In r..- ”nu." ‘h «um zuhnuuu“: hut: \ ‘ . 2.. 9 L . . :5... :5... . .. ; iCHi IIIIIIIIIIIIIIIIIIIIIIIIIII * minimwillImuwnmminimumz 3 1293 00579 9089 *LIBRARY Michigan State , University This is to certify that the dissertation entitled A5567} for TI/FS/n WW, ééfmaf'r/fs/n phi/rim l7 fixafe/n /fl4,é,raz5 ' presented by K€SLUJM LXI/t has been accepted towards fulfillment of the requirements for Pg p degreein F004 fag/’VL’ WA: Major professor Date 5' I873? / / MS U is an Affirmative Action/Equal Opportunity Institution PLACE IN fiETURN BOX to remove this checkout from your record. TO AVOID FINES return on or before date due. DATE DUE DATE DUE DATE DUE MSU Is An Affirmative ActiorVEqual Opportunity Institution ASSAYS FOR TRYPSIN AND CHYMOTRYPSIN INHIBITION BY PROTEIN INHIBITORS by Keshun Liu A Dissertation Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Food Science and Human Nutrition 1989 ABSTRACT ASSAYS FOR TRYPSIN AND CHYMOTRYPSIN INHIBITION BY PROTEIN INHIBITORS by Keshun Liu For determining the antitryptic activity of soybean products, the current colorimetric method has been modified as follows: a) water rather than dilute alkali is used for extracting the inhibitors, b) the aqueous extract is destabilized with Tris buffer and filtered before, rather than after, the reaction, c) porcine rather than bovine trypsin is used, (1) the enzyme, not the substrate, is added last to the reaction mixture, and e) the assay volume is reduced from 10 to 4 ml. The proposed mOdification is more sensitive and reliable than the current method. The relative standard deviation (RSD) was :i: 3.5% (n255). For assaying chymotrypsin inhibitor activity, a colorimetric method is developed, using benzoyl-L-tyrosine-p-nitroanilide as a substrate. Since the inhibition curve (enzyme activity, A385’ vs. concentration of inhibitor, [1]) fits the reverse ratio function, y s l/(a + bx), linearity of l/A385 (l/y) vs. [I] (x) is obtained. Accordingly, one chymotrypsin inhibitor unit (CIU) is defined as a 0.01 increase of (A0 - l), where A0 is the 385/A385 335 enzyme activity when [I]-O. The method, although involves mathematical data conversion, is relatively simple and reliable. The RSD was t 4.8% (n=22). In the assays of trypsin and chymotrypsin inhibition by soybean protease inhibitors, two procedures were used, the common procedure in which substrate is added to mixture of inhibitor and enzyme, and the new procedure in which enzyme is added to mixture of inhibitor and substrate. The inhibition value of the common procedure was either equal to or lower than that of the new procedure, depending on the premix pH and preincubation time, while the value of the new procedure were constant regardless of the premix pH and the preincubation time. When the premix pH was jumped from the acidic or alkaline ranges to near neutral, the sequence effect was abolished completely for trypsin inhibition and partially for chymotrypsin inhibition. These observations are in accordance with the reactive site model proposed by Ozawa and Laskowski, Jr. (1966, J. Biol. Chem. 241, 3955) and suggest an instantaneous binding between inhibitors and enzymes, which may become a complement to the standard mechanism. For assaying protein inhibitors of proteases, the new procedure is preferable to the common procedure. DEDICATION To my parents & Grandparents ii ACKNOWLEDGMENT I feel deeply grateful to Dr. Pericles Markakis, my academic advisor, for his excellent guidance throughout the Ph. D. program at Michigan state University, and for his critical advice in the preparation of this manuscript. Appreciation is expressed to the members of my Guidance Committee: Dr. Thomas Deits of the Department of Biochemistry, Dr. David R. Dilley of the Department of Horticulture, Drs. Denise Smith and Won Song of the Department of Food Science and Human Nutrition, for their advising on the program and reviewing this manuscript. Special thanks are extended to Dr. Clarence Suelter for serving in my Committee at the beginning of the program, and to Mary Schneider, Mary Edington, and Joan Carpenter for their clerical service. I wish to express my gratitude to the Coca-Cola Company for a three-year scholarship to finance my Ph. D. program, and to the Staff of the External Technical Affairs of the Company: Dr. Alex Malaspina, Dr. R. Fenton-May, Ms. Sharon Coleman, Mrs. Rosemary Dcshiell-Young, Mrs. Diane Ewing, Ms. Sarah Allen and Dr. Ardine Kirchhofer, for their friendly help during my 1988 Summer internship at the Company. Special thanks are also expressed to lab f ellows: Hoda El-zeny, Suparmo, Pavlos Aspris, P. Ogun and S. Alani and Dr. Aloisio Antunes, to friends, Jian Pan, Zhonghao Zhang, Mary Sokalski, Thomas Herald, Mr. and Mrs. Wolf, Mr. and Mrs. Morrill (host family), Rex Alocilja and Vangie Alocilja, Sung-Yuan Wang, Zhouji Chen, Huiren Zhou and Wuming Dong, for their friendship, and to my brothers, Kechuan and Kelian, for their encouragement. iii TABLE OF CONTENTS LIST OF TABLES. LIST OF FIGURES . LIST OF SYMBOLS, ABBREVIATIONS 0R NOMENCLATURE . INTRODUCTION. LITERATURE REVIEW . I. II. III. IV. V. VI. VII. VIII. Proteases . . Serine Proteases. . . Protein Inhibitors of Proteases . Protease Inhibitors from Soybeans . A Standard Mechanism of Inhibition. Nutritional Implications of Protease Inhibitors. Current Methods for Trypsin Inhibition Assay. . Current Methods for Chymotrypsin Inhibition Assay . MATERIALS AND METHODS . Part 1. TRYPSIN INHIBITION ASSAY . I. II. Methodology . i. Buffer and solutions. ii. Inhibitor sample preparation. iii. Procedure . iv. Calculating trypsin inhibition. Procedures for Studying the Effect of Reactant Mixing Sequence on the TIA Assay . i. Buffers and solutions . '11. Procedures. iii. Calculating trypsin inhibition. Part 2. CHYMOTRYPSIN INHIBITION ASSAY. I. Methodology . i. Buffer and solutions. . ii. Inhibitor sample preparation. iii. Assay procedure . iv Page vii . viii xi \iO‘WU-D oo 13 15 l7 l7 17 17 18 18 19 20 20 20 20 21 21 21 21 21 iv. v. vi. Chymotrypsin inhibitor unit (CIU) . . Correction for effect of enzyme concentration . Expressing CIA in terms of pure BB inhibitor. II. Procedures for Studying the Effect of Mixing Reactant Sequence on the CIA Assay . . . . . . . . . 1. 1L iii. Buffers and solutions . Assay procedure . . Calculating chymotrypsin inhibition . RESULTS AND DISCUSSION. Part 1 TRYPSIN INHIBITION ASSAY . I. General Assay Conditions. 1. ii. iii. iv. v. Enzyme concentration. Reaction time. . Substrate (BAPA) concentratiOn. Ca ion concentration. pH of the assay buffer. II. Effect of the Reactant Mixing Sequence on the TIA Assay . i. ii. iii. iv. III. Effect of the preincubation time on the reactant sequence effect. . Effect of the premix pH on the reactant sequence effect . Jumping the premix pH. . . The TIA assay as related to limited hydrolysis of inhibitors. . . . . . . . . . . . . . . . Modification of the Current Method for Determining TIA in Soybeans. i. ii. iii. iv. v. vi. vii. viii. Extracting the inhibitors . . Sample cleanup before the reactiOn. Choosing a proper sample dilution . . Using porcine instead of bovine trypsin . Using the E- last test . . . Reducing the volume of the reaction mixture . Expressing TIA. . Applying the modified method to some legume products. Part 2 CHYMOTRYPSIN INHIBITION ASSAY. I. Methodology . i. ii. iii. iv. Enzyme activity vs enzyme concentration . Enzyme activity vs reaction time. Chymotrypsin inhibition curve . . . Linearization of the inhibition curve . 22 22 23 23 23 23 24 25 25 25 25 25 29 29 29 31 31 34 39 39 48 48 50 50 56 56 62 63 65 67 67 67 67 67 71 v. vi. vii. viii. ix. Defining chymotrypsin inhibitor units (CIU) Effect of enzyme concentration on the CIA assay . Inhibitor dilution effect . Presence of acetone . . Application of the new method to some legume products . II. Effect of the Reactant Mixing Sequence on the CIA Assay . 1. Effect of the preincubation time on the reactant sequence effect . . ii. Effect of the premix pH on the reactant sequence effect . iii. Abrupt change of the premix pH. . iv. The CIA assay as related to limited hydrolysis of inhibitor . . . . . . . . . . . . . . . . CONCLUSIONS . RECOMMENDATIONS FOR FUTURE STUDIES. BIBLIOGRAPHY. vi 71 73 78 78 80 80 80 84 87 87 92 93 94 LIST OF TABLES Table . Page 1 Procedure for assaying TIA in legume products. . . . . . . . . 19 2 Extraction of trypsin inhibitors from raw and cooked soybeans by various solvents and different shaking times. . . . . . . . 49 3 Comparison between two clarifiers and two filter papers in the cleanup of a raw soybean extract for the TIA assay. . . . . . 52 4 Curving loci in the line connecting trypsin activity and inhibitor concentration. . . . . . . . . . . . . . . . . . . . 54 5 TIA in some commercial soy products and legume seeds assayed by both the current and the proposed methods . . . . . . . . . 66 6 CIA in some commercial soy products and legume seeds assayed by the new method . . . . . . . . . . . . . . . . . . . . . . 81 7 Effect of an abrupt change of premix pH from 4.0 to 7.0 on assaying the CIA of soybean BB inhibitor . . . . . . . . . . . 88 vii LIST OF FIGURES Figure l The catalytic triad (A) and the catalytic mechanism (B) of a serine protease. . . . . . . . . 2 Structure of the hemiacetal adduct formed between a peptide aldehyde (R-CHO) and the active site serine of a serine protease . . 3 Schematic diagram of the tryptic conversion of virgin into modified soybean trypsin inhibitor . 4 Regulation of the secretion of trypsin by the pancreas . 5 Trypsin inhibitor activity (TUI/ml extract) in relation to level of crude soybean extract . . . 6 Relationship between absorbance at 410 nm and amount of porcine trypsin 7 Effect of porcine trypsin concentration on the assay of soybean TIA. 8 Relationship between absorbance at 410 nm and reaction time in the absence and in the presence of inhibitors 9 Effect of substrate (BAPA) concentration on the assay of soybean TIA. lO Effect.of assay buffer pH on the assay of soybean TIA. 11 Effect of the sequence of mixing the reactants on the assay of soybean Bowman-Birk trypsin inhibition . 12 Relative difference in soybean Bowman-Birk trypsin inhibition values obtained by the S-last and the E-last tests as a function of the preincubation time. 13 Relative difference in porcine trypsin inhibition values obtained by the S-last and the E-last tests as a function of the premix pH . 14 Relative difference in bovine trypsin inhibition values obtained by the S-last and the E-last tests as a function of the premix pH . viii Page 10 12 14 21 27 28 30 32 33 35 37 38 15 l6 l7 l8 19 20 21 22 23 24 25 26 27 28 29 30 31 Jumping the premix pH from 3.5 to 6.5 during assaying porcine trypsin inhibition of soybean BB inhibitor . . . . . . . . . Jumping the premix pH from 9.0 to 6.5 during assaying porcine trypsin inhibition of soybean BB inhibitor . . . . . . . . . Jumping the premix pH from 3.5 to 2.5 during assaying porcine trypsin inhibition of soybean BB inhibitor . . . Effect of cleanup of soy extracts before or after the color reaction on the TIA assay Effect of degree of trypsin inhibition obtained by various dilutions of a raw soybean extract on the estimate of antitryptic activity in soybeans . Comparison of porcine and bovine trypsins for assaying TIA in soybeans . Effect of the sequence of mixing the reactants on the assay of antitryptic activity in soybeans . Relative difference in TIA obtained by the S-last and the E-last tests as a function of the preincubation time . Relative difference in TIA obtained by the S-last and the E-last tests as a function of the premix pH. . . Effect of the reaction mixture volume on the measurement of antitryptic activity in soybeans . Relationship between chymotrypsin activity (A 385) and amounts of enzyme. . Relationship between chymotrypsin activity (A 8 ) and the reaction time in the absence of and in the presence of soybean BB inhibitor . Chymotrypsin activity (A 5) as a function of soybean BB inhibitor concentration. 38 Reversed values of chymotrypsin activity (l/A ) as a function of soybean BB inhibitor concentration . Chymotrypsin inhibitor activity (CIA) as a function of soybean BB inhibitor . . . . . . . . . . . . . . . . . . . . . Effect of Ao , corresponding the amount of chymotrypsin, 385 on the CIA assay . . . . . . . . . . . . The correction factor c as a function of A0385 for CIA assay. . . . . 40 41 42 51 55 57 58 60 61 63 68 69 70 72 74 75 76 32 33 34 35 36 37 Linegrization of the correction factor c as a function of A 385 . Effect of percentage of chymotrypsin inhibition, corresponding various dilutions of the soybean BB inhibitor solution, on the CIA assay. Relationship of A3 5 vs inhibitor concentration in the S-last and the E-last tesés . Relationship of CIA (in terms of CIU) vs inhibitor concentration in the S-last and the E-last tests . Relative difference in CIA of soybean BB inhibitor, assayed by the S-last and the E-last tests as a function of the preincubation time. Relative difference in CIA of soybean BB inhibitor, assayed by the S-last and the E-last tests as a function of the premix pH. 77 79 82 83 85 86 A335 A410 BB BAPA BTpN A CIA CIU IU IUI cat hyd TI TIA TU TUI LIST OF SYMBOLS, ABBREVIATIONS OR NOMENCLATURE Absorbance at 385 nm. Absorbance at 410 nm. Bowman-Birk (soybean inhibitor). Benzoyl-DL-arginine-p-nitroanilidc. Benzoyl-L-tyrosine-p-nitroanilide. Chymotrypsin inhibitory activity Chymotrypsin inhibitor units as is defined under Materials & Methods. Enzyme. Virgin inhibitor with a reactive site peptide bond intact. Modified inhibitor with a reactivesite peptide bond hydrolyzed. Internatioal units. One 10 of enzyme is that amount which catalyzes the formation of l umole of product per min under defined conditions. Internatioal units inhibited. Hydrolysis rate constant for a peptide bond. Equilibrium constant of peptide hydrolysis. Trypsin inhibitor. Trypsin inhibitory activity. Trypsin units as defined under the Materials and Methods. Trypsin units inhibited. xi INTRODUCTION Substances which are capable of inhibiting the proteolytic activity of certain enzymes are ubiquitous (1,2). Some of them are proteins in nature. Protease inhibitors have gained the attention of scientists in many disciplines: (a) nutritionists, because of the possible toxic and antinutritional effects of these inhibitors to humans and animals (2-4), (b) enzymologists, because inhibitors can be used as a natural tool to probe the active center of enzymes (5-7), and (c) protein chemists, because the reaction of these inhibitors with enzymes provides a model system for studying protein-protein interactions (8- 10). However, the greatest amount of research has been directed to soybean protease inhibitors because of their possible influence on the nutritive value of soybean protein, one of the the most important sources of vegetable proteins. (2-4,l 1,12). Most protease inhibitors, such as the soybean Kunitz and Bowman-Birk (BB) inhibitors, are trypsin and/or chymotrypsin inhibitors. To study these inhibitors, we often perform inhibition assays. This research is comprised of two parts. The first part deals with modifications of the currently used method for measuring trypsin inhibitor activity (TIA) in soybean products (13,14). The second part deals with a proposed method for assaying chymotrypsin inhibitor activity (CIA) using a synthetic substrate (15). This thesis challenges the traditional view that preincubation of inhibitor with enzyme is necessary for obtaining equilibrium data in an inhibition assay (1,13,14,16-20) by demonstrating an effect of the reactant mixing sequence on the inhibition assay. This reactant sequence effect is attributed to limited hydrolysis of inhibitors by the very enzyme they inhibit, in accordance with a standard mechanism proposed by Ozawa and Laskowski, Jr. for protein inhibitors of serine proteases (21). LITERATURE REVIEW 1. Proteases Proteases are relatively small proteins (25-35 KD), which cleave other proteins at peptide bonds. All proteases utilize a general acid-base type of cleavage mechanism, but the side chains that act as the acid and base differ. Based on these differences, proteases are grouped into four classes: metallo proteases, carboxyl or acid proteases, thio proteases and serine proteases (22). II. Serine Proteases Serine proteases comprise a large group of proteases which use the hydroxyl group of a serine residue asa Lewis acid during cleavage action towards proteins. A common test for these enzymes is the inhibition of hydrolase activity by the reaction of the serine residue with diisopropyl- phosphofluoridate (DFP) (22). I] l . h . The active site of a typical serine proteinase is made up of two regions: (1) the catalytic site, and (2) the substrate binding site(s). The catalytic . . . 195 . 57 102 . . Site Is composed of residues Ser , HIS and Asp (chymotrypsm numbering). These three residues form a hydrogen binding system often referred to as the catalytic triad or the charge relay system. The catalytic triad and the catalytic mechanism is depicted in Fig. l. The substrate (ester or amide) carbonyl carbon undergoes a nucleophilic attack by the hydroxyl group of Serlgs, 3 Asp 102 His 57 Set 195 O - -0/ ‘ 0 ---- ll ---- ijf N ---- l l ----- u (1 - 5)- V 8‘ .A Set S|er-195 I * . o H ......... “3-57 O H-Hts \ —’ n-coun-n R-fi-NH-R i), D 03 PHZN'R Sb! H HR Sm l "'—' I R-C-OH ' \ I . n/ o 0. k0 RCOZH sf" 0... ........ Hi, B Fig. l The catalytic triad (A) and the catalytic mechanism (B) of a serine protease (22). In B, the catalytic residue Aspm2 is not shown. 5 which leads to the formation of an acyl enzyme intermediate. Hiss7 functions as a catalytic base by assisting in the transfer of a proton from the serine hydroxyl to the substrate leaving group. Asp102 is believed to stabilize the 57 conformation or Hiss7 tautomer (23). His The substrate binding site, which is made up of the primary structure and of the overall three dimentional structure of the enzyme, determines the substrate specificity of a serine protease. T ' h m r i Trypsin and chymotrypsin, the two major proteolytic enzymes of the pancreas, belong to serine proteinase group. They are believed to be similar in terms of catalytic mechanism. The central difference between the two lies in their specificity. Trypsin cleaves a peptide bond most efficiently on the carboxyl side of positively charged amino acids (Lys and Arg). Chymotrypsin favors the peptide bonds on the carboxyl side of large amino acids (Trp, Phe, Tyr and Leu) (24). I] l . E . l .1. . n Thompson (25) initially proposed that the inhibition of porcine pancreatic elastase by some tight-binding peptide aldehydes was a result of the formation of a hemiacetal linkage between the aldehyde carbonyl of the inhibitor and the active site serine of the enzyme. This tetrahedral adduct was presumed to be similar to the tetraaldehydral intermediate formed during peptide or ester bond hydrolysis (Fig. 2). This inhibition mechanism of serine protease was supported by many studies with other serine proteases and their inhibitors (6,7,26). Set R—C—H OH Fig. 2. Structure of the hemiacetal adduct formed between a peptide aldehyde (R-CHO) and the active site serine of a serine protease. Tryosio from gifforoot oiologiool §ooroo§ Trypsin from various biological sources have been found to differ in certain aspects (27,28). Human, bovine, ovine and porcine trypsins are quite similar in pH (8.0-8.2) and temperature (45-50 C) optima, Michaelis-Menten constants and kinetics of esterolytic activity. However, porcine trypsin is found to have a lower isoelectric point and higher resistance to bases than ovine and bovine trypsins. Human trypsin resembles porcine trypsin more than the other two. Another important variation among trypsins is in the extent of inhibition by some trypsin inhibitors. For example, unlike bovine trypsin, human trypsin is not inhibited by chicken ovomucoid, bovine and porcine pancreatic inhibitors, or the soybean Kunitz inhibitor (29). III. Protein Inhibitors of Proteases Protease inhibitors are substances which, when added to a mixture of protease and substrate, bind to the enzyme and produce a decrease in the rate of substrate cleavage. Protein inhibitors of proteases are ubiquitous. All but one of these inhibitors act on serine proteases. The sole exception is the complex between the potato carboxypeptidase inhibitor and carboxypeptidase A, which is a zinc metalloprotease (30). The major common element in the structures of these inhibitors is the primary contact regions or the reactive site loop. The formation of the loop is highly complementary to the surface of the enzymes and resembles that of an oligopeptide substrate. When bound to the serine proteinase active site, these reactive site loops project out from the inhibitor, so that they are accessible to the active site of proteolytic enzymes (30). IV. Protease Inhibitors from Soybeans The protein inhibitors that have been isolated from soybeans fall into two main categories, the Kunitz soybean inhibitor and the Bowman-Birk (BB) inhibitor. i h' ' r The Kunitz soybean inhibitor was first isolated and crystallized by Kunitz (31,32). The isolation involves extracting soybeans with water and precipitating the inhibitor with alcohol. It has a MW between 20-25 KD, with a specificity directed primarily toward trypsin. The inhibitor was shown to combine with trypsin in a stoichiometric fashion, i.e., 1 mole of inhibitor inactivates 1 mole of enzyme. The complete amino acid sequence of the inhibitor was established by Koide et a1. (33). It consists of 181 amino acid residues and two disulfide bonds, with a reactive site at residues Arg63 and lie“. W The soybean BB inhibitor was first described by Bowman (34) as an acetone- insoluble factor in contrast to the alcohol-insoluble factor, which was later recognized as the Kunitz inhibitor. Isolation of the BB inhibitor involves extracting beans with 60% alcohol and precipitating the inhibitor with acetone. Later, Birk (35) and. Birk et al. (36) resumed investigation of the acetone- insoluble factor and succeeded in purifying and characterizing the inhibitor. Cumbersome descriptive terms have then been used to refer to this protein: acetone-insoluble factor, purified AA inhibitor and trypsin and a-chymotrypsin inhibitor. The complete amino acid sequence of the BB inhibitor was determined by Odani and Ikenaka (37). It is a single polypeptide chain of 71 amino acids including seven disulfide bonds. Its MW is about 8 RD. It is capable of inhibiting both trypsin and chymotrypsin at independent reactive sites; the trypsin reactive site being located at residues Lys16 and Ser”, and the chymotrypsin reaCIive site. being located at residues Lea44 and Set“. The BB inhibitor is generally considered more heat stable than the Kunitz inhibitor (35). However, a recent study showed that this is true only for purified forms. In situ, the BB inhibitor appeared to be more heat labile than the Kunitz inhibitor (38). V. A Standard Mechanism of Inhibition Most protein inhibitors of serine proteases appear to interact with the enzyme they inhibit according to a standard mechanism of Laskowski, Jr. (39). They bind to the enzymes as if they were good substrates, but very tightly, and are cleaved very slowly at a peptide bond referred to as the reactive site (21). The model has stemmed from the observations of many workers, especially Michael Laskowski, Jr., its chief proponent. The detailed characteristics of the mechanism are described as follows (1,10). l. Incubation of the inhibitor with catalytic amounts of enzyme leads to specific hydrolysis of one peptide bond, the reactive site peptide bond of the inhibitor (40). Thus, the reaction between inhibitor and trypsin is better represented by the scheme: a: snag-estésn m k-i k2 where E is a serine protease, I is a virgin inhibitor whose reactive site peptide bond is intact, and 1‘ is a modified inhibitor whose reactive site peptide bond has been cleaved. 2. The newly formed COOH terminal in the modified inhibitor was shown to be arginine and the newly formed NHz-terminal, isoleucine. The two peptide chains of the modified inhibitor are strongly held together by one or more disulfide bridges. The equilibrium constant for virgin to modified inhibitor conversion is close to unity at neutral pH (41-43). 3. . Reduction and carboxymethylation of the modified inhibitor produced two fragments. The smaller fragment is composed of 64 amino acids, has the original NHZ-terminal of aspartic acid of the virgin inhibitor and the newly formed COOH- terminal of arginine. The larger fragment, is composed of 134 amino acids, has a newly formed NHZ-terminal belonging to isoleucine and the original COOH-terminal of leucine (Fig. 3). 4. Both virgin and modified inhibitors are active but the modified inhibitor reacts with the enzyme much more slowly than the virgin inhibitor. The stable enzyme-inhibitor complex is the same chemical substance whether formed from virgin or from modified inhibitor. Removal of either the newly formed COOH 10 terminal amino acid residue or the newly formed NH2 terminal amino acid residue form modified inhibitor causes loss of activity (40). ‘ I 30 e3 u “n 00 as 145 137 at _ H3NAsp —-Cys ——Arg - - Iie His Cy: Cy: Cys His —Leu C00 .1. .1 'l_l " Trypsin M5 157 let i 39 63 _ so n as use ‘HSNAsp—Cys—Arqan ’HSNIle—His—Cys—Cys—Cys His—LeuCOO‘ . . l_. Fig. 3 Schematic diagram of the tryptic conversion of virgin into modified soybean trypsin inhibitor according to Ozawa and Laskowski, Jr. (21). The complete amino acid sequence of the inhibitor was determined by Koide et al. (44). 5. The complex involves extremely close fit between the reactive site of the inhibitor and the active site of the enzyme. The conformation of the residue in the inhibitor interacting with the enzyme is closely similar in various inhibitors even through the inhibitors themselves are not conformationally similar. Examples of these inhibitors include the soybean BB inhibitor (45,46), chicken ovomucoid (21), the bovine pancreatic secretory inhibitor (47,48) and the lima bean trypsin inhibitor (18). Of special interest are those protease inhibitors which have the unique capacity to inhibit both trypsin and chymotrypsin at independent, non- overlapping binding sites. They have been termed ”double-headed” (49). Turkey ovomucoid (50), lima bean trypsin inhibitor (51), and BB soybean inhibitor (35) 11 have been cited as examples of double-headed inhibitors. Studies with soybean BB inhibitor have shown that partial proteolysis with trypsin followed by carboxypeptidase B hydrolysis resulted in loss of trypsin inhibitory activity (TIA) without affecting chymotrypsin inhibitory activity (CIA), while partial proteolysis with chymotrypsin resulted in loss of CIA without any effect on the TIA (45,46). This is true also with the lima bean inhibitor'(l8). VI. Nutritional Implications of Protease Inhibitors Osborne and Mendel (52) made the first significant observation that soybeans had to be heated in order to support the growth of rats. An assumption is that trypsin inhibitor (TI) is responsible for growth depression by reducing the digestibility of proteins. Later on, another observation was reported by Desikachar and De (53), that soybean diets containing predigested protein or free amino acids still retard the growth of rats. This was later confirmed by Liener (4). This observation indicated that inhibition of proteolysis by TI was not the sole factor responsible for growth depression. At the same time, the third significant finding was made by Chernick et al. (3). They found that raw soybeans as well as T1. itself could cause hypertrophy of the pancreas of chicks. Nesheim et al (54) made a similar observation with rats. Since the pancreas is responsible for the production of most enzymes required for the digestion of food, any dietary components which affect pancreatic function may markedly influence the availability of nutrients from the diet. Experiments with rats have demonstrated that pancreatic enzyme secretion is controlled by a negative feedback mechanism (Fig. 4). The amount of pancreatic secretion is determined by the level of free trypsin and/or chymotrypsin present in the intestine. As the level of trypsin goes below a 12 threshold level, the pancreas is induced to produce more enzymes. The TI evokes increased pancreatic enzyme secretion by forming inactive trypsin-TI complex. This results in endogenous loss of essential amino acids being secreted by a hyperactive pancreas. The loss of methionine and cysteine in this way would be particularly acute since soybean protein is deficient in these amino acids. On the other hand, intact soybean proteins were found to account for about 60% of the growth inhibitory and pancreatic hypertrophic effects due to their resistance to enzymatic attack unless denatured by heat (56). Therefore, it would appear that the TI and the refractory nature of the soybean protein act through a common mechanism described in Fig. 4 to inhibit the growth of rats. Irypsinogen < [pancreas] \ / {Riff-“l Dietary Trypsin Protein [intestine] Proteolysis Trypsin-TI Fig. 4 Regulation of the secretion of trypsin by the pancreas. CCK, cholecystokinin; TI, trypsin inhibitor (55). More recent studies showed that short-term feeding of raw soy flour and purified TI also caused pancreatic hypertrophy and hyperplasia in certain monogastric animals while prolonged exposure to high levels of T1 in raw soy ultimately led to pancreatic nodular hyperplasia and acinar cell adenoma in rats 13 (12). This confirmed the adverse nutritional effect of TI in food. VII. Current Methods for Trypsin Inhibition Assay Although various methods of column chromatography, affinity chromatography and electrophoresis have proved valuable for isolation and characterization of diverse TI (57,58), these methods are not suitable for quantification. At present, methods for T1 assay are mainly colorimetric although a fluorometric assay (59), immunoelectrophoresis assay (58) and enzyme linked immunoassay (60) have been introduced. The original colorimetric method employing casein, a natural substrate, was first described by Kunitz (32). It involves the spectrophotometric determination of hydrolysis of casein by a given concentration of trypsin in the presence and absence of the inhibitor. However, the rate of hydrolysis was later reported not to follow zero order kinetics under the condition defined by Kunitz (61). Erlanger et a1 (62) introduced a synthetic substrate, benzoyl-DL-arginine- p-nitroanilide HCl (BAPA), for assaying trypsin activity. They found that the hydrolysis rate of BAPA by trypsin not only followed zero order kinetics but also could be followed colorimetrically since the p-nitroaniline released is chromagenic. ' In 1969, Kakade et al. (16) made an evaluation of natural vs. synthetic substrate for measuring TIA in soybean samples and concluded that the use of the synthetic substrate, BAPA, proved to be a convenient and reliable method provided the competitive nature of the inhibition was taken into consideration, that is, trypsin inhibitor activity deviates from linearity at high levels of inhibitor concentration. This A was accomplished mainly by introducing an extrapolation procedure for data interpretation, in which TIA was expressed in l4 arbitrary trypsin units inhibited (TUI) per ml of the extract at zero concentration of the inhibitor (Fig. 5). Questions concerning both the accuracy and reliability of this method led to a collaborative study organized by the American Association of Cereal Chemists and the American Oil Chemists’ Society (63). A modified procedure was reported as a result of this collaborative study (20). Based on the modified procedure the standard AACC method for determining the TIA of soybean products was adopted (64). I c T I I g l- : ‘---- L: o O \N we : a E ‘3 220’ . g .- 0 n 1 1 n n 0 0.2 04 0.6 0.8 1.0 VOLUME OF EXTRACT (ml) Fig. 5 Trypsin inhibitor activity (TUI/ml extract) in relation to level of crude soybean extract. Extrapolated curve, as represented by the broken line, intercepts y-axis (0.0 ml) at 54 TUI/ml extract (16). Although reported separately, Smith et al. (13) and Hamerstrand et al. (14) modified the AACC method in a similar way by using a single inhibitor level instead of serial inhibitor levels. This modification bypasses the cumbersome data manipulation which is done by either extrapolating to zero or averaging over a range of inhibition levels. The reason for their modification is based on two observations: a) the patterns of the relationship between enzyme activity vs. inhibitor concentration are diverse, and b) the extrapolation method of data 15 interpretation uses data that are not in the region in which zero order kinetics is followed. Although another minor modification of the AACC method was also - reported (65), the above two papers established tho oorron; mothoo for TIA assay (6). However, since the modifications are limited only to data interpretation and the reasons behind the diverse patterns of inhibition curves observed remain unknown, the current method still poses questions of accuracy and sensitivity, especially for testing samples with low TIA. VIII. Current Methods for Chymotrypsin Inhibition Assay For measuring chymotrypsin inhibitor activity (CIA), a linear relationship between enzyme activity and enzyme concentration is an important prerequisite for obtaining reliable and reproducible measurements. Use of casein or denatured hemoglobin for measuring chymotrypsin activity was originally proposed by Kunitz (32). It was soon found to give a curvilinear response between enzyme activity and enzyme concentration (66). Several modifications of the method were proposed, including a mathematical transformation (67) and Ca“ incorporation (68). However, Kakade et al. (17) pointed out that these modifications likewise failed to produce a linear relationship and therefore, they modified the casein method by judicious choice of experimental conditions. The method not only involves cumbersome procedures, but also poses a question of reliability. Several simple synthetic substrates have been proposed for assaying chymotrypsin, including acetyl-L-tyrosine ethyl ester (ATEE) (69), N- carbobenzyl-L-tyrosine-p-nitrophenyl ester (CTNE) (70), and N-acetyl-L-tyrosine- p-nitroanilide (ATpNA) and N-benzoyl-L-tersine-p-nitroanilide (BTpNA) (15). Among the methods using a synthetic substrate, the method of Martin et al.(70) is most sensitive. As little as 5 mpg (5 x 10°9 3) enzyme can be 16 detected. However, lack of specificity (trypsin, thrombine, plasmin and papain are also very active against CTNE) and a need for the spontaneous substrate hydrolysis correction restrains it from gaining popularity. The method of Schuert and Takenaka (69) has the same sensitivity to that of Bundy (15) (1.5-15 ug enzyme can be tested), but the latter is simpler since p-nitroaniline released is a chromogenic substance and can be readily followed at 385 nm with spectrophotometers not having an ultraviolet attachment. When used for the chymotrypsin assay, these synthetic substrates have advantages over natural substrates because of simplicity and easy achievement of a linear response, although they are less water-soluble and require the presence of an organic solvent in the reaction mixture. BTpNA is frequently used for CIA assay (38,71), however, so far no detailed report has been given regarding procedure and conditions of the assay, inhibitor titration curve and factors affecting the assay. MATERIALS AND METHODS Reagents Crystalline porcine and bovine trypsins, crystalline bovine a-chymotrypsin, soybean Kunitz and BB inhibitors, benzoyl-DL-arginine-p-nitroanilide hydrochloride (BAPA) and benzoyl-L-tyrosine-p-nitroanilide (BTpNA) were purchased from Sigma Co. (St. Louis, MO). Part 1. TRYPSIN INHIBITION ASSAY I Methodology i. Buffer and solutions The assay buffer was 50 mM Tris buffer containing 10 mM CaClz, pH 8.2. A stock BAPA solution was prepared by dissolving 400 mg BAPA in 10 mL dimethyl sulfoxidei The solution was stable at room temperature. A working BAPA solution was prepared by diluting 0.25mL of stock BAPA solution to a total volume of 25 mL, using the assay buffer prewarmed at 37 C. Fresh running BAPA solution was prepared for each assay. A stock trypsin solution was prepared by dissolving 10 mg of crystalline porcine trypsin in 50 mL of 1 mM I-ICl solution, pH about 2.5, containing 2.5 mM CaClz. The solution was kept at 5 C. For preparing a working trypsin solution, 2 mL of the stock solution was diluted to a total volume of 25 mL, using the above HCI solution. Stock inhibitor solutions were prepared by dissolving 5 mg soybean BB or Kunitz inhibitor in 50 mL water. Working inhibitor solutions were made by diluting 2 mL of the stock solutions to a total volume of 25 mL, using water. 17 18 ii. Inhibitor sample preparation The samples (soy flour, soy protein concentrate, soy‘ isolate, cooked soybeans, raw soybeans, raw cowpeas, raw navy beans and raw pinto beans) were ground, if necessary, and passed through a 50 mesh screen. Half a gram of sample was extracted with 50 mL distilled water for 30 min under mechanical shaking at a speed of 200 RPM. Ten mL of the sample suspension was then destabilized by adding an equal volume of the assay buffer and vigorously shaking for 2-3 min before filtering through a Whatman No. 2 paper. The filtrate was then further diluted with water to the point where 1 mL gave 30-70% trypsin inhibition. This was done to keep the relative standard deviation (RSD) of the TIA measured within s3.5% (see DISCUSSION). A suitable final concentration for raw soybean samples was around 0.1 mg of dry sample per mL, and for heated samples, 0.5-1.5 mg/mL. iii. Procedure The procedure for assaying TIA is shown in Table 1. The reaction was run at 37 C. Exactly 10 min after adding the trypsin solution, the reaction was stopped by injecting 0.5 mL of 30% acetic acid solution with an 1 mL syringe. The absorbance at 410 nm, A";410 (sample reading), was a measure of the trypsin activity in the presence of the sample inhibitors. The reaction was also run in the absence of inhibitors, by replacing the sample with 1 mL water. The corresponding absorbance was symbolized as A0410 (reference reading). Distilled water was used as a blank. 19 Table 1. Procedure for assaying TIA in legume products Sequence of Reactants Concentration in Volume needed Mixing working solution ' for assay lst BAPA 0.92 mM 2.0 mL 2nd Sample Causing 30-70% inhibition 1.0 mL 3rd Enzyme l6 ug/mL 0.5 mL 4th Acetic acid 30% 0.5 mL Total assay volume 4.0 mL iv. Calculating trypsin inhibition Defining a trypsin unit as an A410 increase of 0.01 under the conditions of the assay, the trypsin inhibitory activity is expressed in trypsin units inhibited (TUI) per mg of dry sample and calculated as follows: 0 s . [(A 410 - A 410) x 100]/mL diluted soy extract TUI/mg sample . (mg sample/mL diluted soy extract) Alternatively, for standardization, the TIA can also be expressed in terms of International Units Inhibited (IUI) per g sample. One IU of enzyme is the amount that catalyzes the formation of 1 )1 mole of product per min under difined conditions. One TU is equal to 0.000516 IU on the basis that the molar 20 absorption coefficient (am) at 410 nm is 7760. 11. Procedures for Studying the Effect of the Reactant Mixing Sequence on the TIA assay i. Buffers and solutions Three preincubation buffers were used: 20 mM acetate buffer, 20 mM Tris buffer and mixture of the two to reach pH values from 2.7 to 9.0. Other buffer and solutions are referred to Methodology section (p. 17). ii. Procedures All preincubations of the inhibitors (0-2 pg) with enzymes (8 pg) in the S- last test, or of the inhibitors with BAPA (0.8 mg) in the E-last test, were carried out in one of the three preincubation buffers to reach pH values from 2.7 to 9.0, with total volume of 1.5 mL. After a specified time period of incubation, 2 mL of the assay buffer was added to the premix. This brought the pH of the assay system to 8.11:0.2. Immediately following this step, 20 pL of the BABA solution in the S-last test or 20 pL of the enzyme solution in the E-last test, were added to start the enzymic reaction. The reactions were allowed to proceeded for 10 min and stopped by injecting 0.5 mL of 30% acetic acid solution. iii. Calculating trypsin inhibition Since different doses of the inhibitor were used to measure the activity of the inhibitor by each test, a titration curve (A410 vs. dose of inhibitor) could be plotted. Linearity was generally obtained over lower dose ranges. The slope of the straight line was taken as the inhibition value. 21 Part 2. CHYMOTRYPSIN INHIBITION ASSAY 1. Methodology i. Buffer and solutions Tris buffer, 50 mM,pH 8.2, containing 10 mM CaCl2 was used as an assay buffer. A stock chymotrypsin solution was prepared by dissolving 20 mg of crystalline chymotrypsin in 50 mL of 1 mM HCl solution, pH about 2.5, containing 2.5 mM CaClz. The solution was kept at 5 C. To prepare a working enzyme solution, 2 mL of the stock solution was diluted to a total volume of 25 mL, using the above HCl solution. A stock BTpNA solution was prepared by dissolving 15 mg BTpNA in 25 mL 'acetone. The solution was stored at 5 C. A working BTpNA solution was freshly prepared by diluting 5 m1 of the stock solution to a total volume of 25 mL, using the assay buffer prewarmed at 37 C and kept at that temperature. A stock inhibitor solution was prepared by dissolving 5 mg soybean BB inhibitor in 50 mL water. A working inhibitor solution (4 pg/mL) was made by diluting 2 mL of the stock solution to a total volume of 25 mL using water. ii. Inhibitor sample preparation Refer to the preparation procedure for trypsin inhibition assay (p. 18). iii. Assay procedure One mL of the inhibitor solution, which results in 35-65% chymotrypsin inhibition, was pipetted to a test tube and addition of 2 mL BTpNA solution 22 ' followed. The enzymic reaction was started by adding 0.5 mL of the enzyme solution and allowed to proceed for 10 min at 37 C. The reaction was stopped by injecting 0.5 mL of 30% acetic acid solution with an 1 mL syringe. The absorbance at 385 nm, A385’ was a measure of the chymotrypsin activity. The reaction was also run in the absence of inhibitors, by replacing the inhibitor solution with 1.0 mL water. The corresponding absorbance was symbolized as A0385 Water was used as a blank for all color measurements. iv. Chymotrypsin inhibitor unit (CIU) Under the assay conditions specified in this study (10 min, 4 mL reaction mixture, pH-8.1 at 37 C, with BTpNA as a substrate), one chymotrypsin inhibitor unit (CIU) was defined as a 0.01 increase of (A0 l). The CIA is 385/A385 ' expressed as CIU/mg inhibitor (sample) and calculated as follows: 0 . . . . CIU/mg inhibitor 3 [(A 385/A385 - 1) x 100]/mL Inhibitor solution mg/ml inhibitor solution v. Correction for effect of enzyme concentration The CIA values, when expressed in terms of CIU, were affected significantly by the amount of enzyme used in the assay, which reflected in the A0385 value. Setting the CIA value obtained at A0 =- 0.45, symbolized by CIA 385 0.45' as a reference value, a correction factor c is defined as c - (CIAOAS/CIA - 1), and calculated by the following equation (See DISCUSSION): 3.44 c - 2.05 (A0 + 0.13) - 0.315. 385 . o . Correction of CIA at any other A 385 to CIAO.45 Is done by CIA 5 =(1+ c) CIA. 0.4 23 . vi. Expressing CIA in terms of pure BB inhibitor One ug pure BB inhibitor was shown to have 30.8 CIU. Thus for comparative purposes, CIA can be expressed in terms of pure BB inhibitor per unit sample. If samples contain other inhibitors, we can express CIA in terms of BB inhibitor equivalent, using the same conversion factor. 11. Procedures for Studying the Effect of the Reactant Mixing Sequence on the CIA assay 1. Buffers and solutions Three preincubation buffers were used: 20 mM acetate buffer, 20 mM Tris buffer and mixture of the two to reach pH values from 2.7 to 9.0. Other buffers and solutions are described in the Methodology section (p. 21). ii. Assay procedures All preincubations of the BB inhibitor (0-4 pg) with a-ehymotrypsin (16 pg) in the S-last test, or of the inhibitor with BTpNA (0.4 mL stock BTpNA solution) in the E-last test, were carried out in one of the above three preincubation buffers to reach pH values from 2.7 to 9.0, with total volume of 1.5 mL. After a specified time of incubation, 1.6 mL of the assay buffer was added to the premix. This brought the pH of the assay system to 8.1 4.- 0.2. Immediately following this step, 0.4 mL of BTpNA solution in the S-last test, or 0.4 mL of enzyme solution (40 pg/mL in 0.001 N HCl) in the E-last test, were added to start the enzymic reaction. The reaction was allowed to proceed for 10 min and stopped by injecting 0.5 mL of 30% acetic acid solution. The yellow color of the reaction mixture was read at 385 nm and the A385 value was used as an estimate 24 of chymotrypsin activity. iii. Calculating chymotrypsin inhibition The reader is referred to the Methodology section (p. 22) for a definition ' of chymotrypsin inhibitor unit and data transformations. In the assay, a series of inhibitor levels are used and ‘(A0385/A385-1)x 100 is plotted against inhibitor level. A straight line is obtained, the slope of which is taken as the inhibition value which is further corrected for the enzyme concentration effect RESULTS AND DISCUSSION Part 1 TRYPSIN INHIBITIONASSAY I. General Assay Conditions i. Enzyme concentration As shown in Fig. 6, the quantity of porcine trypsin employed in this test should not exceed that corresponding to A410=0.50, if linearity between absorbance and enzyme level is to be maintained. Within this A range, when 410 two different amounts (6 and 8 pg) of enzyme were used to measure the TIA of the same soy extract, the parallel lines shown in Fig. 7 were obtained. From these lines, the same TIA value, as TUI/mL sample extract, can be derived, indicating that impurity or partial inactivation of the enzyme does not affect the assay. The independence of TIA on enzyme concentration was also addressed in the- current method (13,14). ii. Reaction time Fig. 8 shows the relationship between A and reaction time. Linearity 410 was observed up to 13 min of reaction, both in the absence of inhibitors (0.00 mg raw soybean /mL sample solution) and in the presence of inhibitors (0.10 and 0.15 mg raw soybean /mL). The results indicate that the rate of trypsin inhibition, expressed as TUI per mg dry sample per min, was constant when the reaction time remained within the valid assay time range (0-13 min), while the TIA values, expressed as TUI/mg dry sample, increased linearly with time. For this reason, the reaction time for the TIA assay should be standardized to 10. 25 26 0.60 - A410 P U 0 l q I V T Y T I r 0.0 2.0 4.0 6.0 8.0 10.0 12.0 141.0 Amount of porcine trypsin (pg) Fig. 6 Relationship between absorbance at 410 nm and amount of porcine trypsin. The reaction time was 10 min. 27 0.50 0.40 0.30 — O i '1 0.20 -1 .1 0.10 1 .1 0.00 T r I I T r F r I I 0.0 0.2 0.4 0.6 0.8 1 .0 ML soy extract in 4 ml assay mixture Fig. 7 Effect of porcine trypsin concentration on the assay of soybean TIA. 28 o 0.00 mg/ml 0'50 T I: 0.10 mg/rni " .1 a 0.15 mg/ml ’ .. 0.40 fi - '1 '1 A410 T F1 0000 I I I rfili [II 0 2 4 6 8.101214 Reaction time (Min). Fig. 8 Relationship between absorbance at 410 m and reaction time in the absence and in the presence of inhibitors. 8 pg of enzyme preparation was used in the reaction. The inhibitor samples were aqueous extracts of raw soybeans. 29 min. iii. Substrate (BAPA) concentration The apparent Km value for the porcine trypsin-BAPA reaction was found to be 0.96 mM at 37 C in this study. In the modified TIA assay, the BAPA concentration would be 0.46 mM, corresponding to about 1/2 of the Km. Use of excess BAPA concentration is unfeasible due to its poor solubility. As the BAPA concentration affects the trypsin assay, it also affects the TIA assay. Fig. 9 shows that for two different BAPA concentrations, 0.23 and 0.46 mM, the lines connecting A410 and amounts of inhibitors are not parallel, a fact which emphasizes the significance of standardizing theBAPA concentration in the TIA measurement. In addition, since BAPA decomposed slowly with time, causing variation of the TIA value, it is recommended that a fresh working BAPA solution be used (65, and this study). iv. Ca ion concentration ++ . . . . ++ Ca 1s known to subdue trypsm (27). We observed that when Ca was added at two concentrations, 5 and 10 mM, to the assay buffer, the TIA values were not significantly influenced, but its presence at the 5 mM level is recommended for protection of the enzyme from inactivation. Lehnhardt and Dills (65) observed that the presence of Ca” reduced not only autolytic trypsin inactivation but also the effect of phytate effect on TIA assay. v. pH of the assay buffer The optimum pH for hydrolysis of BAPA by porcine trypsin was found to be 8.1 in this study, which is closed to that of bovine trypsin (Erlanger et al. 1961). In order to determine the optimum buffer pH for the TIA assay, the following 30 0.50 ‘ A410 " a 0.23 mil 0.00 "1 I 0.0 0.2 0.4 l— l I 0.6 0.8 1.0 ML soy extract in 4 ml assay mixture Fig. 9 Effect of substrate (BAPA) concentration on the assay of soybean TIA. 31 five pH levels were tried: 8.5, 8.1, 7.5, 7.0, and 6.5. The results are summarized in Fig. 10, and indicate that the A vs. inhibitor quantity lines 410 were not exactly parallel and the line corresponding to pH 8.1 led to the greatest TIA value (highest slope of the line). 11. Effect of the Reactant Mixing Sequence on the TIA assay In assaying enzyme inhibition, preincubation of inhibit0r with enzyme before addition of substrate is commonly practised (l3-14,16,l9). This is thought to be necessary for obtaining equilibrium data (1). However, while investigating the soybean Kunitz inhibitor, Viswanatha and Liener (72) found that a change in the order of mixing the reactants exerted a considerable influence on the extent of inhibition. In our study, for measuring trypsin inhibition of the soybean BB inhibitor, two procedures were used: the common procedure in which the substrate is added last, after mixing inhibitor with enzyme; being hereafter referred to as "the S-lsst test“, and a new procedure in which enzyme is added last to the mixture of inhibitor and substrate, being hereafter referred to as "the E-last test". The results are presented in Fig. 11 and indicate that the S-last test gave considerably lower inhibition values than the E-last test when the premix pH was 3.5 and preincubation time was as short as 3 min. Under the same preincubation conditions, similar results were reached with the Kunitz inhibitor. The effect of the reactant sequence on trypsin inhibition assay is hereafter referred as "the reactant sequence effect”. i. Effect of the preincubation time on the reactant sequence effect In the E-last test, when the time of incubating the premix of I (inhibitor) with S (substrate) was varied from 0 to 10 min and the premix pH was constant at 32 A410 0.20 -1 q I pH 6 5 0.10— 0 pH 7.0 . ... a pH 7.5 - a pH 3.1 ‘ 0.00- ° H 8'5 . . . l l l 0.0 0.2 0.4 0.6 0.8 1.0 ML soy extract in 4 ml assay mixture Fig. 10. Effect of assay buffer pH on the assay of soybean TIA. 33 0.50 A410 0.10- ‘ 1:1 The S-iast test 000 a ‘ The E—lost test 0.8 0.0 014 Amount of inhibitor (pg) I I T 1 1 1.6 2.0 I. 1.2. Fig. 11 Effect of the sequence of mixing the reactants on the assay of soybean Bowman-Birk trypsin inhibition. The premix p11 was 3.5 and the preincubation time 3 min. Details of the tests are described under Materials and Methods . 34 3.5, the same trypsin inhibition was obtained (data not shown), indicating that the preincubation time in the E-last test had no effect on the trypsin inhibition assay. However, in the S-last test, when the time of incubating the premix of I with E was changed from 0 to 10 min, while the premix pH was fixed at 3.5, the extent of trypsin inhibition varied, indicating that the preincubation time in the E-last test had an effect on the trypsin inhibition assay. At any particular preincubation time, the relative difference between the two tests was expressed as [(Ae-As)/Ae X 100%], where As is the trypsin inhibition obtained by the S-last, and Ae is the trypsin inhibition obtained by the E-last test. Since Ae remained constant regardless of the preincubation time, it was regarded as a reference. The data are presented in Fig. 12 with two pure inhibitors and show that, when the premix pH was constant at 3.5, the trypsin inhibitions obtained by the S-last test were always lower than those obtained by the E-last test, for the porcine trypsin-BB inhibitor system and the bovine trypsin-BB inhibitor system. At the beginning of preincubation, the relative difference between the two tests increased with time. After 3-5 min, the curves leveled off. The data of Fig. 12 also indicate that the extent of this time-dependence feature of the reactant sequence effect varied between porcine and bovine trypsins. The maximum relative difference in BB inhibitor activity between the two tests was about 57% for the porcine trypsin and only 25% for the bovine trypsin. ii. Effect of the premix pH on the reactant sequence effect Like the preincubation time, the premix pH was also found to influence the reactant sequence effect of the trypsin inhibition assay. In the E-last test, 35 60 . .1 o—e Bovine trypsun 5 “+ a—e Porcine trypsin - A _ N 50— ' v .1 <1) .. O - _ c 40— (1) 1.. .1 ID .. ‘4: .3 :6 30- . .. . - - 4 ..>. ' __ :5 20— ., (D . , - m . 10— T 0 - I I T 1 I I I I 1 I I I r 1 I I l I 0 5 10 . 15 20 Preincubation time (min) Fig. 12 Relative difference in soybean Bowman-Birk trypsin inhibition values obtained by the S-last and the E-last tests as a function of the preincubation time. The relative difference was expressed as [(Ae-As)/Ae x 1008] , where Ae is the inhibition value obtained by the E-last test and As is the inhibition value by the S-last test. The p11 of all prenixes was 3.5. Details of the assay are described under Materials and Methods. 36 when the pH of the premix of I with S was varied from 2.7-9.0 and the preincubation time was held at 10 min, the same trypsin inhibition values were obtained, indicating that the premix pH in the E-last test had no effect on the trypsin inhibition assay. While in the S-last, when the pH of the premix of I and E was varied from 2.7 to 9.0 and the time ”of premix incubation was kept constant at 10 min, different trypsin inhibitions were found. The relative differences in porcine trypsin inhibitions measured by the two tests were plotted against the premix pH (Fig. 13). The results indicate that the trypsin inhibitions obtained through the S-last test were either equal to or lower than those through the E-last test, depending on the premix pH. At the pH . 2.7, the S-last test estimated the same inhibition values as the E-last test. When the pH increased to 3.5, a maximum difference was observed, indicating that the S-last test gave the lowest values. Above pH - 4.0, the difference decreased sharply. Over the neutral pH range, the two tests gave the same results again. At slightly alkaline pH range, a second peak was. observed and, at pH around 8.5, the difference between the two tests began to drop again. The two inhibitors, Kunitz and BB inhibitors, showed the same pH-dependence pattern, but the Kunitz inhibitor was affected less by pH and its first peak shifted to the more acidic side. When bovine trypsin was used to measured the trypsin inhibition of the inhibitors, the patterns of the relative difference between the two tests as a function of pH were similar to that with porcine trypsin, except that the acidic pH peak was less pronounced and the difference between the two inhibitors was negligible (Fig. 14). 37 60 e—e Kunitz inhibitor _ " 13—61 88 inhibitor A — N 50- -. v . u d (D .. 0 «nu-I c 40- .. . 0) n L a -I a) T t i H —-d :6 30- 11 EJ _ Q) -I 0 ~ \, .2 _ E 20- a (D . . _ m u 10~ “ — 0" ‘4 T I . ° " ' I 1 I 2.0 4.0 6.0 ’ 8.0 10.0 Premix pH Fig. 13 Relative difference in porcine trypsin inhibition values obtained by the S-last and the E-last tests as a function of the premix p11. The relative difference was expressed as [(Ae-As)/Ae x 1000], where Ae is the inhibition value obtained by the E-last test and As is the inhibition value by the S-last test. The preincubation time was constant at 10 min. Details of the assay are described under Materials and Methods . \ . 38 6C1 e—e Kunitz inhibitor 3 " B—El BB inhibitor A 03 END-d “ v 01 " A 0 r: 40-. — 93 In J ' 3: .4 "6 30— 0) . " .2 _ :6- 20— 0 at at 0: 10- T 0 "" " 1 " ' " I 1 r 1213 I413 ELC) ' ELCI 11343 Premix pH Fig. 14 Relative difference in bovine trypsin inhibition values obtained by the S-last and the E-last tests as a function of the premix p11. The relative difference was expressed as [‘(Ae-As)/Ae x 100s], where Ae is the inhibition value obtained by the E-last test and As is the inhibition value by the S-last test. The preincubation tine was constant at 10 Iain. Details of the assay are described under Materials and Methods . 39 iii. Jumping the premix pH We have shown that the E last test gave the same trypsin inhibition value regardless of premix pH and preincubation time while the S-last test did not. Regarding the premix pH, for the porcine-BB inhibitor system, the trypsin inhibition values obtained by the S-last test were either equal to the reference value (E-last value) when the premix pH was less than 2.7 or near neutral, or lower than the reference value when the pH was 2.7-5.5 or 75-90. Here, premix pHs which are associated with the manifestation of a reactant sequence effect are considered effective pHs, while those resulting in no sequence effect are considered noneffective pHs. A separate study was conducted to see whether jumping the premix pH in the S-last test from effective to nonef f ective levels during preincubation can restore the inhibitory capacity of the S-last test to that of the E-last test. The results of Fig. 15 indicate that in the S-last test, 10 min preincubation at pH 3.5 followed by 10 min preincubation at pH 6.5 restored the inhibition capacity to that of the E-last test (same slopes). So did the 10 min preincubation at pH 9.0 followed by 10 min preincubation at pH 6.5 (Fig. 16). However, Fig. 17 shows that 10 min preincubation at pH 3.5 followed by 10 min preincubation at pH 2.5 did not restore the inhibition capacity to that of the E-last test. Note that jumping the premix pH in the E-last test was not tried since the premix pH has no effect on the trypsin inhibition assay in the E-last test. iv. TIA assay as related to limited hydrolysis of inhibitors Two hypotheses are possible to explain the reactant sequence effect observed in this experiment: (a) that an interaction between I and S occurs in the E-last test, resulting in increased trypsin inhibition in this test over the S-last 40 0.50 0.40 - T 0.30— ‘ " A410 0.20- ‘ T 0.10‘ 0.00 . I 0.0 0.4 Amount of inhibitor (pg) 1 1.6 2.0 I I ' l l . 0.8 1.2 Fig. 15 Jmaping the premix p11 from 3.5 to 6.5 during assaying porcine trypsin inhibition of soybean BB inhibitor. O-—-O S-last test, a 20 min preincubation at pH 3.5; H S-last test, a 10 min preincubation at pH 3.5 followed by a 10 min premix incubation at pH 6.5. pH jumping was carried out by adding 1 ml 40 mM Tris buffer, p11 8.0 to 1.5 m1 of premix (20 Int acetate buffer, pH 3.5) H E-last test, a 20 min preincubation at p11 3.5. All tests were finally run at pH 8.1 :1: 0.2 for the ten-minute enzymic reaction. 4i 0.40 \ . 0.30— ‘ o g; 0.20-J < 0.10- 0-00 'I'I'I'Ifil 0.0 0.4 0.8 1.2 1.6 2.0 Amount of inhibitor (pg) Fig. 16 Juping the premix pH from 9.0 to 6.5 during assaying porcine trypsin inhibition of soybean BB inhibitor. G——€ S-last test, a 20 min preincubation at pH 9.0; H S-last test, a 10 min preincubation at pH 9.0 followed by a 10 min premix incubation at pH 6.5. pH jumping was carried out by adding 1 ml 0.03 R 301. pH 1.9 to the 1.5 ml premix (20 d4 Tris buffer, pH 9.0) H l-last test, a 20 min preincubation at pH 9.0. All tests were finally run at pH 8.1 :h 0.2 for the ten-minute enzymic reaction. 42 0.40 A410 0.10— _ ' ‘- l I I 1.6 2.0 I 0.00 I r I 0.0 0.4 0.8 Amount of inhibitor (ug) I 1 i 1.2. Fig. 17 Jumping the premix pH from 3.5 to 2.5 during assaying porcine trypsin inhibition of soybean BB inhibitor. o——-e S-last test, a 20 min preincubation at pH 3.5; I H S-last test, a 10 min preincubation at phi 3.5 followed by a 10 min premix incubation at pH 2.5. pH jumping was carried out by adding 1 ml 0.02 N 801. pH 2.3 to 1.5 ml of premix (20 ml! acetate buffer, pH 3.5) A-——A B-last test, a 20 min preincubation at pH 3.5. All tests were finally run at pH 8.1 a: 0.2 for the ten-minute enzymic reaction. 43 test; and (b) that an interaction between I and E occurs in the S-last test, resulting in decreased trypsin inhibition in this test compared to the E-last test. Hypothesis (a) is readily rejected by the fact that the E-last test gives the same trypsin inhibition regardless of the premix pH and the preincubation time, under which S and I could interact. Hypothesis (b) remains the only one to explain the lower inhibition values observed in the S-last test and it happens to be in accordance with the reactive site model of Laskowski, Jr. (21), that trypsin is capable of attacking its own inhibitors as if they are substrates. In the S-last test, where I is premixed with E in a near cquimolar ratio, at a relative high temperature (37 C), a conversion of I to I. would occur during the period of preincubation. Thus, the reactant sequence effect observed in this study is attributed to a limited hydrolysis of I by the enzyme it inhibits into 1., which has the same reaction activity as I, but lower affinity (association constant) towards trypsin (1,40). Assuming that [I]o is the concentration of total. virgin inhibitor, the following relationship would exist after preincubation time t: [110 - mt + [1’1t [11 where, [It]t is the concentration of I’ produced during preincubation and [I]t is the concentration of I remained. Also assuming that a is the trypsin inhibition per unit concentration of I and b is the trypsin inhibition per unit concentration of 1‘, then, in the E- last test we measured the total virgin inhibitor activity, (a[I]o), while in the S-last test we measured the activities of both I and It, (a[l]t + b[I’]t). t If b . 1:; 20.0 — . " 2 . - a) a: 10.0-I "‘ 0.0 U L r I I I -I II 0.00 2.00 4.00 6.00 8.00 10.00 pH of premix Fig. 37 Relative difference in CIA of soybean BB inhibitor, assayed by the S-last and the E-last tests as a function of the premix pH. The relative difference is defined as (cue-cmycrx. x 100s, where CIAs is the CIA of the S-last test and CIAe is the CIA of the E-last test. The preincubation time was 10 min. Details of the assay are described under Materials and.Hethods. 87 difference between the two tests, the larger peak on the acidic side (pH about 4.0) and the smaller peak on the alkaline side (pH about 8.5). At the pH - 2.7 or between 6.5-7.5, the S-last test estimated the same inhibition values as the E-last test. iii. Abrupt change of the premix pH In terms of the premix pH effect on the reactant sequence effect, the CIA value of the BB inhibitor obtained by the S-last test was either equal to the reference value (E-last value) when the premix pH was less than 2.7 or near neutral, or lower than the reference value when the pH was 2.7-6.5 or 75-90. Here, premix pHs which result in the sequence effect are considered effective pHs, while those resulting in no sequence effect are considered nonef f ective pHs. A separate study was conducted to see whether an abrupt change of the premix pH from effective to noneffective during preincubation in the S-last test can restore the chymotrypsin-inhibiting capacity of S-last test to that of the E-last test. The results are presented in Table 7. Comparison of the two S-last tests, test No. l and test No. 2 showed that additional lO-min' preincubation at pH 7.0 resulted in a small gain in the chymotrypsin inhibition. Comparison of the two S-last tests, test No. l and test No. 3, showed that abrupt change of premix pH from 4.0 to 7.0 during a 20 min preincubation resulted in a large gain in the inhibition value. iv. The CIA assay as related to limited hydrolysis of inhibitors In Part 1, the effect of the reactant mixing sequence on the assay of trypsin inhibitory activity has been attributed to the limited hydrolysis of inhibitor, in accordance with the reactive model of Laskowski, Jr. (21). Since under the same conditions, chymotrypsin is also capable of attacking its own 88 Table 7 Effect of an abrupt change of premix pH from 4.0 to 7.0 on assaying the CIA of soybean BB inhibitor a Test conditions ‘ CIU/mg CIAe-CIAs x 100 b CIAe test S-last tests 1 a lO-min preincubation at pH 4.0 c plus a lO-min preincub. at pH 7.0 10.0.: 0.4 59.4 2 a 10-min preincubation at pH 4.0 8.8 i 0.4 64.3 3 a 20-min preincubation at pH 4.0 4.4 t 0.2 82.1 4 a lO-min preincubation at pH 7.0 24.3 t 1.2 1.2_ E-last test e 1 a 20-min preincubation at pH 4.0 24.6 t.l.O 00.0 All tests were finally run at pH 8.1 $.0.2 for the 10-min enzymatic reaction. Mean of duplicate measurements :.SD. The pH abrupt change was carried out by adding 1.0 mL 40 mM Tris buffer, pH 8.2, to 1.5 mL of premix (20 mM acetate buffer, pH 4.0). Abruptly changing the premix pH in the E-last test was not done since the premix pH had no effect on the trypsin inhibition assay in the E-last test. 89 inhibitors (45,46,77), the same explanation could be applied to the reactant sequence effect on the CIA assay observed in this Part (Fig. 35). In the S-last test, where I is premixed with E in a near cquimolar ratio, at a relative high temperature (37 C), a conversion of I to I"I would occur during the period of preincubation, while in the E-last teSt, I is premixed with S and no conversion of I to It would take place. Assume that [I]o is the concentration of total virgin inhibitor, at preincubation time t, [110 - [I]t + [1'], [5] where, [1"]t is the concentration of the chymotrypsin-modified inhibitor » produced during preincubation and [l]t is the concentration of remaining 1. Also assume that a is the CIA per unit concentration of I and b is the CIA per unit concentration of I.. Thus, in the E-last test, we measured the CIA of total virgin inhibitor (amo), while in the S-last test, we measured the CIAs of both I and I. (amt + bII'It). Since the chymotrypsin-modified inhibitor is known to be almost inactive towards chymotrypsin (46), that is, b - 0, then (am, + th'It) - am, < amo. [6] indicating that there is a pronounced reactant sequence effect. Regarding preincubation time, the maximum difference between the S-last and the E-last tests for the chymotrypsin-BB inhibitor system (Fig. 36) is larger than the corresponding difference for trypsin-BB inhibitor system (Fig. 12). This might be attributed to the difference in the inhibition capacity between trypsin and chymotrypsin modified BB inhibitors towards their own enzyme. Whereas the trypsin-modified inhibitor acts only more slowly than the virgin inhibitor towards trypsin, the chymotrypsin-modified inhibitor is almost 90 inactive towards chymotrypsin (46). The Y-axis value in Fig. 36 represent the following equation: Y - tatIIo-(at11,+ bll‘ltD/alllo [7] When b-O, substituting eq. [5] into eq. [7] gives Y - tI"I,/IIIo ‘ [8] The maximum relative difference of about 83% shown in Fig. 36 indicates an 83% conversion of I to 1.. This finding is in accordance with the observation by Frattali and Steiner (46), that an 80% conversion is possible. However, it took 48 hrs to reach this conversion in that study, while in our study it took about 20 min. This discrepancy may be due to differences in reaction systems. In their study, the chymotrypsin was used in a catalytic amount (molar, ratio of enzyme to inhibitor was 1:100) at 25 C, while in our study, a near stoichiometric amount of the enzyme was used at 37 C. The effect of the premix pH on the reactant sequence effect can be explained by the fact that both the rate constant kca and the equilibrium constant K t hyd # for the hydrolysis of I into I are pH-dependent (41,42). Here, Khyd is defined as a Khyd ' [1 lC/[IIC [9] t where [I]c and [I 1e are the concentrations of virgin and modified inhibitors at equilibrium, respectively . When t reaches equilibrium time, the Y-axis values in Fig. 37 becomes 0 Y - [I 16/010 [101 Since from eqs. [5], [9] and [10] we know that, when Khyd > 0, Y is a ' monotonically increasing function of Khy d’ Y . Khyd/(HKhyd), [11] the pH dependence pattern of the reactant sequence effect shown in Fig. 37 should f allow the eq. [4] shown in Part 1, that is, Y should rise at both low 91 and high pH levels. The smaller alkaline peak might be due to the fact that Kcat of hydrolysis at alkaline medium is smaller than that at acidic medium (29), resulting in less conversion of I to I‘ in 10 minute preincubation. Under certain conditions, the modified inhibitor can be cleaved and reformed (21,75). Studying the interactions of the BB inhibitor. with both trypsin and chymotrypsin, Frattali and Steiner (46) found that, for both cases, conversion of I to I. occurred at pH 4.0, at room temperature, with catalytic amount of enzyme, while regeneration of I from 1‘ took place upon prolonged exposure at pH 8.0, at 4C in a near stoichiometric amount of enzyme. They also stated that the regeneration of trypsin-modified inhibitor was faster than that of the chymotrypsin-modified inhibitor. In Part I, we showed that pH jumping resulted in almost complete recovery of trypsin inhibitor activity (Figs. 15 and 16) while in Part 2, we observed only a partial recovery of chymotrypsin inhibitor activity, following pH jumping (Table 7). This difference in degree of recovery may be attributed to the difference in regeneration rates for the two cases. Finally, the reaction between trypsin and soybean Kunitz inhibitor is known to be instantaneous. The half life of the reaction is about 4 see with a second- order velocity constant of 2 x 107 L/Mole/sec (78). Although data for interactions betwen other proteinases and their inhibitors are unavailable, the reactant sequence effect an inhibition assays observed in this Part as well as in previous Part could be used as a basis to propose that binding between a protein inhibitor with a serine protease is instantaneous. This hypothesis may be regarded as a complement to the standard mechanism of Laskowski, Jr. (21). CONCLUSIONS The modified method for measuring trypsin inhibition in soybean products has a theoretical basis (the effect of the reactant mixing sequence on the assay is taken into consideration) and a practical significance. It can eventually be used for measuring trypsin inhibitor activity in many other proteinaceous food products. The proposed method for chymotrypsin inhibition assay, although involves mathematical data transformation, is relatively simple and reliable. Regarding the effect of the reactant mixing sequence on the inhibition assays, it was found that the inhibition value of the S-last test (adding substrate last to the reaction mixture) was either equal to or lower than that of the E-last test (adding enzyme last to the reaction mixture), depending on the premix pH and preincubation time, while the values of the E-last test were constant regardless of the premix pH and the preincubation time. These observations are in accordance with the reactive site model for proteinaceous inhibitors of serine proteases (21). The inhibitors bind to the enzyme as if they were substrates, but very tightly, and are cleaved very slowly at a peptide bond referred to as the reactive site. For assaying the aforementioned type of inhibitors, the common practice of sufficiently preincubating inhibitor with enzyme for obtaining an equilibrium data in an inhibition assay is no longer valid, and the new procedure (E-last test) is preferable to the common procedure (S-last test). In addition, the observations suggest an instantaneous binding between the aforementioned type of inhibitors and the enzyme, which may be regarded as a complement to the standard mechanism. 92 RECOMMENDATIONS FOR FUTURE STUDIES As most protease inhibitors are also of protein nature, studies are needed to verify if their assays are affected by the reactant sequence or not. 6 M) of the inhibitors used in Because of the low concentration (about 10' this study, direct evidence by electr0phoresis or by chromatography for the limited hydrolysis of the inhibitors is difficult. However, if relatively larger amounts (10'3 M) of inhibitor and enzyme are tested under the condition similar to the, inhibition assay (37 C and stoichiometric ratio of enzyme to inhibitor), either electr0phoresis or chroatography can then be used to verify if the limited hydrolysis can occur in minutes or not. And finally, the procedure for measuring the reactant sequence effect an inhibition assay 'may eventually be developed as an analytical tool for kinetic study of inhibitor hydrolysis. 93 10. 11. 12. BIBLIOGRAPHY Laskowski, Jr. M. and Sealock, R. W. 1971. Protein proteinase inhibitor —- molecular aspects. in "Enzyme" (Boyer, P. D. ed. ), vol. 3. pp. 376-473. Academic Press, New York/London. Liener,I. E. and Kakade, ML. 1980. Protease inhibitors in ”Toxic constituents of plant f oodstuf f s" (Liener, I. E. ed.), pp. 73-102. Academic Press, New York/London. Chernick, S.S., Lepkovsky, S.S. & Chaikof f, I. L. 1948. 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Biochemistry 14, 275. 78. Green, N. M. 1957. Kinetics of the reaction between trypsin and the pancreatic trypsin inhibitor. Biochem. J. 66. 407-415. HICHIGQN STATE UNIV. LIBRQRIES V lllllllllllllllllllllllllllllllllllllllllllllllllllllll 31293005799089