisourea(III)  (V) i s intramolecular whereas the reactions (III) (VII) are bimolecular (intermolecular),  the  Note was made  to the N-acylurea  -»- (IV) -> (VI) and (IV) •>  the formation of the desired  products should be favored by an increase i n the concentration of the reactants.  Tometsko (1973) i l l u s t r a t e d that the activated carboxyl  groups during peptide synthesis are not stable for long periods of time, since the formation of N-acylurea derivatives r e s u l t s i n a loss of a c t i vated carboxyl component and therefore decrease i n peptide bond formation. The i n a c t i v a t i o n i s a complex process that could also involve solvent impurities, temperature effects,  etc.  One problem associated with the use of the o r i g i n a l carbodiimides, p a r t i c u l a r l y N,N'-dicyclohexylcarbodiimide, was the p u r i f i c a t i o n of the desired products.  On one hand, the N-acylureas have structures and  s o l u b i l i t y rather similar to those of the desired peptides and therefore i t i s sometimes d i f f i c u l t to remove N-acylureas i n order to obtain a pure  11.  product.  On the other hand, as pointed out by Khorana (1955), the  insoluble co-product (dicyclohexylurea) which i s formed during the implication of dicyclohexylcarbodiimide as the condensing agent i n peptide syntheses could sometimes prove undesirable as, for example, in cases involving the synthesis of the less soluble higher polypeptides,  since the co-product and the peptide derivative may have  similar s o l u b i l i t y properties, thus complicating the i s o l a t i o n procedure. In order to overcome the p u r i f i c a t i o n problems caused by the formation of by-product ureas and N-acylureas, other carbodiimides with more favorable s o l u b i l i t y properties have also been investigated and Hlavka, 1956; Sheehan et a l . , 1961).  (Sheehan  With these reagents,  the by-  product ureas and N-acylureas as well as any excess of reagent are easily removed by washing with d i l u t e acid or water.  Peptides have been  synthesized  in high yields and i n very pure forms with these carbodiimides (Sheehan jit al.,  1961).  The most generally useful of them have been found to be 1-  ethyl-3-(3-dimethylaminopropyl) carbodiimide (hereafter  abbreviated to  EDC) and the corresponding hydrochloride s a l t (Sheehan et a l . , 1961). These and the other water-soluble carbodiimides (WSC) are commercially available and the reaction can s t i l l be carried out under mild conditions (Greenstein and Winitz, 1961).  The reaction mechanism of these water-  soluble carbodiimides has been assumed to be similar to that proposed i n 1953 by Khorana (Hoare and Koshland, 1966).  Although a l l water-soluble  carbodiimides react s i m i l a r l y , the smaller reagents might be expected have greater access to p a r t i a l l y buried carboxyl groups. This  to  expectation  appeared correct i n a recent study with tobacco mosaic v i r u s protein (Means and Feeney, 1971), i n which upto three additional carboxyl groups could be  modified with EDC which did not react with l-cyclohexyl-3-(2-morpholinyl4-ethyl) carbodiimide metho-p-toluenesulfonate used water-soluble carbodiimide) .  (the other most widely  Sheehan ejt a l . , (1965) working with  EDC demonstrated that the synthesis of pure oligopeptides was extremely rapid and f a c i l e .  Although coupling was usually about 80% after 1 hr  at room temperature, the time required for complete coupling varied from 24 to 66 hr.  The use of EDC has continued to expand because of  i t s s u i t a b i l i t y to peptide synthesis and i t s high s t a b i l i t y . The reaction between a water-soluble carbodiimide and the functional groups of a protein could be both numerous and complex. interest,  therefore,  It i s of obvious  to review the reactions of these unusual reagents  with proteins. Sheehan and Hlavka (1957) published the f i r s t studies of the reaction between a protein and a water-soluble carbodiimide (in the absence of an added nucleophile), stating that the reaction produced c r o s s - l i n k i n g i n g e l a t i n . This c r o s s - l i n k i n g was presumably due to amide formation occuring between side-chain carboxyl and amino groups of the g e l a t i n molecule. On the other hand, by carrying out the reaction between a protein and a water-soluble carbodiimide at pH values  (pH 4.0),  coupling by nucleophilic groups (such as e-amino group of lysine)  where on the  protein should be t o t a l l y i n h i b i t e d , Franzblau e_t aJL. (1963) have noted that the reaction between g e l a t i n and a water-soluble carbodiimide, i n the presence of hydroxylamine as nucleophile, yielded hydroxamic acid derivatives of side-chain aspartic acid and glutamic acid residues. These workers also reported that a-carboxyl groups reacted more rapidly and quantitatively to produce hydroxamic acid derivatives than 6- or ycarboxyl groups which exhibited lower yields tively).  Of great interest  (about 50% and 40%, respec-  is the work of Previero e_t a l .  (1973), who  13.  reported that EDC could affect  the carboxyl groups of an a c i d i c peptide  i n different ways depending on their environment, namely, they demonstrated that the same reagent could be used for both s e l e c t i v e a c t i v a t i o n of the C-terminal and blocking of the side-chain carboxyl groups i n a single operation.  These workers, thus, presented evidence which indicated  that direct condensation without preincubation of an a c i d i c peptide with EDC resulted i n the coupling of two times as many amino acid ester moles as i n the case of condensation after preincubation with EDC for 90 min. at 40°C.  Their proposed explanation was that, when condensation took  place after preincubation, the O-acylisourea i n i t i a l l y formed isomerized to the inert N-acylurea derivative at the side-chain carboxyl group, while the C-terminal remained activated as an Oxazolinone (Azlactone) which could subsequently react with a suitable nucleophile.  Direct  condensation without preincubation, on the other hand, resulted i n the p a r t i c i p a t i o n of both C-terminal and side-chain carboxyl groups i n the formation of peptide bonds.  Riehm and Scheraga (1966), by studying the  reaction between ribonuclease and a water-soluble carbodiimide (in the absence of nucleophiles), have investigated a l l the possible between a protein and a water-soluble carbodiimide.  reactions  They demonstrated  that the reaction between ribonuclease and carbodiimide, at low pH (4.5) did not involve direct reaction between the reagent and amino or imidazole groups (nucleophilic groups) i n the protein.  This finding might be  expected since at pH 4.5 most ( i f not a l l ) of these groups would be protonated.  On the other hand, the reaction between the protein and  the water-soluble carbodiimide at high pH (9.5), followed by amino acid analyses,  resulted i n a product which contained fewer lysine  residues  14.  than did a ribonuclease hydrolysate.  Therefore, i t was concluded that  the reaction between a protein and a water-soluble carbodiimide, at high pH, results  i n the guanidination of e-amino groups, due to reaction  between carbodiimide and e-amino groups of lysine residues.  The same  authors also excluded any reaction between carbodiimide and side-chain hydroxyl groups of threonine, serine, and tyrosine residues. finding is i n accordance with the results of Sheehan j2t al.  This (1955, 1956),  who reported that the hydroxyl function of threonine, serine, and hydroxyproline does not require masking prior to condensation.  Hoare and  Koshland (1966) have used a water-soluble carbodiimide for the s e l e c t i v e modification of carboxyl groups i n proteins i n the presence of high concentration of nucleophiles.  They have reported a procedure leading  to rapid and quantitative modification of carboxyl groups under mild conditions.  Horinishi et a l . (1968) have also explored the reaction  of water-soluble carbodiimides with proteins i n the presence of nucleophiles.  They reported that with undenatured proteins, p a r t i a l reaction  i s commonly observed and seemed to be an i n d i c a t i o n of the degree of exposure of the carboxyl groups, since only the more accessible or reactive carboxyl groups reacted. (e.g.  In the presence of denaturants  guanidine), however, quantitative modification of protein carboxyl  groups could be obtained.  B. (a)  Physical and chemical properties of soybean proteins Solubility The majority of soy-proteins are c l a s s i f i e d as globulins.  proteins are insoluble i n water i n the region of their  Such  isoelectric  15.  points, but w i l l dissolve i n the i s o e l e c t r i c state when s a l t s such as sodium or calcium chloride are added.  If the pH is above or below the  i s o e l e c t r i c point, a globulin w i l l also dissolve i n aqueous in the absence of s a l t s (Wolf and Cowan, 1975). dispersed i n d i s t i l l e d water at pH 6.5, occurs.  solutions  When defatted meal i s  nearly maximum protein s o l u b i l i t y  Raising the pH with a l k a l i increases  the s o l u b i l i t y  slightly,  but adding acid reduces the s o l u b i l i t y abruptly to a minimum i n the range of pH 4-5, which i s the i s o e l e c t r i c region.  At lower pH values,  the proteins become p o s i t i v e l y charged and redissolve  (Wolf, 1972A).  S o l u b i l i t y of the soybean globulins is also influenced by the co-existence of phytate i n soybeans (Smith and Rackis, 1957).  Another factor that  affects s o l u b i l i t y of soybean proteins is d i s u l f i d e polymerization (Wolf, 1972B).  Commercial soyprotein isolates vary i n s o l u b i l i t y because of  processing variations (Nash and Wolf, 1967).  Part of the i n s o l u b i l i t y  of isolates arises during the i s o e l e c t r i c p r e c i p i t a t i o n step (Nash et al.,  1971).  One reaction responsible for this i n s o l u b i l i t y appears to  be the formation of protein polymers linked by d i s u l f i d e bonds.  When  the isolates are treated with sulfhydryl compounds such as cysteine or mercaptoethanol, depolymerization occurs and p a r t i a l s o l u b i l i t y regained.  is  A portion of the i s o l a t e s , however, remains insoluble even  with added sulfhydryl compounds (Nash e_t a l . , 1971).  S o l u b i l i t y of the  proteins i s often required i n order to obtain desired functional properties, and because soluble ingredients are always easier to formulate into Consequently, almost a l l soy protein concentrates  and isolates are neutra-  l i z e d and sold as proteinates, with sodium proteinate the major form available.  foods.  16.  (b)  Molecular size d i s t r i b u t i o n Undenatured soy proteins are a complex mixture of molecules  d i f f e r i n s i z e , charge, and structure (Wolf, 1975).  that  The d i s t r i b u t i o n  in molecular weight ranges from 8,000 to about 600,000 as demonstrated by ultragentrifugation (Wolf, 1970) et a l .  (1963).  or by gel f i l t r a t i o n (Hasegawa  An ultracentrifuge separates the  water-extractable  proteins of defatted meal into four major fractions. These fractions are designated 2,7,11, and 15S based on their sedimentation rates  (coefficients).  The 7S and 11S fractions comprise more than 60% of the t o t a l protein, and about 80% of the proteins have molecular weights of 100,000 and higher. The 2S f r a c t i o n , representing about 20% of the t o t a l p r o t e i n , contains the low molecular-weight proteins: several trypsin i n h i b i t o r s , cytochrome C, allantoinase,  and 2 globulins with unknown b i o l o g i c a l a c t i v i t y .  The 7S f r a c t i o n makes up more than one-third of the t o t a l protein and contains at least four different types of proteins: four hemagglutinins,  two or more lipoxygenases,  7S globulin (Wolf and Cowan, 1975). (Koshiyama, 1969)  and a protein called  The 7S globulin i s a glycoprotein  and makes up over one-half of the t o t a l 7S f r a c t i o n  (Wolf and Sly, 1967). the 11S f r a c t i o n .  beta-amylase,  About one-third of the t o t a l protein i s found i n  So far only one protein called the 11S globulin or  g l y c i n i n has been found i n this sedimenting f r a c t i o n .  The 11S protein  has a molecular weight of about 350,000, which is t y p i c a l of major globulins of other seeds (Wolf, 1972B). the remainder of the protein.  The 15S f r a c t i o n makes up  This f r a c t i o n has not been isolated and  characterized, but based on i t s sedimentation rate appears to have a molecular weight of over half a m i l l i o n .  Some workers have suggested  17.  that this f r a c t i o n is a polymer of the 11S protein (Wolf, 1975). The wide d i s t r i b u t i o n of molecular sizes among the water-extractable soybean proteins is also observed by gel f i l t r a t i o n .  Seven protein  fractions have been fractionated (Hasegawa e_t a l . , 1963)  as compared  to only four i n the ultragentrifuge. Obara and Kimura (1967), however, working on the gel f i l t r a t i o n fractionation of the  water-extractable  soybean proteins, separated only four protein f r a c t i o n s .  Additional  evidence for the complexity of the soybean proteins has been obtained by hydroxylapatite chromatography (Wolf and S l y , 1965), starch gel electrophoresis  (Puski and Melnychyn, 1968; Shibasaki and Okubo, 1966),  and Immunoelectrophoresis  (c)  (Catsimpoolas et_ al,,  1968).  Association-Dissociation reactions A c h a r a c t e r i s t i c property of the 7S and 11S globulins i s  their  a b i l i t y to undergo rapid and reversible association-dissociation actions under mild changes i n ionic environment (Wolf, 1975). the 7S globulin at pH 7.6,  re-  Thus,  0.5 ionic strength exists as a monomer with  a molecular weight of 180,000 to 200,000, but when the ionic strength i s lowered to 0.1,  the protein dimerizes to a 370,000 mol. wt. species  (Koshiyama, 1968A).  Likewise, the 11S globulin forms a faster-sedimenting  form when ionic strength i s changed from 0.5 to 0.1, but the extent of association i s low (Naismith, 1955). When ionic strength i s reduced furthermore, to 0.01,  the 11S protein dissociates into smaller units  that sediment as 7S and 2 to 3S e n t i t l e s (Wolf and Briggs, 1958).  (d)  Subunit structure The 7S and 11S globulins possess an additional property that adds  to their complexity.  Both proteins are b u i l t - u p of smaller units  (sub-  18.  units) that interact very s p e c i f i c a l l y to form the parent globulins, and there appear to be no sub-units common to the two proteins (Wolf, 1972A).  The 7S globulin contains 9 amino-terminal residues, a fact  that indicates that the molecule contains at least 9 polypeptide chains (Koshiyama, 1968B). The 7S structure is disrupted by the usual reagents capable of dissociating proteins into subunits.  When the protein i s  dissolved i n 8M urea or 4M guanidine hydrochloride, the mol. wt. decreases from 180,000 for the native form to 22,500-24,000 (Koshiyama, 1971) .  Dissociation into subunits also occurs on treatment with the  anionic detergent sodium dodecyl sulfate  (SDS).  In SDS s o l u t i o n ,  molecular weight of 7S globulin is 34,000 (Wolf, 1972A).  the  Binding of  about 40 molecules of detergent per subunit would account for the d i s crepancy i n molecular weight under the different conditions of protein dissociation (detergent versus urea or guanidine hydrochloride).  At high  pH (0.01N NaOH) the 7S protein changes to a form with a sedimentation coefficient  of only 0.4S (Koshiyama, 1968A).  The 11S globulin has a  quaternary structure consisting of 12 subunits.  Isoelectric  focusing  of 11S protein i n urea-mercaptoethanol s o l u t i o n , however, separates only 6 subunits. These results suggest that the 11S molecule (mol. wt. of 350,000) i s actually a dimer of 2 i d e n t i c a l monomers, each consisting of 6 subunits (Catsimpoolas, 1969).  The quaternary structure of 11S mole-  cule i s disrupted under various conditions, including low ionic strength, high or low pH, high concentrations of urea, anionic detergents (e.g. SDS), and temperatures above 80°C (Wolf, 1972B).  For reasons s t i l l unknown,  the 11S.globulin is more susceptible to i r r e v e r s i b l e changes i n structure than the 7S globulin (Wolf, 1972A).  19.  (e)  Amino acid composition The chemical composition of soy proteins i s very important  because of i t s relationship to n u t r i t i o n and reactions of the proteins during processing.  Soy proteins are a good source of a l l the essential  amino acids except methionine and tryptophan (Wolf, 1972A). i s the f i r s t l i m i t i n g amino acid of soy proteins  Methionine  (Berry'et'al.,  1962)  and this deficiency needs to be considered when the proteins are used for n u t r i t i o n a l purposes animal  experiments,  (Coppock, 1974).  Largely on the basis of  i t has been generally assumed that the n u t r i t i v e  value of soybean protein products for human consumption would be enhanced by f o r t i f i c a t i o n with methionine  (Liener, 1972).  The available evidence  indicates that meals of soybean products, f l o u r , or protein concentrates are improved i n protein quality upon f o r t i f i c a t i o n with methionine (Berry e_t a l . , 1962,  1966; Evans and McGinnis, 1946).  Methionine has been added  at 0.5% of soy protein i n infant formulas and such formulas have been judged equivalent  i n protein value to the proteins of meat, egg, and milk  (Fomon e_t a l . , 1973) .  Some studies also suggest that tryptophan or  threonine is the second l i m i t i n g amino acid of soy protein. ment  The improve-  i n protein q u a l i t y , however, brought about by adding methionine  is  so high that any other amino acid added causes only small increases (Bressani, 1975).  Soybean proteins being high i n l y s i n e ,  the  first  l i m i t i n g amino acid i n cereals (Kato and Muramatsu, 1971), are useful as supplementing blends  (Bookwalter et ' a l . , 1971; Horan, 1973).  A  mixture of soy protein concentrate and wheat flour i s n u t r i t i o n a l l y superior to either protein source alone; thus, a soy: wheat protein r a t i o of 3:1 gives an optimum protein efficiency  r a t i o (Wilding et a l . ,  20.  1968).  The various soybean protein fractions d i f f e r i n o v e r a l l amino  acid contents.  Insofar as amino acid composition i s concerned, the  most s i g n i f i c a n t differences  between the 7S globulins and the 11S  globulins are the 5- to 6-fold higher contents of methionine, tryptophan, and %-cystine i n the l a t t e r  (Wolf, 1972B).  MATERIALS AND METHODS  A.  Materials Soy protein i s o l a t e used i n this study was a product of General  Mills,  I n c . , Minneapolis, MN. Pepsin (Hog stomach mucosa 2X : c r y s t a l l i z e d  and l y o p h i l i z e d powder), pancreatin (porcine pancreas grade V I ) , L - t r y p t o phan, and 1-ethy1-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, were a l l products of Sigma Chemical Company.  L-methionine and p-dimethyl-  aminobenzaldehyde (practical) were obtained from Eastman Organic Chemicals. Sodium nitroprusside was purchased from Fisher s c i e n t i f i c (B grade) was from Calbiochem, Los Angeles.  company. Pronase  5 - S u l f o s a l i c y l i c acid (98%)  was obtained from A l d r i c h Chemical Company, Inc.. Sodium dodecyl  sulfate  (SDS), s p e c i a l l y pure 99%, was purchased from BDH Chemicals, Canada. The standard proteins, used i n this study for molecular weight c a l i b r a t i o n , and their sources were: ribonuclease A (bovine pancreas, type I-A, 5X c r y s t a l l i z e d ) , a- chymotrypsin (bovine pancreas, 3X c r y s t a l l i z e d ) , serum albumin (Bovine), and catalase (bovine l i v e r ) were a l l products of Sigma Chemical Company; myoglobin (sperm whale) was from Schwartz/Mann. The Sephadex G-200 gel ( p a r t i c l e diameter 40-120y), and the Blue Dextran 2000, were products of Pharmacia Fine Chemicals, Upsala, Sweden.  21.  B.  Preparation of soy protein hydrolysate 12 g of soy protein i s o l a t e was dispersed i n 500 ml of d i s t i l l e d  water.  The pH of the mixture was adjusted with HCI to 1.6.  To this  dispersion was added 120 mg of pepsin dissolved i n 100 ml of d i s t i l l e d water, and the mixture was incubated at 35°C for a specified time (5 or 16 h r ) .  The hydrolysis was stopped by neutralizing (pH 7.0)  incubation mixture with NaOH.  The r e s u l t i n g product was,  the  then,  centrifuged at 9,150 x g for 15 m i n . , and the supernatant was  freeze-  dried to obtain a powdered soybean protein hydrolysate.  C.  S t a t i s t i c a l analysis  (Selection of i n f l u e n t i a l factors and deter-  mination of their best l e v e l s ) . Various conditions of the carbodiimide condensation reaction were investigated by a 2-level f r a c t i o n a l f a c t o r i a l experiment (Taguchi, 1957; Appendix), i n an attempt to determine the factors affecting amino acid binding to soy protein.  the  To determine the best l e v e l for  each of the selected i n f l u e n t i a l factors a 3-level f a c t o r i a l experiment was used (Taguchi, 1957; Appendix). of the f i n a l product obtained ( i . e .  In order to improve the low y i e l d amino acid-coupled soy protein  hydrolysate), another 3-level f a c t o r i a l analysis of various conditions of the carbodiimide reaction was carried out, thus determining the factors influencing the f i n a l product's y i e l d as well as their best levels.  A l l s t a t i s t i c a l data obtained i n the above mentioned f a c t o r i a l  experiments were analysed by a Monroe Model 1880 programmable c a l c u l a t o r .  D.  Covalent binding of methionine and tryptophan to soy protein hydrolysate A specified amount of soy protein hydrolysate was dissolved i n 15 ml  of d i s t i l l e d water at room temperature.  The pH of the solution was  22.  adjusted to the appropriate l e v e l  (set by the experiment).  amount of 1-ethy1-3-(3-dimethylaminopropyl)carbodiimide  A specified  hydrochloride  dissolved i n 2 ml of d i s t i l l e d water was, then, added to the soy protein hydrolysate solution.  Absolute ethanol (1.1 ml) was also added, i f r e -  quired by the experiment. After the specified time for the a c t i v a t i o n  3  of  the protein carboxyl groups, a specified amount of L-methionine or L tryptophan i n solution^ was added to the reaction mixture. The pH of the mixture was checked and adjusted to the l e v e l set by the experiment. The reaction was allowed to proceed at a specified time.  temperature for a specified  Then, 1.8 ml of g l a c i a l acetic acid was added to the mixture to stop  the reaction (by destroying the excess EDC). dialysed i n the cold (5°C) against  Subsequently,  the sample was  2 changes of 1M acetic acid for 1% day,  against running tap water for % day, and f i n a l l y against one change of d i s t i l l e d deionized water for % day. (pH 7.5), E.  Then, the sample was neutralized  s t i r r e d for 10 min (on a magnetic s t i r r e r ) , and freeze-dried.  Methionine determination The methionine content of various samples was determined by the sodium  nitroprusside colorimetric method of Lunder (1974/1975), with the following modifications: l y s i s tube.  (a) 0.5 g sample was mixed with 2.5 ml HC1 20% into a hydro-  The tube was sealed with vacuum grease, c a r e f u l l y closed with  a metallic clamp, and then placed i n an a i r over at 120°C for 10 hours; (b) 450 mg of activated charcoal (12 x 30 mesh, Witco Chemical, N.Y.) were used to discolourize the protein hydrolysate; and (c) 10 ml of 25% ethyl alcohol was used to wash the activated charcoal and elute the methionine adsorbed on i t .  The remainder of the procedure  If no activation was required by the experiment ( i . e . , a c t i v a t i o n time 0 m i n . ) , then, the addition of amino acid solution to the soyprotein hydrolysate solution preceded the carbodiimide addition, k The amino acid solution was prepared by adding drops of IN HC1 to a s t i r r e d dispersion of a specified amount of amino acid i n 4 ml of d i s t i l l e d water and bringing to the volume (5 ml) by adding d i s t . water.  23.  was carried out exactly as described by Lunder (1974/1975).  F.  Tryptophan determination The tryptophan content of various samples was determined by the  spectrophotometric method of Spies (1967) after hydrolysis of samples with pronase (procedure W). The p-dimethylaminobenzaldehyde (practical) was r e c r y s t a l l i z e d twice from ethanol by adding d i s t i l l e d water to a warm (about 50°C) ethanolic solution of p-dimethylaminobenzaldehyde u n t i l c r y s t a l l i z a t i o n was just i n i t i a t e d and then cooling the solution. A suspension of s l i g h t l y greenish-white crystals was obtained.  These crystals were  harvested by suction f i l t r a t i o n , a i r - d r i e d , and kept i n a desiccator away from l i g h t u n t i l used.  G.  Nitrogen determination The nitrogen content of soy protein i s o l a t e ,  soy protein hydrolysate,  as well as methionine- or tryptophan-bound soy protein i s o l a t e and soy protein hydrolysate samples obtained under best reaction conditions was determined by the rapid micro-Kjeldahl procedure of Concon and Soltess (1973).  The protein content of each sample was then calculated by m u l t i -  plying the amount of i t s nitrogen content by a factor of  H.  6.25.  Amino acid analysis The samples were hydrolysed with p-toluenesulfonic acid according  to the method of L i u and Chang (1971), at 110°C for 24 hr.  Amino acids  were analysed on a single-column system (Durrum Chem. Corp., Palo A l t o , CA.) attached to a Phoenix Model M6800 Amino Acid Analyzer (Phoenix Precision Instrument C o . ) .  24.  I.  Enzymatic digestion  test  The d i g e s t i b i l i t y of various samples was determined by the o r i g i n a l pepsin-pancreatin digest method of Akeson and Stahmann (1964) as modified by Stahmann and Woldegiorgis (1975), with the following change: to ml of digestion mixture was added 5.0 ml of 14% s u l f o s a l i c y l i c The flask was then shaken for 15-30 minutes at 37°C) and removed from the shaker. adding drops of 6N NaOH.  acid.  (in a shaking water bath  The pH was adjusted to 2.2 by  The digest was then transferred to a 15 ml  graduated tube and brought to volume by adding pH 2.2 c i t r a t e diluter.  5.0  sample  The sample was then centrifuged at 1,000 x g for 30 minutes,  f i l t e r e d through a dry Whatman No. 1 f i l t e r paper, then through a sintered glass UF f i l t e r d i r e c t l y into a sample tube, and stored i n a freezer at -20°C or i n a cold room (5°C) for amino acid analysis.  J.  Gel f i l t r a t i o n chromatography  (1)  Preparation and operation Gel f i l t r a t i o n was carried out on Sephadex G-200 i n a glass chromato-  graphic column (Fisher and Porter) with an i n t e r n a l diameter of 2.0 cm and a length of 100 cm.  The packed column was s t a b i l i z e d and equilibrated  with 2 bed volumes of elution buffer (0.1M sodium phosphate buffer, pH 7.0, containing 0.02% sodium azide, to prevent microbial growth, and 0.1% SDS) i n an upward flow d i r e c t i o n .  The operating pressure was adjusted to give  a flow rate of about 10.4 ml per hour during e q u i l i b r a t i o n and subsequent runs.  The column was operated i n an upward flow d i r e c t i o n .  The i n l e t  tubing was joined to a 3-way valve which was connected to a funnel with elution buffer) and a 5 ml syringe.  (filled  Sample containing 150 mg protein  in 5.0 ml solution was applied to the column v i a the syringe.  The valve  25.  was then turned to connect the eluant to the column. was collected by a fraction c o l l e c t o r  The effluent  (Gilson Medical E l e c t r o n i c s ,  Wisconsin) set to take 5.2 ml fractions.  The elution pattern of each  protein sample was obtained by measuring the absorbance of each f r a c t i o n in a 1-cm c e l l at 280 nm by a Beckman DB spectrophotometer, and p l o t t i n g the value against the f r a c t i o n ' s number d i r e c t l y on a recording chart by means of a Varicord recorder Model 43 Corporation, N.Y.) attached to the  (Photovolt  spectrophotometer.  The void volume (V ) and the t o t a l bed volume (V^_) of the column o t were determined from the elution volumes of Blue Dextran 2000 and tryptophan, respectively,  using absorbances at 280 nm.  The column  was calibrated with standards proteins. (2)  S o l u b i l i z a t i o n of samples The methionine- or tryptophan-bound soy protein hydrolysate or  soy protein i s o l a t e samples with the highest coupling yields were insoluble i n the phosphate buffer (eluant).  Thus, different  dissociation  procedures were used to s o l u b i l i z e the amino acid-bound soy protein hydrolysate samples  (which exhibited much greater s o l u b i l i t y than the  corresponding amino acid-bound soy protein i s o l a t e samples).  The  tryptophan-bound soy protein hydrolysate sample was s o l u b i l i z e d as follows:  i n 5.0 ml of elution buffer were added 20 mg SDS and 1 drop  of 2-mercaptoethanol.  To this solution was dispersed 150 mg of  tryptophan-bound soy protein hydrolysate and the mixture was s t i r r e d on a magnetic s t i r r e r . dissolution.  Moderate heat was applied to f a c i l i t a t e  the  The sample after being completely s o l u b i l i z e d was f i l t e r e d  through a dry Whatman No. 1 f i l t e r paper (to remove any i n v i s i b l e microp a r t i c l e ) , and then applied to the column. Although, the soy protein  26.  hydrolysate (control) was soluble i n the elution buffer, the same s o l u b i l i z a t i o n procedure was applied to i t i n order to have a better control. Since the methionine-coupled soy protein hydrolysate sample exhibited s l i g h t s o l u b i l i t y  under the above described conditions,  the following procedure was used for i t s s o l u b i l i z a t i o n : 150 mg of methione-bound soy protein hydrolysate were added to 5.0 ml of d i s t i l l e d water.  The pH of the dispersion was adjusted to 12.0 by NaOH and the  mixture was s t i r r e d at room temperature for 30 min, then centrifuged at 3,090 x g for 15 min and the supernatant was applied to the column. The same method was used to s o l u b i l i z e 150 mg of soy protein hydrolysate (control) and 150 mg of tryptophan-bound soy protein hydrolysate sample. However, by this s o l u b i l i z a t i o n procedure the tryptophan-bound SPH sample was not completely s o l u b i l i z e d , and thus, only the supernatant was applied to the column.  RESULTS AND DISCUSSION  A. Preparation of soy protein hydrolysate Soy protein i s o l a t e was used as the starting material i n this study, in order to eliminate any interference during the carbodiimide-mediated coupling reaction due to the presence of non-protein components. Various l i n k i n g modes are possible during the carbodiimide condensation reaction, i.e.,  linkings between the amino group of the amino acid being bound and -  C-terminal a-carboxyl or side chain aspartic 6- or glutamic y c a r b o x y l groups of the soy protein molecule or between the N-terminal a-amino or e-amino groups of soy protein and the carboxyl group of the amino acid being bound.  However, the n u t r i t i o n a l a v a i l a b i l i t y of 3-a and y-a  linkages  27.  i s uncertain and not well documented i n the l i t e r a t u r e . been reported (Franzblau e_t_ al. , 1963)  Since i t has  that protein a-carboxyl groups  react more readily and quantitatively with carbodiimides i n the presence of nucleophiles  to produce the corresponding protein derivatives  than  protein 3r or y c a r b o x y l groups, the SPI was p a r t i a l l y hydrolysed with pepsin to increase the number of a-carboxyl and a-amino groups in soy protein so as to confine the amino acid binding mainly to the a-a peptide linkages.  B. S t a t i s t i c a l  analysis  Data concerning the reaction characteristics of each amino acid are of fundamental importance for providing optimum reaction and thus obtaining high yields during peptide synthesis. determine, therefore,  conditions  In order to  the factors influencing the methionine and  tryptophan binding to soy protein hydrolysate (SPH), various  conditions  of the carbodiimide condensation reaction were analysed by a 2-level f r a c t i o n a l f a c t o r i a l design (Taguchi, 1957; Appendix). the factors and their assigned levels investigated influence on amino acid binding to soy protein.  Table 1 shows  for their  possible  Tables 2 and 3 show the  analysis of variance of the 2-level f r a c t i o n a l f a c t o r i a l design for methionine and tryptophan binding, respectively,  to SPH.  As i t can be  seen i n Table 2, pH, SPH concentration, reaction time, carbodiimide concentration, activation time and pH x SPH concentration  interaction  were found to s i g n i f i c a n t l y affect the methionine binding  efficiency.  On the other hand, Table 3 shows that pH, reaction temperature, amino acid (tryptophan) concentration, SPH concentration, carbodiimide concentration, and pH x SPH concentration interaction s i g n i f i c a n t l y fluenced the tryptophan binding to SPH.  in-  28.  Table 1 - Factors and their assigned levels investigated for their possible influence on amino acid binding to SPH.  Factor  1. 2. 3. 4. 5. 6. 7. 8. 9.  Reaction temperature (°C) Pepsin hydrolysis time (hr) Amino acid concentration (mg) SPH concentration (mg) Carbodiimide concetration (mg) Activation time (min) Reaction time (hr) P o l a r i t y (% EtOH)  Level  5 22 5 80 220 80 0 1 0  8 40 16 140 660 140 60 24 5  Table 2 - Analysis of variance of the 2-level f r a c t i o n a l f a c t o r i a l experiment for methionine binding to SPH.  Source of Variation  S.S.  D.F.  M.S.  F-value  pH (A)  1.93  1  1. 93  64. 33**  SPH concentration (E)  4.48  1  4. 48  149. 33*?v  Reaction time (F)  0.87  1  0. 87  29. 00**  Carbodiimine concentration (G)  1.00  1  1. 00  33. 33**  Activation time (H)  0.20  1  0. 20  6. 67*  A x E  0.35  1  0. 35  11. 67**  0.27  9  0. 03  9.10  15  Error  3  Total  The sum of square values for reaction temperature (B), pepsin hydrolysis time (C), amino acid concentration (D) and p o l a r i t y (I) were very low and, therefore, incorporated into the error S.S. *F  0 > 0 5  F  (1,9) ( 1  ** 0.01 '  = 5.12. 9 ) =  1  0  -  6  0  -  Table 3 - Analysis of variance of the 2-level f r a c t i o n a l f a c t o r i a l experiment for tryptophan binding to SPH.  Source of Variation  S.S.  D.F.  M.S.  F-value  pH (A)  9.30  1  9.30  35.77**  Reaction temperature (B)  2.10  1  2.10  8.08*  Amino acid concentration (D)  1.62  1  1.62  6.23*  SPH concentration (E)  7.02  1  7.02  27.00**  Reaction time (F)  1.00  1  1.00  3.85  Carbodiimide concentration (G)  3.24  1  3.24  12.46*  Activation time (H)  1.00  1  1.00  3.85  Polarity  0.64  1  0.64  2.46  4.20  1  4.20  1.54  6  0.26  31.66  15  (I)  A x E Error  3  Total  16.15**  The sum of square value for pepsin hydrolysis time (C) was low and, therefore, incorporated into the error S.S. *F . (1,6) 0  F  0 5  ** 0.01  ( 1 , 6 )  = 5.99. =  1 3  -  7 0  «  31.  In order to find the l e v e l  (of the two assigned) for each of the  determined i n f l u e n t i a l factors, which more favourably affected  the  binding of each amino acid to SPH, the response curve for each factor should be constructed.  F i g s . 2,3,4,5 and 6 show the effect of pH, SPH  concentration, carbodiimide concentration, activation time, and reaction time, respectively,  on the rate of methionine binding to SPH, whereas  F i g s . 2,3,4,7 and 8 show the effect of pH, SPH concentration, carbodiimide concentration, tryptophan concentration, and reaction temperature, t i v e l y , on the rate of tryptophan binding to SPH.  respec-  As i t can be seen i n  these figures, a pH 5 was found to be more favourable than pH 8 for the binding of both amino acids  (Fig. 2).  Moreover, a low SPH concentration  (220 mg) and high carbodiimide concentration (140 mg) were found to favor the rates of binding of both amino acids (Figs. 3 and 4,  respectively).  Figs. 5 and 6 show respectively that, activation time of 0 m i n . , namely, no a c t i v a t i o n (direct condensation),  and long reaction time (24 hr) were  more favourable for the binding of methionine to SPH than a c t i v a t i o n time of 60 m i n . , and a short reaction time (1 h r ) .  On the other hand, a high  amino acid (tryptophan) concentration (140 mg) and a high reaction temperature (40°C) were found to favor the tryptophan binding to SPH (Figs. 7 and 8,  respectively).  In order to determine the best l e v e l for each of the selected i n f l u e n t i a l factors an optimization of the carbodiimide reaction was conducted by carrying out a 3-level f a c t o r i a l analysis of variance (Taguchi, 1957; Appendix).  Table 4 shows the three (3) l e v e l s assigned  for each factor affecting the binding of methionine and tryptophan to SPH. Footnotes a and b indicate the l e v e l for each of the n o n - i n f l u e n t i a l  factors  4  5  6  7  8  pH Effect of pH on the rate of methionine (solid line) and tryptophan (broken line) binding to SPH. The v e r t i c a l lines represent the 95% confidence l i m i t s . The confidence l i m i t was calculated as follows: c  L  ± t ( 0 . 0 5 ) a t D . F . error no. of data averaged  / S . S . error D . F . error  <  33.  1  °0  300  SPH  500  concentration  700  (mg)  F i g . 3. Effect of SPH concentration on the rate of methionine (solid line) and tryptophan (broken line) binding to SPH.  EDC Fig. 4.  concentration  (mg)  E f f e c t of EDC c o n c e n t r a t i o n on the r a t e of methionine l i n e ) and t r y p t o p h a n (broken l i n e ) b i n d i n g to SPH.  (solid  35.  £  3  L  Acti vation Fig.  5.  E f f e c t of a c t i v a t i o n to SPH.  time (min )  time on the r a t e of methionine b i n d i n g  36.  Reaction Fig.  6.  time  ( hr)  Effect of reaction time on the rate of methionine binding to SPH.  37.  « 40  80  Amino Fig.  7.  acid  12 0  16 0  c o n e e n t r a t i o n ( mg )  E f f e c t of amino a c i d (tryptophan) c o n c e n t r a t i o n on the r a t e of t r y p t o p h a n b i n d i n g to SPH.  38.  X  3  QL (f)  o a  o o  a  1  r-  a  H  0  20  Reaction  40  temperature  (°C )  F i g . 8. E f f e c t of reaction temperature on the rate of tryptophan binding to SPH.  which was kept constant i n a l l experiments.  The 3 levels assigned for  each influencing factor were not chosen a r b i t r a r i l y , but selected so as to approach the optimal l e v e l of the factor found by the f r a c t i o n a l f a c t o r i a l design.  2-level  Tables 5 and 6 show the analysis of  variance of the 3-level f a c t o r i a l experiment for methionine and tryptophan binding, respectively. significant  (P<0.01) differences  As i t can be seen i n Table 5, highly i n methionine binding were brought  about by the three assigned levels of pH, SPH concentration, carbodiimide concentration, and activation time, whereas no s t a t i s t i c a l l y (P>0.05) differences  significant  i n methionine binding were caused by the three  levels of reaction time tested or by the interaction of pH x SPH concentration.  Table 6 indicates that highly s i g n i f i c a n t  (P<0.01) d i f f e r -  ences i n tryptophan binding were produced by the tested three levels of carbodiimide concentration and reaction temperature, whereas no statistically  significant  (P>0.05) differences  i n tryptophan binding  were caused by the three levels of the other i n f l u e n t i a l factors or by the pH x SPH concentration i n t e r a c t i o n . In order to find the best l e v e l of each influencing factor among the assigned three levels the response curves of these factors should be constructed.  F i g . 9 shows the effect of pH on the rate of methionine  and tryptophan binding to SPH.  It can be seen that, pH 6.5 was found to  be the best among the three levels for the e f f i c i e n t amino acids.  binding of both  This factor (pH) has been mentioned i n several papers  using carbodiimides (Hoare and Koshland, 1966; Means and Feeney, Riehm and Scheraga,  1971;  1966), as having an optimum at 4.75, namely, a pH  which should normally l i m i t the reaction to the protein carboxyl groups,  40.  Table 4 - Assigned levels ' binding to SPH.  for factors influencing the amino acid  Factor  Methionine Level  Amino Acid Tryptophan Level  1  2  3  1  2  3  1.  pH  3.5  5.0  6.5  3.5  5.0  6.5  2.  SPH concentration(mg)  160  220  400  160  220  400  3.  Carbodiimide  concentration(mg)100  140  200  100  140  200  4.  Amino acid concentration(mg)  100  140  200  5.  Activation time(min)  6.  Reaction time (hr)  7.  Reaction temperature (°C)  30  40  50  0  20  40  12  24  30  Level for each of the non-influencing factors which was kept constant in a l l experiments of methionine-binding reaction to SPH: pepsin hydrolysis time (16 h r ) , amino acid concentration (120 mg), p o l a r i t y (0% EtOH), and reaction temperature ( 2 2 ° C ) . Level for each of the non-influencing factors which was kept constant i n a l l experiments of tryptophan-binding to SPH: pepsin hydrolysis time (16 h r ) , p o l a r i t y (0% EtOH), activation time (0 min), and reaction time (16 h r ) .  Table 5 - Analysis of variance of the 3-level f a c t o r i a l experiment for methionine binding to SPH.  Source of Variation  SS.  D.F.  MS.  F-value  pH(A)  19.29  2  9.65  16.16**  SPH concentration(B)  21.31  2  10.66  17.86**  Carbodiimide concentration(C)  9.37  2  4.69  7.86**  Activation time (D)  9.38  2  4.69  7.86**  Reaction activation time(E)  3.66  2  1.83  3.07  A x B  5.09  4  1.27  2.13  Error  7.16  12  0.60  Total  75.26  26  *  F  0.05  ( 2  '  ** o . o i ' F  ( 2  V  1 2 )  1 2 )  "  3  '  - 6  8  9  9 3  F  '  0 .05  > o F  >01  ( 4  '  1 2 )  <4,12)  -  3.26.  , 5.41.  Table 6 - Analysis of variance of the 3 - l e v e l f a c t o r i a l for tryptophan binding to SPH.  Source of variation  S.S.  D.F.  experiment  MS.  F-value  pH(A)  1. 97  2  0,,99  1,,30  SPH concentration(B)  4. 06  2  2.,03  2.,67  71. 04  2  35. ,52  2. 08  2  1.,04  44. 21  2  22. ,11  A x B  4. 25  4  1.,06  Error  9. 12  12  0.,76  Total  136. 73  26  Carbodiimide Amino acid Reaction  concentration(C)  concentration(D)  temperature(E)  F  0 > 0 5  ( 2 , 1 2 ) = 3.89,  F  0 ( ) 5  F  0 > 0 1  ( 2 , 1 2 ) = 6.93,  F  0 0 1  ( 4 , 1 2 ) = 3.26.  ( 4 , 1 2 ) = 5.41.  46. ,74** 1..37 29,.09** 1.,40  43.  44.  since at this pH most ( i f not a l l ) of the protein nucleophilic groups (such as amino or imidazole groups) would be protonated (Riehm and Scheraga, 1966).  On the other hand, H o r i n i s h i e_t a l . (1968) reported  that the WSC-promoted reaction between b a c i t r a c i n A (a c y c l i c dodecapeptide having two carboxyl groups of aspartic and glutamic acids i n the double ring structure) and glycine ethylester  (nucleophile),  well as the reaction between lysozyme (egg white) and glycine both had a broad f l a t optimum between pH 5.2 and 6.9.  as  ethylester,  These workers also  ascribed the drop of the degree of reaction at the a c i d i c side to decomposition of WSC, and on the a l k a l i n e side to hydrolysis of glycine ethylester with a l k a l i and to deprotonation from the t e r t i a r y amino group of WSC ( i . e . , to the drop of the r e a c t i v i t y of the  reagents).  Holbek (1976), furthermore, working on the EDC-mediated binding of lysine to wheat gluten, has noted that pHs 7.5 and 10.0 resulted i n considerably greater coupling y i e l d s than pH 5.0, the best among these three levels tested.  pH 7.5 found to be  The finding that at pH 6.5  the amino acid coupling was higher than at pH 5.0 can be explained by taking into account the fact that the i s o e l e c t r i c point of soy protein f a l l s i n the range of pH 4.0-5.0. soy protein at pH about 5.0,  Therefore, with the p r e c i p i t a t i o n of  there w i l l be less a c c e s s i b i l i t y  for both  the WSC to be bound to soy protein carboxyl groups to form the reactive intermediates  (0-acylisoureas)  and the amino acid to attack these and  other reaction intermediates to form the desired protein derivatives (products).  Moreover, i t was recently suggested by Addy et a l .  (1973A)  that the pH used i n the carbodiimide-mediated synthesis of peptides  in  aqueous medium should leave the amino group of the amino acid (being bound)  45.  unprotonated, although the carbodiimide and postulated O-acylisourea should be protonated.  Therefore, the y i e l d of peptide bond formation  should r i s e to a maximum when this condition exists and f a l l quickly when the pH is low enough to protonate the amino group of the amino a c i d , thus preventing i t from acting as nucleophile (since the unprotonated form of the a-amino group of the amino acid being bound i s the r e a l nucleophile). The pK values for the amino groups of methionine and tryptophan are 9.21 and 9.39,  respectively,  (West and Todd, 1961),  that i s , much higher than the recommended pH of 4.75. however,  both c o n f l i c t i n g requirements, i . e .  At pH 6.5,  low pH to l i m i t the  reaction to protein carboxyl groups and pH which should leave the amino group of the amino acid being bound unprotonated, are partly met. this pH (6.5)  Since  l i e s between the i s o e l e c t r i c point and the pK values of  the amino groups of both amino acids, therefore,  there w i l l be a certain  amount of unprotonated amino groups, although low, available for nucleop h i l i c attack.  On the other hand, at this pH the reaction between soy  protein and EDC did not involve d i r e c t reaction between EDC and amino or imidazole groups i n the protein, as w i l l be demonstrated from the amino acid analysis below.  Figures 10, 11 and 12 show,  respectively,  that low SPH concentration, high carbodiimide concentration, and high amino acid concentration (although not s t a t i s t i c a l l y  significant)  in higher amino acid coupling yields than the other two assigned  resulted levels  of these influencing factors. These findings are i n accordance with the o r i g i n a l note made by Khorana i n 1955 which stated that an increase i n the concentration of reactants should favor the formation of desired protein products over the formation of undesirable side-products (N-  46.  CO  e o I  a  o i  CD  O  a>  o o v.  o •*->  CD  100  200  SPH  300  concentration  400  (mg )  F i g . 10. E f f e c t of SPH c o n c e n t r a t i o n on the r a t e of methionine ( s o l i d l i n e ) and tryptophan (broken l i n e ) b i n d i n g to SPH.  47  120  80  EDC F i g . 11.  160  concentration  20  (mg)  E f f e c t of EDC c o n c e n t r a t i o n on the r a t e of methionine ( s o l i d l i n e ) and t r y p t o p h a n (broken l i n e ) b i n d i n g to SPH.  48.  Amino  acid  concentration  (mg)  F i g . 12. E f f e c t of amino a c i d (tryptophan) c o n c e n t r a t i o n on the r a t e of tryptophan b i n d i n g to SPH.  acylurea d e r i v a t i v e s ) . Horinishi et a l .  (1968) have demonstrated that  the WSC-mediated coupling between carboxyl groups of acetylglycine or proteins and a l i p h a t i c amines at room temperature can be enhanced by increasing the amine or WSC concentrations. On the other hand, i t is well known, that an excess of carboxyl component favors the formation of symmetrical anhydrides (Schroder and Lubke, 1965).  Delayed addition  of the nucleophiles, i n p a r t i c u l a r , has been found to decrease  the  importance of O-acylisourea and increase the extent of anhydride's p a r t i c i p a t i o n during peptide synthesis (Rebek and F e i t l e r , 1973). Symmetrical anhydrides, however, have been claimed to be undesirable intermediates from the racemization point of view (Bodanszky and Ondetti, 1966). F i g . 13 shows the effect of a c t i v a t i o n time on the rate of methionine binding to SPH.  As i t can be seen, a c t i v a t i o n time of 0 m i n . , that  is,  direct condensation without pre-incubation of the SPH with EDC, resulted i n higher methionine coupling than activation (condensation after preincubation of SPH with EDC) for 20 or 40 min.  This finding may be ex-  plained by r e c a l l i n g the note of Previero _et'al.  (1973), who reported  that direct condensation without preincubation of an a c i d i c peptide with EDC resulted in the coupling of two times as many amino acid ester moles per mole of peptide as i n the case of condensation after preincubation of the same a c i d i c peptide with EDC for 90 min. at 40°C. On the other hand, the extent of condensation of amino acid esters with non-acidic peptides was constant and did not change during the incubation with EDC.  These workers suggested that peptides containing a carboxyl  group only at the C-terminal position rapidly reach a degree of activation  Q.  6  B»  Activation F i g . 13.  E f f e c t of a c t i v a t i o n to SPH,  time  (min)  time on the r a t e of methionine  binding  51.  which remains constant, while peptides containing a side-chain carboxyl group show an i n i t i a l maximum of a c t i v a t i o n which subsequently decreases to about one half of i t s value.  Taking into consideration the fact  that  a c i d i c amino acids (glutamic and aspartic) represent about 33% of the t o t a l amino acid composition of soy protein, i t might be expected that at least a few of the side-chain aspartic and glutamic carboxyl groups participated i n the formation of peptide bonds i n the cases at which direct condensation was used.  These groups, however, did not contribute  to coupling when condensation after pre-incubation was used.  Another  factor which may also explain the higher binding y i e l d s observed when no activation took place is the formation of the undesirable stable N-acylureas.  As i t has already been mentioned, N-acylureas are formed  during the carbodiimide condensation r e a c t i o n , when no nucleophiles are present i n the reaction mixture or the reaction of those present slow or hindered (Kopple, 1966).  is  The formation of N-acylurea, therefore,  should be expected to be much higher when condensation after pre-incubation is used, and moreover, i t should be anticipated that the longer the preincubation time the higher the formation of N-acylurea would be.  N-  acylurea, however, is not an acylating agent and thus, i t s formation would lead to lower coupling y i e l d s . Figure 14 shows the effect of reaction time on the rate of methionine binding to SPH.  As i t can be seen, the three assigned reaction time  levels did not cause any s i g n i f i c a n t difference i n methionine binding to SPH.  The decrease i n methionine binding, however, corresponding to  the 24 hr reaction time l e v e l could not be explained.  Hoare and Koshland  (1966) stated that the reaction between a WSC and a protein i n the presence  52.  X CL  (/>  6  -  C 3  Reaction F i g . 14.  time  ( h r)  Effect of reaction time on the rate of methionine binding to SPH.  53.  of nucleophiles is quite rapid.  Moreover, Sheehan et a l . (1965)  working with EDC demonstrated that during the synthesis of o l i g o peptide derivatives coupling was extremely rapid (about 80% after 1 hr).  However, the reaction time required for complete coupling varied  from 24 to 66 h r .  Therefore, i t seems from F i g . 14 that the optimum  reaction time (time required for complete coupling) varies not only with the nature of carbodiimide used (Greenstein and Winitz, 1961), but also with the carboxyl component involved (starting protein or peptide). (efficiency)  F i g . 15 represents the effect of temperature on the rate of tryptophan binding to SPH.  As indicated i n this  figure,  a reaction temperature of 50°C resulted i n a s i g n i f i c a n t l y higher tryptophan binding than a temperature of 40° or 30°C.  This finding  can be easily explained by taking into account the fact that the synthetic reaction of a peptide-bond formation from a peptide and a free amino acid i s a strongly endergonic (endothermic) (Yamashita et a l . , 1970).  process  Therefore, when a temperature of 50°C i s  used during the WSC condensation reaction, there w i l l be more energy available to the system to help overcome the energy barrier with concomitant higher y i e l d of peptide bond formation.  Holbek (1976) has  also noted that a reaction temperature of 40°C resulted i n higher lysine coupling to gluten than a temperature of 10° or 25°C.  The carbodiimide-  mediated peptide synthesis i s usually carried out at room temperature, because i t i s easier to handle and also to reduce the formation of undesirable N-acylurea derivatives.  However,since the formation of N-  acylureas would be expected to result i n lower tryptophan coupling yields (for N-acylureas are not acylating agents), the fact that the tryphophan binding was more e f f i c i e n t  at 50°C than at 40° or 30°C might indicate  54.  8  r  Reaction F i g . 15.  t e m p e r a t u r e (°C )  Effect of reaction temperature on the rate of tryptophan, binding to S P H .  55.  that the N-acylureas formation at 50°C was perhaps not s i g n i f i c a n t i n this reaction.  The data presented i n the above mentioned response  curves are summarized i n Table 7, which shows the factors  affecting  the binding of each amino acid to SPH, and their best levels of those tested. One problem encountered during this study was the low y i e l d of the f i n a l product, namely, of the amino acid-bound SPH.  Thus, i n order to  improve the f i n a l product's y i e l d another 3-level f a c t o r i a l analysis of the carbodiimide reaction conditions was carried out.  Table 8 shows the  factors and their assigned levels analysed for their possible on the f i n a l product's y i e l d .  influence  Footnote a of Table 8 indicates the  reaction conditions (factors) which were held constant i n a l l experiments of this f a c t o r i a l design. Tables 9 and 10, show the analysis of variance of this 3-level f a c t o r i a l experiment for methionine and tryptophan, respectively, used for selection of the factors influencing the f i n a l product's y i e l d and optimization.  As shown i n Table 9, pH, SPH concen-  t r a t i o n , reaction temperature and reaction time s i g n i f i c a n t l y affected the methionine-bound SPH y i e l d s .  On the other hand, Table 10 shows  that only SPH concentration s i g n i f i c a n t l y influenced the tryptophanbound SPH y i e l d s .  Table 11 indicates the factors, which were found to  s i g n i f i c a n t l y affect  the yields of methionine- or tryptophan-bound SPHs,  and their best l e v e l s .  The highest f i n a l product's y i e l d obtained  (in this f a c t o r i a l design) was 36%, whereas the lowest one was 17%. The methionine and tryptophan contents of the samples with the highest y i e l d (36%) were 2.3% and 4.3% ( i . e . , 2.5 times and 4.6 times as much as those of SPI), respectively.  Table 7 - Factors affecting amino acid binding and their best l e v e l  Factor Methionine  Amino acid Tryptophan  pH  6.5  6.5  SPH concentration(mg)  160  160  Carbodiimide concentration(mg)  200  200  -  200  Amino acid concentration(mg) Activation time(min)  0  Reaction time(hr)  12  Reaction t e m p e r a t u r e ( ° C )  -  50°  Table 8 - Assigned levels for factors investigated by a 3-level f a c t o r i a l experiment for their possible influence on f i n a l product's y i e l d .  Factor  Level 1  2  3  SPI concentration(%)  2.4  3.6  4.8  Pepsin concentration(mg)  50  100  150  PH  5  6  7  SPH concentration(mg)  200  350  500  Reaction temperature (°C)  25  35  50  Reaction time(hr)  6  12  16  Constant factors: pepsin hydrolysis time (16 h r ) , amino acid concentration (140 mg), carbodiimide concentration (140 mg), activation time (0 min).  Table 9 - Analysis of variance of the 3 - l e v e l f a c t o r i a l experiment for selection of the factors influencing the methioninebound SPH yields and optimization.  Source of Variation  s.s.  D.F.  M.S.  F-value  SPI concentration(A)  8.06  2  4.03  0.94  Pepsin concentration(B)  2.32  2  1.16  0.27  268.90  2  134.45  31.49**  SPH concentration(D)  192.93  2  96.47  22.59**  Reaction temperature(E)  207.51  2  103.76  24.30**  Reaction time(F)  60.02  2  30.01  7.03*  A xB  97.85  4  24.46  5.73  A xC  27.45  4  6.86  1.60  Error  25.61  6  4.27  Total  890.65  26  PH  (C)  F  0 > 0 5  (2,6)  = 5.14,  F  0 > 0 5  (4,6)  = 4.53.  F  0 > 0 1  (2,6)  = 10.43, F  0 > 0 1  (4,6)  = 9.15.  Table 10 - Analysis of variance of the 3 - l e v e l f a c t o r i a l experiment for selection of the factors influencing the tryptophanbound SPH yields and optimization.  Source of Variation  S.S.  D.F.  MS.  F-value  4.25  2  2.13  0.33  Pepsin concentration(B)  17.89  2  8.95  1.37  pH  29.05  2  14.53  2.23  SPH concentration(D)  71.92  2  35.96  5.52*  Reaction temperature(E)  59.72  2  29.86  4.58  Reaction time(F)  18.46  2  9.23  1.42  A xB  169.67  4  42.42  6.51  A xC  7.75  4  1.94  0.30  Error  39.11  6  6.52  Total  417.82  26  SPI  concentration(A)  (C)  *  F  0 i 0 5  ( 2 , 6 ) = 5.14.  ** o . o i ' F  ( 2  6 )  =  1 0 , 4 3  *  Table 11 - Factors affecting levels.  f i n a l product's y i e l d and their best  Factor  Amino Acid Methionine  Tryptophan  pH  5  -  SPH concentration(mg)  500  500  Reaction time(hr)  16  Reaction t e m p e r a t u r e ( ° C )  50°  -  61. Since the y i e l d of the f i n a l product could not be markedly improved by the above 3-level f a c t o r i a l design, i n which peptic SPH was used as the starting material i n the carbodiimide reaction, another attempt to further improve the y i e l d was done by using SPI i t s e l f  ( i . e . , without  preliminary pepsin hydrolysis) as the s t a r t i n g material and the same reaction conditions under which the amino acid-bound SPH samples with the highest coupling yields were obtained. 95% and 99% (on a weight basis) SPI samples, respectively.  The y i e l d so obtained was  for the methionine- and tryptophan-bound  Moreover, the efficiency of methionine and  tryptophan binding to SPI was s t i l l excellent,  as w i l l be demonstrated  i n the amino acid analysis section below. Amino acid analysis The amino acid composition of SPI, and methionine- and tryptophanbound soy protein samples with the highest amino acid binding efficiency is reported i n Table 12.  These results show that the L-methionine content  in the methionine-bound SPH sample was 7.22g/16gN, whereas i t s content i n the SPI (control) was only 0.94g/16gN.  Furthermore, i t can be seen that  the L-tryptophan content i n the tryptophan-bound SPH sample was 17.05g/16gN, whereas i t s content i n the SPI was only 0.95g/16gN.  On the other hand,  Table 12 shows that the L-methionine content i n themethionine-bound SPI sample was 5.92g/16gN, and the L-tryptophan content bound SPI sample was 10.74g/16gN.  i n the tryptophan-  The above r e s u l t s , therefore, very  c l e a r l y demonstrate that the L-methionine and L-tryptophan added have been very e f f i c i e n t l y bound to SPHs and SPIs. The fact that the L-tryptophan has been more e f f i c i e n t l y bound i n both cases ( i . e . , when SPH or SPI was used as the s t a r t i n g material) than the L-methionine may be attributed to the  higher  r e a c t i v i t y of L-tryptophan (due to the presence of the indole ring i n i t s molecul  62.  Table 12 - Amino acid composition (g/16gN) of SPI andS amino a c i d bound soy protein samples with the highest coupling y i e l d s _b  Amino Acid  Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Lysine Histidine Tryptophan Arginine Cystine  Control SPI  11.41 3.67 5.31 19.40 5.10 3.76 3.75 3.67 0.94 4.27 7.20 3.58 5.30 5.83 2.53 0.95 7.38 0.92  Met-bound SPH  10.57 3.66 5.75 18.21 5.54 3.72 3.16 3.54 7.22 3.61 6.20 3.34 4.50 4.45 2.77 0.94 6.82 1.00  Trp-bound SPH  8.82 3.09 4.25 15.37 5.52 3.26 2.83 3.09 0.94 3.68 5.22 3.07 4.37 5.17 2.63 17.05 5.64 0.99  Met-bound SPI  10.84 3.53 5.00 18.34 4.84 3.52 3.56 3.23 5.92 3.97 7.13 3.30 5.00 5.71 2.43 0.90 6.93 0.87  Trp-bound SPI  10.32 3.39 4.73 17.50 4.68 3.32 3.46 2.96 0.83 3.69 6.61 3.15 4.72 5.25 2.25 10.74 6.56 0.84  Each value is the average of duplicate analyses. Experimental conditions: (a) methionine binding: pH 6.5; SPH or SPI concentration, 160 mg; EDC concentration, 140 mg; a c t i v a t i o n time, 0 min; reaction time, 12 hr; pepsin hydrolysis time, 16 hr; methionine concentration, 120 mg; reaction temperature, 22°C; (b) tryptophan binding: pH 6.5; SPH or SPI concentration, 160 mg; EDC concentration, 200 mg; a c t i v a t i o n time, 0 min; tryptophan concentration, 140 mg; reaction temperature, 50°C; pepsin hydrolysis time, 16 hr; reaction time, 16 hr.  since i t is known that the coupling reaction, i n general, depends upon the r e a c t i v i t y of each amino acid (Tometsko, 1973).  A selectivity  in  aqueous peptide synthesis mediated by EDC was also reported by Addy et al.  (1973B),  which was suggested to be due not only to chemical r e a c t i -  v i t y of the side-chains of the amino acids but also to size and geometry of their side-chains.  However, the higher efficiency  of tryptophan  binding observed here i s apparently due to the former reason  (i.e.,  higher r e a c t i v i t y ) . It i s noteworthy that as indicated i n Table 12, the methionine-and tryptophan-bound SPI samples possessed amino acid compositions  (except  for the bound amino acids) which were similar (or closely resembled) that found for the SPI (control).  to  Therefore, i t may be concluded that  the reaction between SPI and EDC i n the presence of added amino acid (methionine or tryptophan), at pH 6.5,  did not involve direct reaction  between the EDC and amino or imidazole groups in the protein, since  the  reaction between the EDC and a nucleophilic group (amino or imidazole group) i n the protein should y i e l d amino acid derivatives which would be stable to acid hydrolysis (Riehm and Scheraga, 1966). Although soybean protein contains about 33% a c i d i c amino acids (aspartic and glutamic), the possible lesser involvement of side-chain carboxyl groups i n the EDC-mediated reaction could be due to amidation of aspartic and glutamic acid residues i n the soy protein i n the forms of asparagine and glutamine residues.  The amide groups of these residues,  however, have been shown to be very resistant  to the action of carbo-  diimides (Liberek, 1962; Bodanszky'et a l . , 1976), since the formation of the obligatory active intermediate  (O-acylisourea) with carbodiimides  64.  requires that the carboxyl groups be free (Franzblau et^ a l . , 1963). Therefore, these groups would not participate i n the EDC condensation reaction.  Another factor which could also explain the lesser p a r t i -  cipation of side-chain carboxyl groups to EDC reaction is the finding of Franzblau et a l .  (1963), who reported that the unstable  reactive  intermediate O-acylisourea - i n addition to generally accepted  possi-  b i l i t i e s of formation of a stable N-acylurea, a symmetrical anhydride or a peptide derivative - could c y c l i z e . Thus, i n the case of an aspartic acid residue (in an a-peptide linkage) c y c l i z a t i o n would y i e l d a succinimide r i n g , and a glutamic acid residue (in a-peptide linkage) could form either a glutarimide or acylpyrrolidone r i n g .  If present,  succinimide  structures,  on subsequent acid hydrolysis, would give aspartic acid and glutarimide or pyrrolidone rings would y i e l d glutamic a c i d .  Therefore, the cyclized  residues would again appear as unreacted amino acids.  A t h i r d factor  which could also p a r t l y explain the lesser involvement of  side-chain  carboxyl groups is the formation of undesirable N-acylurea as a byproduct of EDC reaction, which (since N-acylurea i s not an acylating agent) r e s u l t s i n lower coupling y i e l d s .  N-acylureas, however, have been  suggested to be a c i d - l a b i l e (Riehm and Scheraga, 1966), thus, the amino acid involved on acid hydrolysis.  liberating  Undoubtedly a certain amount  of N-acylurea can accompany the formation of desired peptide  derivatives  in the EDC reaction. D. Enzymatic digestion  test  It i s well known that the n u t r i t i v e value of a protein depends not only on the pattern of i t s amino acid composition, but also on the physiological a v a i l a b i l i t y of i t s amino acids.  Of the several characteristics which  65.  determine the n u t r i t i o n a l quality of food proteins, their d i g e s t i b i l i t y is of paramount importance.  Although i n the f i n a l analysis the n u t r i t i v e  quality of a protein must be evaluated by feeding t r i a l s , i n v i t r o enzymatic methods for protein quality evaluation have been u t i l i z e d for preliminary quality evaluations  (Stahmann and Woldegiorgis, 1975). Even  though, the enzymatic hydrolysis may not be complete, the amount of the l i m i t i n g amino acid released by enzymatic digestion i s a good indicator of the amino acid a v a i l a b i l i t y and hence protein q u a l i t y .  Table 13 reports  the amount of each amino acid liberated by an In v i t r o pepsin followed by pancreatin digestion from the SPI (control) and the amino acid-bound soy protein samples with the highest binding yields  (efficiencies).  As i t  can be seen, 2.18g/16gN of L-methionine were released from the methioninebound SPH sample as compared to only 0.25g/16gN of L-methionine released from the SPI, whereas 4.55g/16gN of L-tryptophan were released from the tryptophan-bound SPH sample as compared to only 0.47g/16gN of L-tryptophan released from the SPI.  On the other hand, Table 13 shows that 1.56g/16gN  of L-methionine was released from the methionine-bound SPI sample and 2.06g/16gN of L-tryptophan was released from the tryptophan-bound SPI sample.  These r e s u l t s , therefore, c l e a r l y indicate that the bound  methionine and tryptophan can be e f f i c i e n t l y released from both the SPH and SPI treated samples by enzymatic digestion i n v i t r o .  66.  Table 13 - Liberated amino acids (g/16gN) from SPI and amino a c i d bound soy protein samples with the highest coupling yields^ by pepsin-pancreatin digestion jln v i t r o .  Amino Acid  Threonine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Lysine Histidine Tryptophan Arginine  Control SPI  0..05 1..46 0.,25 0..95 2..74 1..98 2..65 1..51 0..36 0..47 2,.17  Met-bound SPH  0. 20 0. 59 2. 18 0. 73 1. 58 1. 29 1. 58 1. 24 0. 57 0. 54 3. 36  Trp-bound SPH  Met-bound SPI  0.,20 0.,50 0. 09 0.,63 1.,34 1.,09 1.,40 1.,09 0.,53 4.,55 2.,28  0,.18 0..51 1..56 0,.75 2..62 1..75 2..76 1..38 0,.53 0,.49 3,.16  Each value i s the average of duplicate analyses. N u t r i t i o n a l l y important amino acids only were shown. For experimental conditions see footnote b on Table 12.  Trp-bound SPI  0. 19 0. 44 0. 13 0. 56 2. 19 1. 16 1. 68 0. 97 0. 49 2. 06 2. 64  Gel f i l t r a t i o n chromatography Gel f i l t r a t i o n chromatography was used to study the e f f e c t , i f any, of the carbodiimide condensation reaction on the molecular weight d i s t r i b u t i o n pattern of soy protein.  In order to determine the molecular  weights of the different protein fractions the Sephadex G-200 column was calibrated with the standard proteins l i s t e d i n Table 14.  A linear  standard curve was obtained by p l o t t i n g the logarithm of the molecular weights of the standard proteins against the ratios of their elution volumes to the void volume of the column (Whitaker, 1963). shows the c a l i b r a t i o n curve of this column. elution p r o f i l e of SPH s o l u b i l i z e d with SDS.  Figure 16  Figure 17 represents  the  As i t can be seen,  three  protein fractions were separated, the molecular weights of which were estimated  (from the c a l i b r a t i o n curve) to be 158,490, 70,000, and 4,924,  respectively  (in order of increasing elution volume). Figure 18 shows  the elution pattern of tryptophan-bound SPH sample with the highest binding efficiency  s o l u b i l i z e d with SDS. This pattern was obtained  af ter d i l u t i n g (1:5 d i l u t i o n ) each collected fraction (5.2 ml) with eluting buffer.  This d i l u t i o n was necessary i n order to obtain absor-  bance values being within the spectrophotometer's (0.0-2.0).  absorbance range  The f i r s t - e l u t e d f r a c t i o n of the Figure 18 appeared at the  void volume of the column and thus i t s molecular weight could not be estimated.  It is assumed to be very high, since the peak of catalase  curve, whose molecular weight is 244,000, was collected inthe f r a c t i o n (tube) 35, whereas the peak of this protein fraction appeared at the tube 29, namely, was eluted 31.2 ml (6 tubes) before the catalase. The molecular weights of the two other protein fractions of Figure 18 were estimated to be 97,700 and 15,550.  F i g . 19 represents a comparison of  the elution p r o f i l e s of SPH (Fig. 17, used as control) and tryptophanbound SPH (Fig. 18).  It can be c l e a r l y seen that the elution pattern  from the peptic SPH shifted towards the high-molecular weight side as a  Table 14 - Proteins used i n c a l i b r a t i o n of Sephadex G-200 column eluted with 0.1M sodium phosphate buffer, pH 7.0, and containing 0.1% SDS .  Proteins  V /V e o  Mol.Wt.  (Ref.)  1.  Catalase  1.10  244,000  (102)  2.  Bovine serum albumin (Monomer)  1.52  70,000  (112)  3.  a-Chymotrypsin  2.03  22,500  (112)  4.  Sperm whale myoglobin  2.14  17,500  (*)  5.  Ribonuclease A  2.17  13,600  (112)  6.  L-tryptophan  2.38  204  (*)  * Mol. Wt. provided by supplier.  69.  Log.  Molecular  F i g . 16. C a l i b r a t i o n curve of Sephadex G-200.  Weight  Fraction  number  F i g . 17. Gel f i l t r a t i o n pattern of SPH s o l u b i l i z e d with SDS on Sephadex G-200.  71.  Fraction  number  F i g . 18. Gel f i l t r a t i o n pattern of tryptophan-bound SPH s o l u b i l i z e d with SDS on Sephadex G-200. The elution pattern was measured after d i l u t i n g (1:5) each f r a c t i o n with the eluant .  72.  Fraction Fig.  number  19.. Gel f i l t r a t i o n patterns of SPH (solid line) and tryptophan-bound SPH (broken l i n e ) on Sephadex G-200.  73.  result of the EDC condensation reaction.  Figure 20 shows the gel  f i l t r a t i o n pattern of SPH solublized with NaOH (pH 12.0).  Three  fractions were, again, obtained, the molecular weights of which estimated from the c a l i b r a t i o n curve were 158,490, 70,000 and 6,300, respectively  (in order of increasing elution volume).  Figure 21  represents the elution pattern of the methionine-bound SPH sample with the highest binding e f f i c i e n c y ,  s o l u b i l i z e d with NaOH (pH 12.0).  i t can be seen four fractions were separated.  The f i r s t - e l u t e d  As fraction  appeared at the void volume of the column, and thus, the estimation of i t s molecular weight was not possible.  The other fractions were estimated  to have molecular weights of 91,200, 28,900, and 13,600, (in order of increasing elution volume).  respectively  Figure 22 shows a comparison  of the elution p r o f i l e s of SPH (Fig. 20, used as control) and methioninebound SPH sample (Fig. 21).  It can be seen, again, that the EDC conden-  sation reaction caused an increase i n the molecular weights of soy protein fractions.  This finding is i n accordance with the results of several  papers dealing with the use of carbodiimides i n peptide synthesis. Sheehan and Hlavka (1957), have reported that the reaction of a commercial gelatin with a WSC (in the absence of nucleophiles)  resulted i n very rapid  gelation attributed to interchain c r o s s - l i n k i n g by amide bond formation occuring between side-chain carboxyl and amino groups.  Blout and Des  Roches (1959), then, reported that through the use of carbodiimides as condensing agents the molecular weights of polypeptides could be increased significantly,  and moreover, that i n several instances they observed four-  fold or greater increases  i n molecular weights.  These workers also suggested  that this increase i n molecular weight occurred because of terminal i n t e r -  1.0  .  0.8  L  E c  O  CO CM  I  30  40  Fraction Fig..20.  50  60  70  80  number  G e l f i l t r a t i o n p a t t e r n of SPH s o l u b i l i z e d w i t h NaOH (at pH on Sephadex G-200.  12.0)  75.  Fig.  21.  G e l f i l t r a t i o n p a t t e r n of methionine-bound SPH w i t h NaOH (at pH 12.0) on Sephadex G-200.  solubilized  3 0  40  Fraction  5 0  60  70  8 0  number  F i g . 2 2 . G e l f i l t r a t i o n p a t t e r n s o f SPH ( s o l i d l i n e ) and m e t h i o n i n e bound SPH (broken l i n e ) on Sephadex G-200..  77.  molecular peptide bond formation, but d e f i n i t e proof was d i f f i c u l t obtain.  to  However, indirect evidence that linear peptide bond formation  was involved could be deduced from their observation that  polypeptides  with one terminal amide group ( i . e . , the carboxyl end of the polypeptide chain was blocked by amide formation) and one terminal amine group did not undergo molecular weight increases when treated with carbodiimides, and also from the results obtained with p o l y - L - p r o l i n e . peptide no inter-molecular condensations  With this poly-  other than terminal may occur  and p o l y - L - p r o l i n e did show a d e f i n i t e molecular weight increase when treated with carbodiimides.  More recently, Mukerjee and S r i Ram (1965),  by carrying out the reaction between bovine serum albumin (BSA) and 1ethyl-3-(3-dimethylaminopropyl)  carbodiimide i n the absence of nucleo-  p h i l e s , and based on subsequent  sedimentation studies,  some aggregation of the protein had occurred.  reported that  These workers also  reported that, on reaction of EDC with BSA i n the presence of d i g l y c i n e , part of the peptide apparently was conjugated to the protein. In order to determine i f there was any s e l e c t i v i t y  regarding the  binding of added amino acid to different soybean protein fractions during the EDC reaction, the elution p r o f i l e s of tryptophan-bound SPH 3  sample, s o l u b i l i z e d with NaOH (pH 12.0), at 280 nm (Fig. 23) and 210 3  nm' (Fig. 24) were obtained, and consequently, separated fractions  the r a t i o of the three  (areas under the eluted peaks) for each elution  p r o f i l e was determined.  The area r a t i o (%) of the three fractions of  Absorption maximum of proteins due to the presence of aromatic amino acids (tryptophan, tyrosine, and phenylalanine). k Absorption of the peptide bond (Pomeranz and Meloan, 1971).  Fraction  number  F i g . 23. Gel f i l t r a t i o n pattern (A280nm) ° f tryptophan-bound SPH s o l u b i l i z e d with NaOH (at pH 12.0) on Sephadex G-200. The elution pattern was measured after d i l u t i n g (1:5) each fraction with the eluant.  79.  20  30  40  50  Fraction Fig.  60  70  number  24. Gel f i l t r a t i o n pattern (A2in ) of tryptophan-bound SPH s o l u b i l i z e d with NaOH (at pH 12.0) on Sephadex G-200. The elution pattern was obtained after d i l u t i n g (1:20) each fraction with 0.1 M sodium phosphate buffer, pH 7.0. nm  80  80. F i g . 23 was (44.79):(36.46):(18.75), i n order of descending molecular weight, as compared to the r a t i o of (45.91):(38.26):(15:83) of corresponding fractions of F i g . 24. close (the small differences  the  Since these two ratios are very  between the corresponding areas % of  F i g s . 23 and 24 are n e g l i g i b l e and can be attributed to the d i l u t i o n effects),  i t is concluded that there was no selective amino acid binding  among the different  soy protein fractions during the EDC reaction.  GENERAL DISCUSSION From the results of Table 12, i t appeared that the L-methionine and L-tryptophan added could be very e f f i c i e n t l y by the EDC condensation reaction. digestibility  bound to both SPH and SPI  Moreover, Table 13 indicated a high  i n both cases ( i . e . , amino acid bound SPH and SPI).  The  use of SPI (instead of SPH) as the starting material i n this reaction would be more advantageous  for the following reasons:  (1)  elimination  of the pepsin hydrolysis step required for the production of SPH. Thus, the covalent binding procedure would be s i m p l i f i e d to one-step process which would permit direct binding of amino acids to soy protein;  (2)  prevention of the loss of some amino acids which is associated with the pepsin hydrolysis step (see Table 12); and (3) very high product y i e l d s (95-99%) as compared to the low y i e l d s  (maximum y i e l d 36%) obtained when  SPH i s used as the starting material. The results of Table 12 showed that the L-methionine content i n the methionine-bound SPI sample was 5.92g/16gN; this value i s 6.3 times the methionine l e v e l ,  0.94g/16gN, of the unmodified SPI.  On the other hand,  the L-tryptophan content i n the tryptophan-bound SPI sample (Table 12) was 10.74g/16gN, namely 11.3 times the tryptophan content,  0.95g/16gN, of the  81.  SPI  (control).  Although such contents (5.92g/16gN methionine and  10.74g/16gN tryptophan) are considered to be a surplus for optimum amino acid n u t r i t i o n , i t is possible,  however,  to obtain a protein  product with a satisfactory methionine or tryptophan content i f factors affecting  the  the binding of each amino acid to soy protein ( e . g . ,  amino acid concentration, carbodiimide concentration, pH, etc.) properly controlled upon the application of EDC reaction.  are  A more  p r a c t i c a l way to decrease the content of these amino acids i s to mix the products with SPI.  A 12:20:1 mixture, for example, of methionine-  bound SPI sample: SPI:tryptophan-bound SPI sample w i l l contain 2.7g/16gN methionine,  0.9g/16gN cystine,  and l.lg/16gN tryptophan.  Such a mixture  w i l l resemble the i d e a l protein proposed by the FAO/WHO Expert Committee (1973), i n respect to the l e v e l s of sulfur-containing amino acids 16gN) and tryptophan (1.0g/16gN).  It is well known that,  (3.5g/  i f not properly  considered, the addition of an excess of the f i r s t l i m i t i n g amino acid may result in a secondary deficiency and, thus, there w i l l be no improvement i n the o v e r a l l quality of the diet  (Bressani, 1975). Consequently,  the f i n a l mixture should include both the f i r s t and second l i m i t i n g amino acids i n the amounts needed to maintain the proper r a t i o according to requirements of the reference pattern (FAO/WHO Expert Committee - suggested pattern). A major obstacle i n the application of chemical modification to food proteins is the potential health hazards. (Whitaker, 1977)  Although enzymatic procedures  offer advantages with regard to potential health hazards,  chemical procedures are frequently more advantageous due to a low cost (Feeney,  1977).  for different  products  The major advantage of EDC used i n this  study i s the f a c i l e removal of excess reagent and the corresponding urea  82.  by washing with d i l u t e a c i d or water. (Aumuller, 1963)  Moreover, i t has been reported  that the mammalian t o x i c i t y of carbodiimides  ( L D ^ Q of dicyclohexylcarbodiimide was reported to be 2.6 g/kg  i s low in rats).  However, the p o s s i b i l i t y that some E D C and urea d e r i v a t i v e s were s t i l l remained i n the f i n a l product, a f t e r i t s p u r i f i c a t i o n , could not be excluded.  I t i s obvious, therefore, that a thorough b i o l o g i c a l  evaluation i s needed to assess the e f f e c t of chemical m o d i f i c a t i o n of soy proteins by E D C on t h e i r i n vivo d i g e s t i b i l i t y as w e l l as  any  e f f e c t that m o d i f i c a t i o n might have on the t o x i c i t y of t h e i r d e r i v a t i v e s (methionine- or tryptophan-bound soy p r o t e i n s ) .  CONCLUSION  A c c o r d i n g t o the data presented condensation  i n t h i s study, the c a r b o d i i m i d e  r e a c t i o n appeared t o be a v e r y e f f e c t i v e means f o r  c o v a l e n t l y b i n d i n g methionine thus improving  and tryptophan  i t s nutritional quality.  product w i t h a s p e c i f i e d methionine  t o soy p r o t e i n  and  I t i s f e a s i b l e to o b t a i n a  or tryptophan  content by simply  c o n t r o l l i n g the f a c t o r s a f f e c t i n g the b i n d i n g of each o f these amino a c i d s t o soy p r o t e i n d u r i n g the c a r b o d i i m i d e r e a c t i o n . the methionine  and tryptophan  I t was found  that  added c o u l d be v e r y e f f i c i e n t l y bound to  both soy p r o t e i n h y d r o l y s a t e and s o y p r o t e i n i s o l a t e by the c a r b o d i i m i d e reaction.  Moreover, a 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 demonstrated a  high d i g e s t i b i l i t y  i n both c a s e s .  Soy p r o t e i n i s o l a t e , however, was  found  to be more advantageous as s t a r t i n g m a t e r i a l i n the c a r b o d i i m i d e  (EDC)  r e a c t i o n than soy p r o t e i n h y d r o l y s a t e , because o f the s i m p l i f i c a t i o n  of  the c o v a l e n t b i n d i n g procedure  t o o n l y one-step  process,  prevention  of  the l o s s of some amino a c i d s a s s o c i a t e d w i t h the p e p s i n h y d r o l y s i s  s t e p , and above a l l , because of the v e r y h i g h product  yields  i n t h i s case.  o b t a i n e d , as  determined of  The n u t r i t i o n a l q u a l i t y of the product  by amino a c i d c o m p o s i t i o n , was found  soyprotein i s o l a t e Gel f i l t r a t i o n  to be s u p e r i o r t o t h a t  (control).  chromatography e l u c i d a t e d the f o r m a t i o n o f h i g h e r -  m o l e c u l a r weight substances a result  from p e p t i c h y d r o l y s a t e s of soy p r o t e i n as  of the EDC r e a c t i o n .  Gel f i l t r a t i o n  chromatography, moreover,  r e v e a l e d t h a t t h e r e was not any s e l e c t i v i t y c o n c e r n i n g added amino a c i d to t h e d i f f e r e n t reaction.  obtained  the b i n d i n g of  s o y p r o t e i n f r a c t i o n s d u r i n g the EDC  The carbodiimide r e a c t i o n may be of s p e c i a l importance from the viewpoint of food processing i n p r a c t i c e .  Further research i s required  to determine the commercial and economic f e a s i b i l i t y of t h i s method as a means f o r improving the n u t r i t i o n a l and f u n c t i o n a l properties of food proteins.  Moreover, i t should be emphasized that the safety and  a c c e p t a b i l i t y of the product as a human food must be c a r e f u l l y evaluated.  LITERATURE CITED  1.  Addy, M. E . , Steinman, G . , and M a l l e t t e , M. F . 1973A. A problem in the mechanism of carbodiimide-mediated synthesis of peptides in aqueous medium. Biochem. Biophys. Res. 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What i s soy protein?  chemical  Food Technol. 26 (No. 5):44.  In "Fabricated Westport.  Soybeans as a food source.  95.  APPENDIX  96.  Fractional f a c t o r i a l experiment (Taguchi,  1957)  A 2-level experiment for selection of the factors influencing the amino acid binding to SPH.  Fractional f a c t o r i a l design  3  Run No.  1  2  1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16  1 1 1 1 1 1 1 1 2 2 2 2 2 2 2 2  1 1 1 1 2 2 2 2 1 1 1 1 2 2 2 2  3  3  See Table 1.  4  5  6  1 1 2 2 1 1 2 2 1 1 2 2 1 1 2 2  1 1 2 2 1 1 2 2 2 2 1 1 2 2 1 1  1 1 2 2 2 2 1 1 1 1 2 2 2 2 1 1  Factor l e v e l 7 8 9 10 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2  11  12  1 2 1 2 .2 1 2 1 2 1 2 1 1 2 1 2  1 2 2 1 1 2 2 1 1 2 2 1 1 2 2 1  13 1 2 2 1 1 2 2 1 2 1 1 ' 2 2 1 1 2  14  15  97.  A scheme to detect the  1.  interactions  Assign the nine factors (to be tested for their possible influence on amino acid binding to SPH) to columns 1,2,4,5,6,8,11,12,and 13:  Column 1 • 2 4 5 6 8 11 12 13  PH reaction temperature pepsin hydrolysis time amino acid concentration SPH concentration reaction time carbodiimide concentration a c t i v a t i o n time polarity  The. columns shown on the connecting lines of the scheme are l e f t blank for detection of interactions. The effect of interactions w i l l appear in those columns, i . e . : Column 3 7 9 10 14  pH x reaction temperature pH x SPH concentration pH x reaction time reaction temperature x reaction time carbodiimide concentration x amino acid concentration  The unused column(s) w i l l be used for the c a l c u l a t i o n of the error term.  98.  2.  Assign 2 d i f f e r e n t levels  to each f a c t o r .  3.  C a r r y out runs 1 to 16 i n random o r d e r .  4.  Determine i n f l u e n t i a l f a c t o r s and i n t e r a c t i o n s by c a r r y i n g the a n a l y s i s of v a r i a n c e (see T a b l e s 2 and 3 ) .  out  99.  Fractional f a c t o r i a l (Taguchi,  experiment  1957)  A 3-level experiment for optimization of amino acid binding to SPH. Fractional f a c t o r i a l  Run No.  1  1 2 3 4 .5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27  1 1 1 1 1 1 1 1 1 2 2 2 2 2 2 2 2 2 3 3 3 3 3 3 3 3 3  a  See Table 4.  2  3  4  5 1 2 3 1 2 3;. 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3  6  Factor l e v e l 7 8 9 1 2 3 2 3 1 3 1 2 2 3 1 3 1 2 1 2 3 3 1 2 1 2 3 2 3 1  design  10 1 2 3 2 3 1 3 1 2 3 1 2 1 2 3 2 3 1 2 3 1 3 1 2 1 2 3  11  12 1 2 3 3 1 2 2 3 1 2 3 1 1 2 3 3 1 2 3 1 2 2 3 1 1 2 3  13  100.  A scheme to d e t e c t the  interactions  9 e  1.  10 •  12 e  13 •  A s s i g n the f i v e i n f l u e n c i n g f a c t o r s , which are to be to columns 1,5,9,10 and 12:  optimized,  (a) methionine b i n d i n g Column  1 5 9 10 .12  (b) tryptophan Column  1 5 9 10 12  pH SPH c o n c e n t r a t i o n carbodiimide concentration a c t i v a t i o n time r e a c t i o n time  v  binding pH SPH carbodiimide concentration amino a c i d c o n c e n t r a t i o n r e a c t i o n temperature  A l l of the unused columns w i l l be used f o r the c a l c u l a t i o n of the term. 2.  A s s i g n 3 d i f f e r e n t l e v e l s to each f a c t o r .  3.  C a r r y out runs 1 to 27  i n random order.  4.  Carry out  of v a r i a n c e  5.  C o n s t r u c t response curves by p l o t t i n g theaverage of the c a l c u l a t e d f o r each l e v e l of the f a c t o r s .  the a n a l y s i s  (see T a b l e s  5 and  6). data  error  

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