This is to certify that the thesis entitled Isolation of Trypsin Inhibitor from Navy Beans (Phaseolus vulgaris L.) by Affinity Chrorn atography presented by Jose Carlos Gomes has been accepted towards fulfillment of the requirements for v.3. . Food Science and degree 1n Human Nutrition Major professor Date 4/24/78 0-7639 - Emma. mmN STATE UNNERS" EAST LANSING. MICH. 43323 ISOLATION OF TRYPSIN INHIBITOR FROM NAVY BEANS (Phaseolus vulgaris L.) BY AFFINITY CHROMATOGRAPHY By Jose Carlos Gomes A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Food Science and Human Nutrition 1978 ABSTRACT ISOLATION OF TRYPSIN INHIBITOR FROM NAVY BEANS (Phaseolus vulgaris L.) BY AFFINITY CHROMATOGRAPHY By Jose Carlos Gomes Legume grains are relatively high in protein content. Common beans (Phaseolus vulgaris L.) are one of the major sources of protein in the world. Among undesirable nutritive factors in beans are the proteinase inhibitors. A trypsin/chymotrypsin inhibitor present in the albumin fraction of navy beans _ sanilac cultivar - was separated by affinity chromatography on trypsin immobilized on agarose beads. Molecular weights calculated by SDS-PAG electro- phoresis were 16,600 and 33,800 for the major and minor bands, respectively. Molecular weight estimated from inhi- bition measurements was ll,900; minimum molecular weight, based on methionine as limiting amino acid was 12,2l4. Dissociation constant of trypsin-inhibitor complex was 7.5 x 10'“) M. Isoelectric pH's were at 4.40 and 4.45 for the major and minor bands, respectively. Amino acid analysis showed that the inhibitor has a high content of half-cystine, a low amount of methionine and no tryptophan. ACKNOWLEDGMENTS The author wishes to express his sincere gratitude to his major professor, Dr. J.R. Brunner, for his advice and excellent guidance during the course of this study and for his aid in the preparation of the thesis manuscript. Appre- ciation is also extended to Dr. P. Markakis and Dr. L. Dugan of the Department of Food Science and Human Nutrition; to Dr. F.C. Elliott of the Department of Crop and Soil Sciences for serving on the guidance committee and for reviewing the thesis. Acknowledgment is due to U. Koch for performing the amino acid analyses for this study. The author feels grateful to the Departamento de Technologia de Alimentos at Universidade Federal de Vicosa (Brazil) and to the Programa de Ensino Agricola Superior (PEAS-Brazil) for the opportunity and financial assistance granted to him. ii TABLE OF CONTENTS LIST OF TABLES. LIST OF FIGURES LIST OF APPENDIX TABLES . . . . INTRODUCTION. LITERATURE REVIEW . Isolation and Characterization of Navy Bean Trypsin Inhibitors. . . . Nutritional Importance. . . Significance in Plants. . . EXPERIMENTAL. Chemicals and Materials . . Apparatus and Equipment . Methods . Synthesis of ll- aminoundecanoate Methyl Ester . . . Immobilization of Trypsin on Porous Glass Immobilization of Trypsin on Agarose. Determination of Unsound Trypsin. . Activity Measurements of Agarose- -Trypsin. Protein Extraction. . . Raw beans . Heat treated beans. Nitrogen Determination. Moisture Determination. Inhibition Measurements Inhibition measurement with trypsin Inhibition measurement with o-chymo- trypsin . Preparation of Affinity Columns Experimental glass- trypsin column Experimental agarose-trypsin column Preparative column. Separation of the Navy Bean Trypsin Inhibitor . . . . . . O O O I O Page SDS-polyacrylamide Gel Electrophoresis. Isoelectric Focusing. Amino Acid Analysis Tryptophan determination. Amino acid composition. . Methionine and cystine analysis RESULTS AND DISCUSSION. Immobilization of Trypsin Protein Extraction. Separation of Inhibitor Inhibition Measurements Inhibition Measurement with SBTI. Inhibition Measurement with NBTI. . Molecular Weight Determination by SDS- PAGE. Isoelectric Focusing. . Amino Acids Determination CONCLUSIONS REFERENCES CITED. APPENDIX. iv Page Table II III IV LIST OF TABLES Page Amount of Trypsin Bound to Agarose. . . . . . 43 Activity of Immobilized Trypsin . . . . . . . 44 Protein Content of Albumin and Globulin Fractions Isolated from Navy Beans. . . . . . 44 Amino Acid Composition of Navy Bean Trypsin Inhibitor O 0 O O O O O O O I 9 O O O O I ° 0 45 Figure TO LIST OF FIGURES Mechanisms involved in the immobilization of trypsin on porous glass. . . . . . . . . . . Immobilization of trypsin on agarose . . . Chromatogram showing nonspecific binding of QIaSS-trypSin COIUmn o o o . o g o o o o Chromatogram showing specific binding of agarose-trypsin column . . . . . . . . . pH-stat plot showing the esterase activity of agarose-trypsin. . . . . . . . . . . . . . . Chromatographic separation of navy bean tryp- Sin inhibitor. 0 o g g o Q Q o g g 0 I I 0 O Trypsin activity on hydrosis of casein . . . Polyacrylamide-SDS gel electropherogram of the navy bean trypsin inhibitor. . . . . . . . . Standard curve for determination of molecular “'81th by SDS'PAGE o o o a 9 I o o o o o o o Ferguson plot derived from PAG-SDS electro- pherograms for navy bean trypsin inhibitor vi Page 17 20 47 47 SI 53 54 56 56 LIST OF APPENDIX TABLES Table I Inhibition of Trypsin Activity by Navy Bean Trypsin Inhibitor. . . . . . . . . . II Inhibition of Trypsin Activity by Soybean Trypsin Inhibitor. . . . . . . . . . . III Inhibition of o-Chymotrypsin Activity by Navy Bean Trypsin Inhibitor . . . . . . . . . . . IV Equations Derived from Ferguson Plots. ._. . vii Page 66 67 67 68 INTRODUCTION A significant proportion of the protein in the human diet, on a worldwide basis, comes from legumes. It is well known that common beans (Phaseolus vulgaris L.) are one of the main sources of protein in Latin America and particularly in Brazil where several dietary surveys have shown an average daily consumption of 60 g of beans per person (De Souza _t 31., 1973). Unfortunately they contain a great number of antinutritional factors which, if not destroyed, have undesirable effects on the nutritive value of these seeds. Raw beans when fed in an experimental diet are toxic to animals (Jaffe, 1972). Among these undesirable factors are the proteinase inhibitors. Trypsin inhibitors from navy beans (Phaseolus vulgaris L.) have been prepared by fractional precipitation, gel filtration and ion-exhcange chromatography or combinations of these techniques. Conventional procedures of protein separation and purification are generally based on small differences in physiochemical properties of proteins. Generally these techniques are tedious, yield poor resolution and selecti- vity. Affinity chromatography exploits the biological specificity of the protein-ligand interaction. In principle, molecules with appreciable affinity for the ligand will be retained and others will pass through the column unretarded. The adsorbed proteins are eluted by altering the composition of the solvent to favor dissociation of the liquid-protein complex (Cuatrecasas and Anfinsen, 197l). It was the purpose of this study to separate a trypsin inhibitor from navy beans on the basis of the specific binding with trypsin immobilized on an insoluble support. LITERATURE REVIEW Isolation and Characterization of Navy Bean Trypsin Inhibi— tors Read and Haas (1928) were among the first workers to recognize the presence of a trypsin inhibitor in plant material. They reported that an aqueous extract of soybean flour inhibited the ability of trypsin to liquify gelatin. No attempt was made to separate the inhibitor. Bowman (l944) described the presence of trypsin inhi- bitors in navy beans, soybeans, wheat and corn. He was the first to suggest that the presence of a heat labile protein in aqueous extracts of navy beans and soybeans, which inhi- bited in 11319 digestion of casein by trypsin, might account for the low nutritive value of raw legumes. He reported that the digestion retarding fraction from navy beans could be concentrated by precipitation with acetone or alcohol while satisfactory precipitation from soybean extract could be achieved with acetone only. Kunitz (l945, l946) succeeded in crystallizing a trypsin inhibitor protein of globulin type from the extracts of soybean which later became the most extensively studied inhibitor of plant origin. Soybean and the navy bean trypsin inhibitors were differentiated by Bowman (1948) in regard to their solubilities and activities. He found that the crude navy bean inhibitor was considerably more active and water soluble than the crystalline, globulin soybean trypsin inhibitor. Additional studies were conducted on the trypsin inhi- bitor of navy beans. Wagner and Riehm (l967) isolated a trypsin inhibitor from navy beans (California small white bean) by first extracting the ethanol washed ground seeds with low concentration hydrochloric acid followed by ammo- nium'sulfate fractionation, gel filtration and ion-exchange chromatography on DEAE-cellulose. Important aspects of its amino acid composition were the low level of methionine, the absence of tryptophan and the high content of half- cystine. The inhibitor contained 2 moles of hexose per mole of protein and no thiol groups. Gel filtration of the oxi- dized protein indicated that the native protein was a single polypeptide chain. Ultracentrifugation data gave a molecu- lar weight of 23,000. The stoichiometry of the reaction between inhibitor and trypsin suggested an inhibitor to enzyme molar ratio of 1:2. Bowman (l97l) chromatographed the water extract of navy bean (Sanilac) on a DEAE-cellulose column with NaCl gradient. Except for the first peak eluted without the aid of NaCl, the entire chromatographic elution pattern repre- sented inhibiting material. One of the components was further investigated. This component was homogeneous on disc electrophoresis and inhibited the proteolytic and esterolytic activities of both trypsin and a-chymotrypsin. The constant apparent specific activity of this navy bean inhibitor in reaction with trypsin, containing varying pro- portions of active and inactive enzyme, suggested that the inhibitor reacts with active as well as inactive trypsin. In a subsequent experiment, Whitley and Bowman (1975) investigated the water-extractable proteins of navy beans. The fraction containing inhibitors was eluted from a DEAE- cellulose column with 0.13 M NaCl. This fraction was rechromatographed on the same column using NaCl and pH gradients. Four components were separated of which one was shown to be homogeneous and one nearly so. The molecular weight for the homogeneous component calculated by reaction with trypsin was 7,900. Based on this value and in the content of characteristic amino acids in both components, the authors place them as members of a general class of low molecular weight proteins known as Bowman-Birk inhibitors. They explained that the possibility of self-association was the reason why Wagner and Riehm (1967) reported a much higher molecular weight for one inhibitor with an amino acid composition similar, on a relative basis, to the two compo- nents investigated. It should be noted that the methods thus far described for calculation the molecular weight of the navy bean inhi- bitor did not take advantage of dissociating agents like a combination of sodium dodecyl sulfate (SDS) and mercaptoethanol. Oxidation of dissulfide bonds as performed by Wagner and Riehm (1967) does not eliminate the possibility of association through other residues. Intermolecular asso- ciation for the Bowman-Birk inhibitor from soybeans does not involve disulfide bonds (Birk, 1976). Nutritional Importance Early in this century Osborn and Mendel (1912) showed that phaseolin and raw navy bean meal were not capable of supporting animal growth. McCollum gt al. (1917) fed navy bean meal as a source of protein and the animals failed to grow. These authors attribute the failure of navy beans to promote growth to the presence of hemicellulose. Johns and Finks (1920) supplemented the fraction phaseolin and navy bean meal with cystine after heat treatment to obtain normal growth. They suggested that the beneficial effect of heat alone could be destruction of toxic material or improvement in digestibility of the proteins. Waterman and Johns (1921) tested the later hypothesis. They found that heat treatment of the navy bean protein increased the in 11319 digestion by trypsin and pepsin. Everson and Heckert (1944) studied the biological value of several legume seeds. including navy beans. They reported appreciable differences in the growth-promoting qualities between raw and cooked beans. Kakade and Evans (1965a) isolated five protein frac- tions from navy beans containing trypsin inhibitor and hemagglutinin activity in different proportions. These fractions were combined with autoclaved beans, or with raw beans followed by autoclaving. All of those fractions were found to inhibit the growth of rats. The growth inhibitory effect was attributed neither to the hemagglutinin nor the trypsin inhibitor, but to a toxic material scattered through- out all navy bean fractions. In a subsequent experiment. Kakade and Evans (1965b) suggested that the low nutritive value of navy beans was due to the presence of a heat labile growth inhibitor and methionine deficiency. Autoclaving - 121°C; 5 min. - destroyed nearly all trypsin inhibitor and totally the hemagglutinin activity; Even when supplemented with the deficient amino acids, raw navy beans did not sup- port the growth of experimental animals. This growth inhi- bitor factor was later isolated and characterized (Evans t 1., 1973). It was identified as a phytohemagglutinin Z ith leucocyte stimulating activity. Its molecular weight was 110,000 and possessed an isoelectric point at pH 5.2. This protein appears to be similar to the phytohemagglutinin pH A=a' (Dahlgren gt 31., 1970) in respect to molecular weight and amino acid composition. Soaking the beans was found to decrease both trypsin inhibitor and hemaglutinin activity. Germination had the opposite effect on the inhibitor and did not change the hemagglutinin (Kakade and Evans, 1966). Although comprising only 2.5% of the total protein, the trypsin inhibitor, present in a fraction isolated by Kakade and Evans (1965a), contributes 40% of the total cys- tine of the navy beans. Since the navy bean trypsin inhi- bitor is poorly attached by digestive enzymes unless modified by heat, Kakade gt gt. (1969) suggested that this disproportionate distribution of cystine was the major fac~ tor involved in the low nutritive value of raw navy beans. This resistance to enzymatic attack is probably due to the stability of molecule produced by a large number of disulfide bonds. The effect of heat is to cause an unfol- ding of the molecule resulting in the exposure of peptides bonds susceptible to enzymatic cleavage (Birk, 1968). In a related experiment Kakade gt gt. (1970) fed rats with a synthetic inhibitor - p-aminobenzalmidine - and navy bean trypsin inhibitor. The effects of antibiotic and cystine supplementation were investigated. Both inhibitors caused growth retardation, pancreatic enlargement, and exaggerated secretion of pancreatic enzymes. Cystine supplementation failed to bring the growth to the same level obtained with antibiotics. They explained the results on the basis of conversion of methionine to cystine to meet the demand created by abnormal secretion of pancreatic enzymes, and the increase in intestinal absorption of cystine promoted by antibiotics. The existence of trypsin inhibitors in legume seeds seemed to offer a reasonable explanation for the observation made by the early investigators that heat treatment improved the nutritional value of these seeds. From the results of a study on the physiological response of rats to raw or treated soybean meal diets, it was concluded that the tryp- sin inhibitors are responsible for the pancreatic hypertro- phy and from 30 to 50% of the growth-inhibiting effect (Steiner and Frattali, 1969). In a review, Birk (1968) affirmed the pancreatic hypertrophic effect, but concluded that these inhibitors have a minor role in growth depression. For a more comprehensive review of trypsin inhibitors of plant origin, the reader is referred to the reviews by Pusztai (1967) and Birk (1968). Distribution, occurrence, chemistry, structure and preparation of several plant pro- teinase inhibitors were covered by Birk (1976). Significance in Plants The function of plant proteinase inhibitors has been a subject of speculation. Various proteinase inhibitors are easily extracted in active form from their source, which indicates that they are not bound to or associated with proteolytic enzymes. Some of these proteins inhibit pro- teolytic enzymes of insects, but rarely proteolytic enzymes of plant origin. The arguments regarding plant proteinase inhibitors as specific metabolic defense mechanism agents against insects are based on their ability to inhibit insect digestive proteinases. This hypothesis is further supported by the finding that wounding of the leaves of potato or tomato plants by insects or by mechanical means induces an accumulation of proteinase inhibitors in both damaged and adjacent leaves (Applebaum t 1., 1964; Birk, 1968; Green and Ryan, 1972; Birk, 1976 and Steffans t al., 1978). EXPERIMENTAL Chemicals and Materials Navy beans, Sanilac cultivar (B. vulgaris), obtained from the 1976 harvest, were supplied by the Bean and Beet Research farm - Saginaw. The principal chemicals used in this research are listed here: 11-aminoundecanoic acid which was used to synthetize the ll-aminoundecanoate methyl ester employed as spacer in the immobilization of trypsin, was obtained from Pfaltz and Bauer Inc. Amino propyl porous glass, containing 0.053 mmole of amino group/g of material, mean pore diameter of 518A°, surface area of 60.2 mZ/g, 80- 120 mesh, was purchased from Electro-nucleonics, Inc. Cyanogen bromide and hydrazine (anhydrous) were acquired from Pierce. Sepharose 4B-200, trypsin (from bovine pan- creas, 2x crystallized, dialysed and lypophilized, salt free), a-chymotrypsin (from bovine pancreas, 3x crystallized, 1yophilized, salt free), soybean trypsin inhibitor (type I-S), p-tosyl-L-arginine methyl ester (TAME), tris-hydroxy- methyl amino methane (Trizma Base) and coomassie brilliant blue R-250 were obtained from Sigma. Acrylamide and N'N' methylenebisacrylamide were obtained from the Ames Company and recrystallized from acetone before use. The N,N,N'N' tetramethyl ethylenediamine 11 12 and riboflavin were purchased from Eastman Organic Chemicals. Reagent grade ammonium persulfate was purchased from Baker Chemical Company. Ampholine pH 4-6, 40% w/w was obtained from LBK. All other chemicals were of reagent grade. Agparatus and Equipment pH values were monitored with an Instrument Labora- tory Inc., Model 245 pH meter or a digital, Chemtrix Model 60A equipped with glass electrodes. For most laboratory weighings, a top loading, direct reading Mettler type K-7 balance was used. Analytical weighings were performed on a Sartorius analytical single pan balance. Centrifugation was performed in a sorvall model RC2-B centrifuge using either the large capacity model GSA or the superspeed SS-34 rotors. A recording pH stat manufactured by E.H. Sargent and Company was used for activity measurements of immobilized trypsin on agarose. The immobilized trypsin on agarose (Sepharose 4B) was packed in a Pharmacia 50 x 2.5 cm glass column. The eluates were monitored at 254 nm with a recording ultraviolet analyser, model US-2 and in some cases fractions were col- lected and absorbances measured at 280 nm. The U.V. monitor, the recorder and a fraction collector model 1100 were manu- factured by Instrumentation Specialities Company. A Beckman DK-2A spectrophotometer equipped with silica cells - 1 cm path length - was used for all spectrophotometric 13, measurements. An electrophoresis apparatus manufactured by Buchler Instruments and a Bi-Rad Laboratories model 400 power supply (voltage range 0-500V, and current 0-100 mA) were used for all 505 gel electrophoresis and isoelectric focusing runs. The excess dye was removed from the electrophoretic gels by means of a Bio Rad Model 170 diffusion destainer. Protein solutions were freeze-dried on a laboratory- constructed 1yophilizer. A micro-Kjeldahl apparatus was used for nitrogen determination. Amino acid analysis was performed on a Beckman Model 120C Amino Acid Analyser. Methods Synthesis of ll-aminoundecanoate Methyl Ester Methylation of ll-aminoundecanoic acid was performed as described by McKay gt gt. (1958):50.0 g of ll-amino undecanoic acid was added to 1250 ml of 3.5 N hydrochloric acid in methanol. Crystallization of derivative was induced by allowing this solution to stand at room temperature for 16-20 hours. After one third of the methanol was removed in vacuum the remaining mixture was cooled (4°C) and the crystals formed were recovered by filtration on Whatman No. 1 paper and air dried at room temperature. Immobilization of Trypsin on Porous Glass Immobilized trypsin on glass was prepared by the tech- nique described by Loeffler and Pierce (1973): 1.5 g of amino propyl porous glass was suspended in 20 ml of distilled water and 2.0 ml of 4.0 M KC1 was added. pH was adjusted to 10.0 with 5 N NaOH. With continued stirring at room tem- perature under a hood, a solution of 3.0 g of cyanogen bro- mide in 3 ml of tetrahydrofuran was added. 5 N Na0H was added dropwise to maintain the pH at 10.0 for about 10 min. The suspended glass was filtered and washed thoroughly with 0.1 M sodium bicarbonate at pH 9.0. The "activated" glass was added to a cold solution of ll-amino undecanoate methyl ester - 3.90 g in 50 m1 of 14 15 0.1 M sodium bicarbonate (pH = 9.0) and 50 ml of methanol. The suspension was adjusted to pH 9.0 and stirred at 4-5°C for 20 h. Following this treatment it was filtered on a coarse sintered glass funnel and washed stepwise with methanol, 0.1 M HCl and, finally, water. Forty milliliters of methanol and 3 m1 of hydrazine were added to the treated beads, stirred for 3 h at 4-50C, filtered on coarse sintered glass, washed stepwise with methanol, 0.1 M hydrochloric acid and water to neutrality and, finally, with methanol. The product was air dried before continuing the next step in the derivatization. The glass-hydrazide derivative was suspended in 5.0 ml of 0.1 M hydrochloric acid. With continuous stirring, a solution of 1.50 g of sodium nitrite in 5.0 ml of water was added and reaction continued for 2 min at 0°C. The product was washed with cold water and 6.0 m1 of a pH 4.1 buffer (0.1 M CaCl 0.001 M HCI, 0.02 M H 303) was added. 2’ 3 The glassaazide derivative obtained by this procedure was suspended in a solution of 5.0 ml of buffer, pH = 4.1, containing 300 mg of trypsin. The system was adjusted to pH 9.0 with 5 N NaOH and stirred for 20 h at 0°C. The pH was checked twice and maintained at pH 9.0 during this operation. The reaction mixture was filtered on coarse, sintered-glass and washed with small amount of buffer pH = 4.1. Five milliliters of buffer pH 9.0 (0.1 M NH Cl, 4 0.1 M NH 0H, 0.1 M CaClz) was added to the washed glass- 4 trypsin derivative and stirred at 0°C for 4 h. The 16 suspension was filtered and washed with a solution of pH = 3.8 (0.1 M CaCl 0.001 M HCl) at 0°C. The product was 2! stored in this medium at 4-5°c with sodium azide added as a preservative against microbial growth. The immobilization steps described above are represented schematically in Fig. l. Immobilization of Trypsin on Agarose Activated agarose was obtained by the method of Cuatrecasas and Anfinsen (1971). An experimental prepara- tion using 20 m1 of agarose-Sepharose 4B- was performed before the preparative immobilization. One hundred milliliters of decanted agarose was mixed with 100 ml of distilled water. Twenty grams of finely divided cyanogen bromide was added in one addition to the stirred suspension. Immediately, the reaction was raised to pH 11 with 5 N NaOH. The temperature was maintained at about 20°C by adding pieces of ice as needed. The reaction was completed in 10 min as indicated by the cessation of base uptake. A large amount of ice was added to the sus- pension which was transferred to a coarse sintered-glass funnel and washed under suction with 300-400 ml of a cold solution of NaHCO3 (0.1 M, pH 9.0). Following a procedure described by Loeffler and Pierce (1973), the activated agarose was added to a cold solution of 9 g of ll-amino undecanoate methyl ester in 150 ml of 0.1 M NaHCO3 pH = 9.0, the final volume adjusted to 250 ml 17 .Am~s_ .aetaaa new gmpmwwon ”mmmp .p—mummzv cwum owocmumucz ocmsmtpp mo muovumEmecP mnPNm may mw> mmmpm maosoa co :wmgzgu mo cowamNPFwaoEEw mg» cw um>~o>cw mamvcmnomz .F meamwd I I o I . I o I . a IPmast - . a . .meI o~.uaoso.muI . a . . e Imuu-o_A~Iuvz-u-z-mANIuv-mmapu A», camaxe» mz.mu -o_A~Is-z-u-z-mANIUV-mmaPI .Iae N sues Nozaz M_UI I o q I I I . : .mL; m mu cm . . - 3:85:93-z-u-z-£NI8-mmaS N ONVNIEI10.3-0:NIUTISIANISISE 9 1212 I = I .III om ”gem mo.auIa MIuoou-o_A~Isv-zNI mma_m Passes cease I u I zmu-z-mA~:uvtmmm_w zmwmmm chsmzu-mzu-mxuum o _ -o-em-o- . o . 18 with the same solution of NaHCO3 and stirred overnight at O-ZOC. The product was filtered on a coarse sintered-glass funnel, washed stepwise with water, 1 M HCl and water to neutrality. The activated gel was washed with six 50 ml portions of methanol, adjusted to 300 ml with methanol, and 10.0 g of hydrazine added. The suspension was stirred for 6-7 h at room temperature. The hydrazide derivative was filtered on a coarse sintered-glass funnel, washed thoroughly with methanol and water to neutrality, and finally adjusted to 200 g with water. Twenty milliliters of 1.0 M HCl at 0°C was added to the cold suspension followed by 3.0 g of NaNO2 dissolved in 10 ml of water. After stirring for 20 min at 0-20C, the gel was filtered, washed thoroughly with water and then made to 200 g with buffer, pH 4.0 (0.1 M CaClz, 0.001 M HCl, 0.02 M H3B03). Two and half grams of trypsin dissolved in 100 ml of buffer pH = 4.1 were added and pH adjusted to 9.0 with 5 N NaOH. The suspension was stirred at 0°C in a beaker covered with plastic film for 20 h. The pH was checked twice during this operation. The reaction suspension was filtered and washed with small portions of buffer at pH = 4.1. Filtrate and washings were collected, volume adjusted to 500 m1 (Filtrate I) with water and absorbance read at 280 nm. To the washed gel, 100 m1 of buffer pH = 9.0 (0.1 M NH CT, 0.1 M HN OH, 0.1 M 4 4 CaClz) was added and the suspension stirred at 0°C for 4 h. 19 The suspension was filtered and washed with a cold solution at pH = 3.8 (0.1 M CaClz, 0.001 M HCl). As before, fil- trate and washings were collected for absorbance readings (Filtrate II). Washed gel was made up to 200 ml with the same solution (pH 3.8) and stored at 4-5°C with sodium azide added. The immobilization procedure is schematically represented in Fig. 2. Determination of Unbound Trypsin Absorbances of trypsin solutions ranging from 0.05 mg.m1" to 0.5 mg.m1" in buffer at pH = 4.0 were read at 280 nm - 1 cm - with the buffer serving as reference. Amount of unbound trypsin was estimated by reading the absorbance of filtrates and washings after addition of trypsin in the immobilization procedure. Activity Measurements of Agarose-trypsin Measurements of esterase activity of agarose-trypsin were Performed using p-tosyl-L-arginine methyl ester as substrate as indicated by Walsh and Wilcox (1970). Seven milliliters of 0.01 M p-tosyl L-arginine methyl ester (TAME) in buffer at pH = 7.95 (0.1 M KC1, 0.05 M CaClZ, 0.01 M tris) was transferred to the sample cell of a Sargent pH-Stat. When the temperature of solution reached 37 i 0.5°C, with the pH selector set at 8.0, the function dial was turned to "RUN". After the base line was stable. volumes ranging from 0.050 to 0.100 ml of agarose-trypsin 20 .mump .mocmea w Impmemoo mommp .cmx< a sumgoqv mmogmam co cNmamsa mo coNNNNPFPnoEEH .N mgzawu I I o :Pmaxshuh d m .mI; om muoomc.m.:Q mz/ oF N _ = VIIINIUTINI 1 Emma sea. I INTINI o ewe oN NueN PUI NOIII I . I m I o .NII I mu INmIomIN N -b o IN-o, OF N Ir a o a N NI. II /u-o_ANINI-b-%-o I l l I \ INN A II II I I o\ .SI IN SIN moNhIaw N Is-o I OF N u m can- A Iuv z I mmogmmm \b TepuIa - o + I-qu All Imu-o A1. INNII Io 21 suspension or from 0.020 to 0.050 ml of 0.1% trypsin solu- tion in 0.001 M HCl were added with continuous stirring. During the first 3-5 min, the volume of 0.1 M tris base necessary to maintain the pH at 8.0 was recorded, and the initial slopes were determined for use in enzymatic acti- 1., 1957). vity calculations (Jacobsen gt Protein Extraction Raw beans. The protein extraction procedure was adapted partially from Danielson (1950), Seidl _t _t. (1969), and Goa and Strid (1959). Two hundred grams of Navy bean (Sanilac) powder (50 mesh) was added to 1 liter of l M NaCl, homogenized with a Super Dispax (Tekmar Co. Mod. SD45K), adjusted to pH 7 with sodium hydroxide and stirred overnight at 4-5°C. The suspension was centrifuged at 16,000 x g for 30 min at 4-5°C. The precipitate was washed with l M NaCl, centrifuged again and discarded. The supernatant was centrifuged at 35,000 x g for l h at 4—5°C and the resulting precipitate was discarded. The classified supernatant was dialysed for 4-5 days against distilled water with periodic changes of water. The precipitated globulins were separated by centrifugation at 16,000 x g for 2 h at 4-5°C, washed with distilled water and centri- fuged. The globulin fraction was freeze-dried and stored at 4-5°C. The supernatant containing the albumins was pervapo- rated in dialysis bags overnight at 4-5°C until the volume 22 was reduced to about half of the initial volume, freeze- dried and stored at 4-5°C. Heat treated beans. Before extracting the protein, 500 g of navy beans were treated at 121°C for 30 min. The cooked beans were air dried at 40°C overnight and ground in a Wiley mill to pass 50 mesh screen. Two hundred grams of this powder were treated similarly to the raw beans. There was no water-insoluble fraction in the l M NaCl extract. Nitrogen Determination Duplicate samples ranging from 4 to 15 mg of dried protein or about 100 mg of the 50 mesh powder were digested with 4 ml of the digestion mixture over a gas flame for l h. The digestion mixture consisted of 5.0 g of CuS04'5 H20 and 5.0 g of Se02 in 500 ml of concentrated sulfuric acid. After cooling the digestion mixture, 1 m1 of 30% H202 was added to each flask and digestion continued for another hour. After cooling, the sides of the digestion flask were rinsed with small volumes of distilled water. The digestion flasks were connected to the distillation apparatus. Then, following neutralization with 25 ml of 40% sodium hydroxide solution, the released ammonia was steam distilled into 15 m1 of 4% boric acid solution containing five drops of indi- cator consisting of 400 mg of bromocresol green and 40 mg of methyl red in 100 ml of 95% ethanol. Distillation was continued until a final volume of 50 to 50 ml of distillate was collected. The resulting ammonium borate was titrated 23 with 0.0193 N HCl. Hydrochloric acid was standardized with tris-hydroxymethyl amino methane (Sigma-Trizma base). Titrations were performed with a 10 m1 burette. Recoveries of tryptophan standards performed in each run varied from 93.1 to 101.0% with an average of 97.9%. Moisture Determination The procedure consisted in weighing accurately, to the nearest 0.1 mg, a 4-5 9 sample of 50 mesh navy bean powder into a previously dried and tared weighing bottle. The samples were dried at 70°C for 24 h. in a vacuum oven. At the end of drying time the vacuum was released with sul- furic acid dried air. The samples were cooled to room temperature in a desiccator and reweighed. Weight loss was assumed to be water. Inhibition Measurements Inhibition measurement with trypsin. Activity measure- ments of trypsin in the presence of inhibitor isolated from navy beans or other proteic fractions were performed by a modification of the method employed by Kunitz (1947) as proposed by Kakade gt gt. (1969). In order to check the effective range of enzyme con- centrations, 0.2 to 1.0 m1 of 50 ug/ml of trypsin solution in 0.001 M HCl was pipetted into a triplicate set of test tubes - one set for each level of trypsin - and the final volume was adjusted to 2.0 ml with 0.1 M phosphate buffer 24 at a pH of 7.6. To one of the triplicate tubes (blank), 6.0 m1 of 5% trichloroacetic acid was added and the tubes were set in a water bath at 37°C. Then 2.0 ml of a 2% casein solution, previously adjusted to 37°C, was added to each tube. The casein solution consisted of 2 g of casein dis- solved in the phosphate buffer by heating on a steam bath for 15 min and made up to 100 ml. After exactly 20 min, the reaction was stopped by adding 6.0 ml of 5% TCA to the sam- ple tubes. After standing for 1 h at room temperature, the suspension was filtered through Whatman No. l, and absor- bance of the filtrate was monitored at 280 nm against the blank for each level of enzyme. Hydrolysis time was checked with 1.0 ml of trypsin solution in intervals varying from 2 to 25 min of reaction. All conditions were the same as described for measurement of the effective range of concen- trations. For inhibition measurements, solutions of 50 ug/ml of navy bean trypsin in hibitor (NBTI), 50 ug/ml of soybean trypsin inhibitor (SBTI), 500 ug/ml of globulin fraction, 200 ug/ml of albumin fraction and 1000 ug/ml of heat treated navy bean protein were employed. All solutions were pre- pared in phosphate buffer. Volumes from 0.0 to 1.0 ml of protein solution were pipetted into a triplicate set of tubes and adjusted to 1.0 m1 of final volume with phosphate buffer. One milli» liter of a 50 ug/ml of trypsin solution was added to each tube; the tubes were placed in a water bath at 37°C for 25 10 min. Finally, 6.0 m1 of 5% TCA were added to one of the triplicate tubes which served as a blank for each level of protein examined. The remainder of the procedure was the same as that described above. In all cases, absorbances 3/2 as originally proposed by Miller were transformed to A and Johnson (1951). Inhibition measurements with o-chymotrypsin. The pro- cedure for determination of the activity of o-chymotrypsin in the presence of navy bean trypsin inhibitor (NBTI) was adapted from the method described by Rick (1974). Volumes from 0.0 to 1.0 m1 of NBTI solution in the borate buffer were pipetted into a triplicate set of tubes and the volumes adjusted to 1.0 ml with the borate buffer. The borate buffer was prepared by addition of 6.06 g of H3803 and 50 ml of 1 N NaOH in 650 ml of distilled water, pH adjusted to 8.0 with hydrochloric acid and the volume completed to 1 1. One milliliter of solution containing 48 ug/ml of o-chymotrypsin in 0.001 M HCl was added to each tube which were incubated at 35°C for 10 min. Then, 6.0 m1 of 5% TCA was added to one of each triplicated set of tubes which served as blank. Two milliliters of casein solution, pre- viously adjusted to 35°C, was added to each tube. The casein solution was prepared by suspending 2.0 g of casein in 95 ml of the borate buffer and heated on a steam bath for 10 min. One and one tenth milliliters of a 5% CaCl2 solution was added and the final volume made to 100 m1 after 26 cooling at room temperature. After 20 min, hydrolysis was stopped by the addition of 6.0 ml of a 5% TCA solution to the experimental tubes. After standing for 1 h at room temperature, the hydrolysis mixture was filtered and the absorbance of the TCA-soluble products was measured at 280 nm against the blank for each level of inhibitor. Preparation of Affinity Columns Experimental glass-trypsin column. One gram of glass- trypsin was mixed with 1.5 g of amino propyl glass and placed into a glass laboratory-made column. The packed column measured 3.50 x 1.25 cm. The column was washed with 1 M acetic acid, followed by tris buffer, pH 8.0, composed of 0.05 M tris-hydroxyimethyl amino methane, 0.10 M potas- sium chloride, 0.02 M calcium chloride and adjusted to pH 8.0 with hydrochloric acid. Solutions of soybean trypsin inhibitor (5 mg.m1'1) were added to the column in two 1 m1 portions. Protein solutions were prepared in tris buffer, pH 8.0. The charged column was washed with tris buffer, pH 8.0, until the absorbance (254 nm) was reduced to a low level. At this point the solvent was changed to l M acetic acid to elute the bound protein. Experimental agarose-trypsin column. A glass column was packed with agarose-trypsin (4.5 x 1.25 cm) representing 5.0 m1 of original Sepharose-4B. The column was washed successively with 1 M acetic acid and tris buffer, pH 8.0 (Filtrate III). Seven portions of 1.0 ml each of soybean 27 trypsin inhibitor and two of ovalbumin were added to the washed column. Both proteins were in a concentration of l mg.ml'1. The remainder of the procedure was the same as described for the glass-trypsin column. Preparative column. The preparative affinity-column was prepared by pouring agarose-bound trypsin, obtained from the initial 100 ml of agarose, into a 50 x 2.5 cm glass column. A sample applicator basket was placed on top of the column and the column was washed with about 300 m1 of buffer, pH 2.0. The buffer consisted of a 0.2 M KCl solu- tion which was adjusted to pH 2.0 with hydrochloric acid. Finally, the column was washed with 2 column volumes of tris buffer, pH 8.0, before applying the sample. Separation of the Navy Bean Trypsin Inhibitor Albumin solutions were prepared by dissolving the freeze-dried albumin fraction in the tris buffer, pH 8.0, to give 1% solutions. These solutions were filtered through a 5 u-Millipore filter before applying to the affinity column. Following a procedure employed by Mitchell gt gt. (1976), volumes of from 50 to 250 ml of the 1% albumin solu- tion were percolated through the agarose-trypsin column at a flow rate of 2 ml/min. The column was washed with the tris buffer, pH 8.0. Washing was continued until absorbance of the eluate measured at 254 or 280 nm was reduced to a low level, thus eliminating non—inhibitor proteins. The 28 protocol for eluting the inhibitor was adapted from Mosolov and Fedurkina (1974). The eluting solvent was changed to a pH 2.0 buffer to enhance the dissociation of the trypsin- inhibitor complex. The inhibitor was eluted in a volume ranging from 20 to 50 ml. After the inhibitor peak was eluted, the column was washed with a pH 3.8 solution, con- taining sodium azide as a preservative against microbial growth. The column was stored at 4-5°C and reused for 10 preparations of inhibitor. The collected inhibitor fraction was dialysed for 1-2 days against distilled water, freeze dried and stored at 4-5°C. SDS-polyacrylamide Gel Electrophoresis Polyacrylamide, sodium dodecyl sulfate (PAG-SDS) gels were prepared according to the method of Weber and Osborn (1969) with two modifications. 1. The proteins were incubated at 100°C for 5 min. in 0.01 M sodium phosphate buffer, pH 7.0, con- taining 1% SDS and 1% Bsmercaptoethanol. 2. Four concentrations of acrylamide were used to facilitate data reduction to Ferguson plots. Acrylamide and N,N' methylene bisacrylamide were re- crystallized from acetone before preparation of gel solu- tions. All proteins were prepared in a concentration of 1 mg/ml except chymotrypsinogen and the navy bean trypsin inhibitor which were adjusted to 2 mg/ml. The density of 29 the samples was increased with sucrose and about 5 ul of bromophenol blue/50 ul of solution was added as tracking dye. 20 to 40 ul of sample was layered over the gels. Electrophoresis was carried out at room temperature with a constant current of 8 mA/tube until the tracking dye migrated to within 5 mm of the lower end of the gel. The lower electrode served as the anode. Gels were soaked in 5% trichloroacetic acid for 30 min followed by staining with Coomassie Bri-liant Blue R-250 for 1-2 h at room tempera- ture. Gels were destained by diffusion (circulation) in acetic acid-methanol-water (15:10:175). Isoelectric Focusing Isoelectric focusing gel electrophoresis was performed according to the method described by Wrigley (1971). The gels were prepared by mixing 7.70 ml of distilled water and 0.30 ml of navy bean trypsin inhibitor solution with 0.30 ml of carrier ampholyte solution - pH 4-6, 40% w/w - and 3.0 ml of acrylamide solution. After addition of 0.70 ml of 0.015% aqueous solution of riboflavin, this mixture was transferred to 75 x 5 mm glass tubes. Water was layered on top and the tubes were exposed to a fluorescent light for polymerization. The acrylamide solution consisted of 30 g of acrylamide and 1.2 g of N,N' methylene bisacrylamide, both cyrstallized from acetone and dissolved in water to a volume of 100 ml. 30 The tubes were placed in a Buchler disc electrophor- esis apparatus cooled with water at 0°C. Sulfuric acid (0.2%) was used as the anodic solution and 0.4% ethanolamine as the cathodic solution. A current of 2 mA per tube was applied until the voltage required to yield this current increased to 400 v. The voltage was then stabilized at this level for 4-5 h. After focusing, the pH gradient was determined. Two or three gels were cut in 2.4 mm pieces which were macerated in 0.5 ml of freshly boiled and cooled water. The pH of the water extract was measured and plotted vs. length of the gel. Comparison gels were stained by the method of Malik and Berrie (1972) or by the procedure employed in SOS-PAGE. Amino Acid Analysis Tryptophan determination. Tryptophan was deter- mined colorimetrically after a partial hydrolysis with pronase as described by Spies (1967) in procedure W. A 3.0 mg sample was weighed into a small glass vial fitted with a screw cap. To each vial, 100 pl of pronase solution was added. The pronase hydrolytic solution was prepared by adding 100 mg of pronase to 10 ml of 0.1 M phosphate buffer, pH 7.5. The suspension was shaken and clarified by centrifugation. The vials were closed and incubated for 24 h at 40°C. Following incubation the vials were opened and 0.9 ml of phosphate buffer was added. These uncapped vials were 31 placed into Erlenmeyer flasks containing 9.0 ml of 21.2 N sulfuric acid and 30 mg of dimethylaminobenzaldehyde and their contents mixed by rotating the flasks. The reaction mixture was kept in the dark at room temperature for 6 h. Following the addition of 0.1 ml of 0.045% sodium nitrite solution, the reaction mixture was shaken and the color was allowed to develop for 30 min in the dark at room tempera- ture. Absorbances were measured at 590 nm. Duplicate blanks of the pronase solution were run similarly, and the trypto- phan content of pronase was subtracted from the total tryp- tophan contents. A standard curve from zero to 120 ug of tryptophan was prepared according to Spies and Chambers (1948). Absorbance and tryptophan levels were related by linear regression. Amino acid composition. Amino acid analyses were performed on 24 and 72 h acid hydrolysates employing a Beck- man Amino Acid Analyser, Model 120°C according to the pro- cedures of Moore gt _t. (1958). Eight milligrams of navy bean trypsin inhibitor was weighed into 10 m1 glass ampoules and 5 m1 of 6 N HCl was added to each of the ampoules. The content of the ampoules was frozen in dry ice-ethanol bath, evacuated with the 1yophilizer vacuum pump, and allowed to melt slowly under vacuum to remove dissolved gases. The content was again frozen and sealed with an air-propane flame. The sealed ampoules were placed in an oil bath at 110°C for 24 and 72 h. 32 The ampoules were broken on top and 1 m1 of norleu- cine solution containing 2.5 umoles/ml was added to each as a standard for transfer losses. The content of each ampoule was quantitatively transferred to an evaporating flask. The hydrochloric acid was removed on a rotatory evaporator. The dried sample was washed with a small amount of deionized water and again taken to dryness. In all, three washings were performed to remove residual HCl. The acid-free hydrolysate of each ampoule was transferred to 5 ml volu- metric flask with citrate-HCl buffer, pH 2.2. An aliquot of 0.2 ml was applied to the analyser column. The chromato- grams were quantitated by peak integration using a Spectra Physics Autolab System AA. Methionine and cystine analyses. The methods of Schram gt _t. (1954) and Lewis (1966) were used. These methods involve performic acid oxidation of methionine and cystine to methioine sulfone and cysteic acid, respectively. Ten milligrams of navy bean trypsin inhibitor was weighed into 25 m1 pear-shaped flask. The protein was oxidized for 15 h with 10 m1 of performic acid at 4°C. After oxi- dation, 1 ml of norleucine (2.5 umoles/ml) was added and the performic acid removed in a rotatory evaporator. The dried sample was quantitatively transferred to a 10 m1 ampoule with 5 ml of 6 N HCl. Hydrolysis and amino acid analyses were performed as previously discussed. RESULTS AND DISCUSSION Immobilization of Trypsin Immobilized trypsin on glass, via the azide of the ll-aminoundecanoic acid, retained a portion of its original activity for the hydrolysis of p-tosyl L-arginine methyl ester. Because this experiment represented a preliminary evaluation of the principle of immobilization, no quantita- tive measurements were made. The column prepared with this form of immobilized trypsin bound soybean trypsin inhibitor (SBTI) as well as ovalbumin and casein when these proteins were percolated separately through the affinity column. Chromatograms are recorded in Figure 3. Nonspecific reten- tion of proteins by the affinity column was a-tributed to ionic forces which exist between amino groups of proteins and the dissociated silanol groups on the glass surface (Messing, 1976). Trypsin immobilized on agarose beads - Sepharose 4B - retained a large portion of its original activity and exer- ted specific binding of SBTI as shown by the data presented in Figure 4. Commercial preparations of SBTI are contami- nated with other minor inhibitors present in soybean. The presence of small amounts of other proteins than the major inhibitor was verified by SOS-gel electrophoresis. Two 33 34 peaks observed during elution of bound SBTI from the affi- nity column may be attributed to the presence of these proteins with different degrees of association with trypsin. The amount of trypsin bound to agarose was measured by the difference between the initial amount added and that recovered in the washings. The esterase activity of the immobilized trypsin was measured from the data obtained with a pH-stat using p-tosyl L-arginine methyl ester as substrate. Data relating to these assays are shown in Tables I, II and Figure 5. Agarose beads both before and after activation and coupling exhibit very little nonspecific adsorption of proteins provided the ionic strength of the buffer is 0.05 M or greater (Cuatrecasas and Anfinsen, 1971). This proper- ty, among other considerations (Srere and Uyeda, 1976), made agarose the supporting material of choice for the purpose of this study. Regained enzymatic activity was calculated by divi- ding the activity of the immobilized species by that of native trypsin. A value of 19.5% retention was obtained. Activity measurements performed four months later revealed no significant loss of activity (i.e. 19.1%). The pre- parative scale affinity column made with 100 m1 of agarose retained 17.4% of initial actiVity. 35 Protein Extraction The extraction of proteins from dry navy bean with l M NaCl pH 7.0 was 45.6% efficient based on the crude pro- tein content of the beans as estimated by Kjeldahl nitrogen determination (N% x 6.25 = protein %) and the dry powder resulting from the extraction. The water soluble fraction - i.e., albumin - compressed 18% and the salt-soluble frac- tion - i.e., globulins - 82%, respectively, of the extrac- table proteins (on the Table III). Osborne (1894) reported that a globulin fraction accounted for about 85% of the bean seed protein (Phaseolus tulgaris) - Ishino and Ortega (1975) found that globulins represent 85% of black bean protein. Heat treated beans yielded only water-soluble proteins - albumins - by extraction with NaCl solution. The globulins which precipitated during dialysis of a NaCl extract of raw beans against water, were absent in the extract of cooked beans, after extensive dialysis. The freeze-dried, water—soluble, extract was low in protein, i.e. 31.5%. Extraction efficiency lowered from 45.6% for raw beans to 6.5% for heat treated beans. Heat causes drastic modifications in the structure of proteins, leading to physical and chemical changes (Joly, 1965). The severe reduction in proteins extractable by NaCl may be attributed to changes in protein solubility. 36 Separation of Inhibitor Figure 6 shows the result obtained when the albumin fraction was percolated through the preparative affinity column. The peak eluted with 0.2 M KC1 at pH 2.0 represents the inhibitor. This fraction, after dialysis and freeze- drying, contained 59.4% protein and comprised 2.7% of the albumin fraction. The content of inhibitor in the albumin fraction was calculated by the relation between the weight of the freeze-dried inhibitor to the weight of the albumin fraction on protein basis, applied on the column. Inhibition Measurements The rate of hydrolysis of casein by trypsin does not follow zero order kinetics under the conditions defined by Kunitz (1949). This aberration has been attributed to limited substrate concentration (Bundy and Mehl, 1958). The modification proposed by Kakade gt gt. (1969) employs 2% casein solution as substrate, instead of 1% in the Kunitz method, and the mathematical transformation of absorbance reading (A) of TCA soluble products to A3/2 as proposed by Miller and Johnson (1951). This modified procedure gives 3/2 and enzyme concentration a linear relationship between A over a large range. The activities of trypsin and o-chymo- trypsin in the presence of inhibitors and protein fractions of navy bean, shown in Figure 7 and Appendix Tables I, II, III, were calculated using this transformation. 37 Inhibition Measurement with SBTI The amount of inhibitor necessary for complete inhi- bition of the activity of a given amount of enzyme can be determined by extrapolation of a plot of "remaining acti- vity" 1g "inhibitor added" to zero enzymatic activity. An estimation of the inhibitor molecular weight can be obtained from this plot (Green and Work, 1953a). Based on a molecu- lar weight of 23,300 for trypsin, the method yielded a molecular weight of 12,000 for SBTI. Assuming a molecular weight of 21,500 for the SBTI as reported by Wu and Scheraga (1962) it was calculated that the trypsin was 55.5% active. This correction factor was used in calculating the amount of active trypsin in the inhibition assays. Inhibition of trypsin by SBTI is shown in Appendix Table II. Inhibition Measurement with NBTI The navy bean trypsin inhibitor (NBTI) strongly de- creased the proteolytic activity of trypsin. The action of NBTI, albumin, globulin and heat treated bean protein on hydrolysis of casein by trypsin is shown in Figures 7a, b, c, d, respectively. Extrapolation to zero activity of the enzyme indicated that 5.850 mg of albumins or 0.515 mg of NBTI was necessary to inhibit 1 mg of trypsin, thus resulting in an increase of 11.3-fold in the specific activity of the isolated inhibitor. The golbulins and heat-treated bean protein had little or no effect on the trypsin activity. The fact that the NBTI is an effective 38 inhibitor of o-chymotrypsin reported by Bowman (1971) was verified. The NBTI strongly inhibited the hydrolysis of casein by o-chymotrypsin (Appendix Table III). Soybean trypsin inhibitor strongly reduced the acti- vity of proteinases present in baker's yeast - Saccharamyces cerevisae, and NBTI had little or no effect on such pro- teinases (Dalilottojari, 1978). That the trypsin inhibitor is concentrated in the aqueous extract of navy bean was shown by Bowman (1944) and was isolated as a proteinase inhibitor (Bowman, 1971). A water-soluble protein fraction had the highest trypsin inhibitor activity of all fractions obtained from navy beans by Kakade and Evans (1965a). Similar results had been reported for other varieties of Phaseolus vulggris. Moso- lov and Fedurkina (1974) separated trypsin inhibitors from aqueous extracts of the Shirokostruchnaya variety. Seidl _t _t. (1969) found that a specific trypsin inhibitor from black kidney beans remained in aqueous solution while a non- specific proteinase inhibitor precipitated with the globulin fraction. The absence of anti-tryptic activity in the proteins extract from heat treated beans agrees with the observa- tion made by Bowman (1944) that the trypsin inhibitor from navy bean is heat labile. Also, Kakade and Evans (1965b) reported that a heat treatment for 121°C for 30 min destroyed nearly all the trypsin inhibitory activity of navy beans. 39 Assuming a trypsin-NBTI complex formed in 1:1 molar ratio, a molecular weight of 11,900 was obtained for the NBTI from the plot of "remaining activity" 1; "inhibitor added". The dissociation constant of the trypsin-inhibitor complex computed from the residual trypsin activity at the equivalence point by the method of Green and Work (1953b) -10M was 7.6 x 10 . Values of the same order of magnitude had been reported for effective inhibitors of trypsin. For ']°M was obtained for both pan- example, a value of 2 x 10 creatic and soybean trypsin inhibitors (Green, 1953); and a value 5 x 10']°M for an inhibitor isolated from Phaseolus vulgaris seeds (Mosolov and Fedurkina, 1974). Molecular Weight Determination by SDS-PAG Figure 8 shows the electropherogram obtained when the navy bean trypsin inhibitor was applied to 10.8% 505 gel and stained with coomassie blue. The molecular weight estimated from the standard curve of log MW vs. mobility (Figure 9) were 16,600 i 1,200 and 33,800 i 3.700 for the major and minor bands, respectively. Although the molecular weights obtained are informa- tive, the results must be analysed carefully. The basic assumption for the accuracy of molecular weight determina- tions on SDS gels is that the SDS molecule binds to the protein with a constant ratio and that separation is due to size (Fish gt a1., 1970; Weber and Kuter, 1971; Svasti and Parrijpan, 1977). Abnormal binding of $05 by some proteins 40 was shown by Kogen gt gt. (1972). Anomalous binding of SDS, atypical conformation of the protein-SOS complex, or unusual properties of the native protein-SOS complex main- tained in a $05 solution can affect the mobility of proteins in SDS gels (Banker and Cofman, 1972). The ideal situation where the presence of highly charged ionic detergent results in a uniform charge density for all proteins, including both standards and unknowns, is only rarely achieved in practice (Rodbard, 1976). In an attempt to use a protein which resembles the NBTI, at least in respect to origin, soybean trypsin inhi- bitor was included in the set of known molecular weight proteins. The resulted mobility was much higher than expected for its molecular weight. Considering that the major band of NBTI possessed a mobility very Close to that of SBTI, a molecular weight of about 21,000 should be ob- tained instead of the much lower value which was observed. The molecular weight of SBTI calculated from its relative mobility was 17,400. Because of its peculiar behavior, SBTI was not considered in the linear regression used to derive the calibration curve. Ferguson plots for NBTI, SBTI and four other proteins are shown in Figure 10. Equations derived from this plot are given in Appendix Table IV. The mobilities (Yo) at 0% total gel concentration (%T) for both NBTI and SBTI were very close, 1.20 and 1.21, respectively. All other proteins had considerable systematic variations in Y0; i.e., Y0 41 increases with increasing molecular weight (Appendix Table IV). Equality of Yo's and no systematic trend between Y0 and molecular weight or between Y0 and retardation coeffi- cient (Kr) is a condition for molecular weight estimates from a single value of %T (Frank and Rodbard, 1975). When these conditions are not achieved, estimation of molecular weight by a relationship between Mw and Kr is preferable since it combines information from several gel concentra- tions (Rodbard, 1976; Ugel gt gt., 1971; Kawasaki and Ash- well, 1976). The plot of molecular weight versus retardation coef- ficient yielded a linear relationship given by the equation ? = 608.98 x +3886.40 R2 = 0.9882, where 9 is the estimated molecular weight and x is 1000 Kt. The molecular weight calculated by this equation for SBTI was 19,300, reflecting the abnormal mobility of this protein in all gel concentra- tions studied. This equation yielded a molecular weight of 17,650 for the NBTI. Isoelectric Focusing The major band of the navy bean trypsin inhibitor showed an isoelectric pH at 4.40, whereas the minor band possessed an isoelectric pH of 4.45. The bands were visible only after staining with coomassie blue since no precipita- tion occurred at isoelectric pH as it was observed for SBTI. 42 Amino Acids Determination Table IV shows the amino acid composition of the navy bean trypsin inhibitor. Amino acids which are subject to degradation during hydrolysis were extrapolated to zero time of hydrolysis. Assuming a first order decomposition, the relationship 109 A0 = [tz/(tz-t])] log A1 - [t1/(t2-t1)] log A2 was applied; where A0, A1 and A2 are the concentrations of each amino acid after 0, 24 and 72 hours of hydrolysis, respectively. Values of 72 h hydrolysis only were consi- dered for those amino acids which are more resistant to hydrolysis. Important aspects of the amino acid composition of the navy bean trypsin inhibitor were the absence of tryp- tophan, the low amount of methionine and the high content of half-cystine. The minimum molecular weight, calculated on the basis of methionine as the limiting amino acid, was 12,214. The last two columns of Table IV compare the values obtained from this study with those reported by Wagner and Riehm (1967). Systematic trends in the amino acid composition pointed out by Birk (1968) for other trypsin inhibitors from plant origin were verified with the navy bean trypsin inhibitor: namely, a remarkably high and constant content of proline, acidic and basic amino acids; a high percentage of serine and threonine associated with a high content of half-cystine; low content or the absence of tryptophan and 43 the small contribution of methionine. Table I. Amount of Trypsin Bound to Agarose (20 m1) Volume (ml) Dilution Alcm Amount of tryps1n* 280 (mg) filtrate I 200 1:10 0.205 300.0 filtrate II 100 - 0.030 2.2 filtrate III 100 1:4 0.085 25.2 Total 327.4 Trypsin added 500.0 Bound trypsin 172.6 *Calculated from absorbance by a standard curve at 280 nm. 44 Table II. Activity of Immobilized Trypsin Vol. of suspensiona div/minb Trypsin (pg) activityc 0.05 ml 19 215.7 88.1 0.07 26 302.0 86.1 0.08 31 345.2 89.8 0.10 39 431.5 90.4 Average 88.6 Trypsin 450.0* a - total volume of 40 m1 (original 20 m1 of agarose) b - taken from initial slopes c - arbitrarily defined as chart div./min./mg protein * - average of three demoninations Table III. Protein Content of Albumin and Globulin Frac- tions Isolated from Navy Beans Fraction g/200 g NBa protein (%)b g prot/200 g NB % ext. prot. Albumins 5.15 66.2 3.40 18.0 Globulins 19.30 80.2 15.50 82.0 a - navy bean: 10.9% moisture and 23.2% protein - corrected for moisture b - content of protein (N x 6.25) in the resulting freeze- dried fractions; average of 4 determinations 46 ~ Table IV. Amino Acid Composition of Navy Bean Trypsin Inhibitor Amino acid mmole/mg prot. Residues mole Ref. 24 h 72 h Ext.1 % value3 lysine 0.518 0.493 0.531 6 5.17 5.27 histidine 0.448 0.420 0.463 5 5.04 4.95 arginine 0.330 0.313 0.338 4 3.30 3.50 trypt0phana - - 0.0 0 0.00 0.00 aspartic acid 1.459 1.419 1.480 16 14.43 14.39 threonine 0.622 0.575 0.647 7 6.32 6.49 serine 1.492 1.232 1.643 18 16.02 16.64 glutamic acid 0.930 0.886 0.953 10 9.29 8.30 proline 0.723 0.715 0.728 8 7.10 7.79 glycine 0.247 0.243 0.250 3 2.44 2.17 alanine 0.388 0.387 0.388 4 3.77 3.72 a cystineb - - 1.267 14 12.36 14.38 valine* 0.193 0.231 0.231 3 2.26 1.04 methionineb 0.092 1 0.90 0.62 isoleucine* 0.453 0.495 0.495 5 4.83 4.33 1eucine* 0.362 0.374 0.374 4 3.65 2.92 tyronine 0.161 0.131 0.179 2 1.75 1.70 phenylalamine 0.193 0.191 0.194 2 1.90 1.79 1Values extrapolated to zero time of hydrolysis or the 72 h results* considered only. 2Based on methionine as limiting amino acid. 3Wagner and Riehm (1967). aDetermined by the procedure of Spies (1967). bDetermined as cysteic acid and methionine sulfone, respec- tively. Figure 4. 46 Figure 3. Chromatogram showing non—specific binding of glass-trypsin column (3.5x1.25 cm); soybean trypsin inhibitor (SBTI) and ovalbumin- 5 mg.m1'1; casein-4 mg-ml‘ . Arrows indicate change in solvent from tris buffer, pH=8.0, to 1 M acetic acid. Chromatogram showing specific binding of agarose-trypsin column (4.5x1.2 cm); SBTI and ovalbumin- 1 mg-ml‘ . Arrows indicate the same change in solvent as indi- cated in Figure 3. 47 2:: 03:6 On On 0 $63.20 On On 0 ON _ :96 .600 ( £63.25 :95 in 8 o .96 4.0.. V a .88 ..o.N r L :3 25V muogw 8 0 ON 0 _ :30 :30 a :08 :08 9 5:8 .63 £3426 48 Figure 5. pH stat plot showing the esterase activity of agarose-trypsin (0.05 ml suspension) on p-tosyl b-arginine methyl ester as substrate (pH=8.0; 37°C). 49 A. .55 _ 1V 0.0 00. .0 00N.0 00nd 00¢0 0000 000.0 COMO 9309 818.1. 1° IW Figure 6. Chromatographic separation of navy bean trypsin inhibitor from the albumin fraction column: 18.5x 2.5 cm-aragose—trypsin. Flow rate: 2 m1~min' . Sample: 50 m1 1% albumin solution in tris buffer pH 8.0. Fractions: 5 ml each. Arrow indicates change in solvent from tris buffer pH 8.0 to 0.2 M KC1 pH 2.0. 51 l l l l o. 0. o. 0. 0. ID V '0 N — UJU 092 to eouoqiosqv Frociion Number Figure 7. Trypsin activity on hydrolysis of casein in rela- tion to the level of: A—navy bean trypsin inhibi- tor; B-albumin; C-globulins; D-proteins from heat-treated seeds. 50.0... 00.02... .00: 01 05.5.0.4 oi 53 2.8 0m... 2.... can 00.N 0m. 00 % @— bml 0 8 0. a 5:0 as. 2 m. 8 003.0. x8000.“ 8 m. 8 % . 6 8 . . . 00 v 0.. m o 1 MW 0 o l_8_w a... M .M 8 M v 2. w 00$.Ouum 0 W. 08.8 + 308.0- .0 x 00 m 00. :02 at 8.8 Na: 8.... 8... .8 3.» SN 0 3:306 01 . . . . . . 0 bmomduum o. 00.. 00m 08 00. 0 . . . . 80.0.8080- AI. 8 a 0n % e/ H 2. w... 0v m . w 00 m. m. m. 880.0 "am .8 m. 8m . . u % ON 0.03 + 8.0000 . .8 w 800...... 8 m. a rum 000.5 + £8040 .w. qllbllldlld. .09.». 00 0 < 8. 54 ~-.:.~c‘ .o’v- - - wowmn n N".~‘.'o “I“ "W"" a... I -o o ‘ a» ‘. Figure 8. Polyacrylamide-SDS gel (10.8% total concentration) electropherogram of the navy bean trypsin inhibi- tor. Figure 9. Figure 10. 55 Standard curve for determination of molecular weight by SOS-PAGE (10.8%). 1 - bovine serum albumin; 2 - ovalbumin; 3 - chymotrypsinogen; 4 - trypsin; 5 - lysozyme. SBTI not included in the linear regression. mobilities for the navy bean trypsin inhibitor. Points are the average of five duplicate gels. Ferguson plot derived from PAG-SDS electro- pherograms for navy bean trypsin inhibitor (NBTI), soybean trypsin inhibitor (SBTI) and four reference proteins referred to in Figure 7. :3 a) O O O :3 s1 0 O O :5 01 O O 0 09 p .0 O O O :5 N 0 O 0 :2 o o 0 Relative Mobility E V 56 9.4192011 + 5.0203 R2 = 0.9860 01.2 03 6.4 0.5 0.6 of? 08 Relative Mobility 5 0 won - SBTI O . 3 2 1- ° ' O 5 75 IO 12.5 CONCLUSIONS Percolation of albumin fraction from navy beans - Sanilac cultivar - through an affinity column prepared with immobilized trypsin on agarose - Sepharose 4B - and elution with acidic solution yielded a protein fraction which strongly inhibited the enzymatic activity of trypsin and o-chymotrypsin. The navy bean trypsin inhibitor showed two bands under isoelectric focusing. Isoelectric pH was 4.40 for the major band whereas the minor band was at 4.45. Molecular weights calculated from the relative mobil- ity on SDS-PAG electrophoresis were 16,600 3 1,200 and 33,800 1 3,700 for the major and minor bands, respectively. Molecular weight estimated from inhibition measurements with trypsin was 11,900, and the minimum molecular weight, on the basis of methionine as limiting amino acid, was 12,214. The dissociation constant of the trypsin-inhibitor complex computed from the residual trypsin activity at the equivalence point was 7.6 x lO'IOM. The fact that soybean trypsin inhibitor possessed a relative mobility under SDS-PAG electrophoresis higher than expected for its molecular weight can not be extrapolated for the navy bean trypsin inhibitor. Whether or not the navy bean trypsin inhibitor possess a mobility compatible with its molecular weight could not be established, but the 57 58 possibility of a molecular weight of 7,900, as proposed by Whitley and Rowman (1975), can be excluded. Important aspects of the amino acid composition of the navy bean trypsin inhibitor were the absence of tryp- tophan, the low amount of methionine and the high content of half-cystine. The amino acid composition is similar to the inhibitor isolated from navy bean seeds by Wagner and Riehm (1967). The proteins present in the globulin fraction of raw navy beans and the heat-treated bean protein had little or no effect on the hydrolysis of casein by trypsin. REFERENCES CITED Applebaum, S.W., Birk, Y., Harpaz, I. and Bondi, A. 1964. Comparative studies on proteolytic enzymes of Tenebrio molitor t. Comp. Biochem. Physiol. tt:85. Banker, G.A. and Cotman, C.W. 1972. Measurement of free electrophoretic mobility and retardation coefficient of protein-sodium dodecyl sulfate complexes by gel elec- trophoresis. J. Biol. Chem. ggt:5856. Birk, Y. 1968. Chemistry and nutritional significance of proteinase inhibitors from plant origin. A. N.Y. Acad. Sci. 146:388. Birk, Y. 1976. Chapters: 56, 57, 58, 59, 60, 61, 62, and 63. In: Methods in Enzymology. Vol. g§:695, L. Lorand ed. Academic Press Inc. Publishers, New York. Bowman, D.E. 1944. Fractions derived from soybeans and navy beans which retard tryptic digestion of casein. Proc. Soc. Exp. Biol. Med. gt:l39. Bowman, D.E. 1947. Further differentiation of bean trypsin inhibiting factors. Arch. Biochem. Biophys. tg:109. Bowman, 0 E. 1971. Isolation and properties of a proteinase inhibitor of navy beans. Arch. Biochem. Biophys. 144: 541. Bundy, H.F. and Mehl, J.W. 1958. Trypsin inhibitors of human serum. 1. Standardization, mechanism of reaction, and normal values. J. Clin. Invest. gtz947. Cuatrecasas, P. and Anfinsen, C.B. 1971. Affinity chromato- graphy. In: Methods in Enzymology. Vol. gg:345, W.B. Jakoby ed. Academic Press Inc. Publishers, New York. Dahlgren, K., Porath, J. and Lindahl-Kiessling, K. 1970. On the purification of phytohemagglutinins from Phaseolus vulgaris seeds. Arch. Biochem. Biophys. 137:306. Dalilottojari, H. 1978. Personal communication. Michigan State University, East Lansing, MI. 59 60 Danielson, C.E. 1950. An electrophoretic investigation of vicilin and legumin from seeds of peas. Acta Chem. Scand. 5:762. De Souza, N., Santo, J.E. and Oliveira, J.E.D. 1973. Clini- cal and experimental studies on common beans. In: Nutritional Agpects of Common Beans and Other Legumes Seeds as AnimaT and Human Foods. W.G. Jaffe ed. Pro- ceedings of a meeting held in Ribeirao Preto—Brazil in Nov. 1973. Evans, R.J., Pusztai, A., Watt, W.B. and Bauer, D.H. 1973. Isolation and properties of protein fractions from navy beans (Phaseolus vulgaris) which inhibit growth of rats. Biochim. Biophys. Acta tQ§:175. Everson, G. and Heckert, A. 1944. The biological value of some leguminous sources of protein. J. Am. Diet. Assoc. 20:81. Fish, W.W., Reynolds, J.A., Tamford, C. 1970. Gel chroma- tography of proteins in denaturating solvents. J. Biol. Chem. 24545166. Frank, R.N. and Rodbard, D. 1970. Precision of sodium dodecyl sulfaterpolyacrylamide-gel electrophoresis for the molecular weight estimation of a membrane glycopro— tein: studies on bovine rhodopsin. Arch. Biochem. Biophys. ttt:l. Goa, J. and Strid, L. 1969. Amino acid content of leguminous proteins as affected by genetic and nutritional factors III. Arch. Microbiol. 11:253. Green, N.M. 1953. Competition among trypsin inhibitors. J. Biol. Chem. 205:535. Green, N.M. and Work, E. 19538. Pancreatic trypsin inhibitor 1. Preparation and properties. Biochem. J. ggzzs7. Green, N.M. and Work, E. 1953b. Pancreatic trypsin inhibitor 2. Reaction with trypsin. Biochem. J. pg 347. Green, T.R. and Ryan, C.A. 1972. Wound-induced inhibitor in plant leaves: A possible defense mechanism against insects. Science 175:776. Ishino, K. and Ortega, D. M.L. 1975. Fractionation and characterization of major reserve proteins from seeds of Phaseolus vulgaris. J. Agric. Food Chem. gg:529. 61 Jacobsen, C.F., Leonis, J., Linderstrom-Lang, K. and Otten- sen, M. 1957. The pH- stat and its use in biochemistry. In: Methods of Biochemical Analysis. Vol. 3:171. D. Glick ed. Interscience Publishers, Inc. New York. Jaffe, W.G. 1972. Factors affecting the nutritional value of beans. In: Nutritional Improvement of Food Legumes py Breeding. Proceedings of a symposium held at the Food and Agriculture Organization, Rome, Italy in July 1972. John Wiley and Sons. New York. 1975. Johns, C.O. and Finks, A.J. 1920. Studies in nutrition. 11. The role of cystine in nutrition as exemplified by nutrition experiments with the proteins of navy beans, Phaseolus vulgaris. J. Biol. Chem. 51:349. Joly, M. 1965. A physico-chemical_gpproach to the dena- turation of proteins. Molecuiar biology. An interna- tional series of monographs and textbooks. B. Horecker. N. Kaplan, and H. Scheraga ed. Academic Press Inc. Publishers, New York. Kakade, M.L., Arnold, R.L., Liener, I.E., and Waibel, P.E. 1969. Unavailability of cystine from trypsin inhibitors as a factor contributing to the poor nutritive value of navy beans. J. Nutr. ggz34. Kakade, M.L., and Evans, R.J. 1965a. Toxic factors in beans: Growth inhibition of rats fed navy bean frac- tions. J. Agr. Food Chem. tg:450. Kakade, M.L., and Evans, R.J. 1965b. Nutritive value of navy beans (Phaseolus vulgaris). Brit. J. Nutr. tgz269. Kakade, M.L. and Evans, R.J. 1966. Effect of soaking and germinating on the nutritive value of navy beans. J. Food Sci. gt:781. Kakade, M.L., Simons, N., and Liener, I.E. 1969. An eval- uation of natural vs synthetic substrates for measuring the antitryptic activity of soybean samples. Cereal Chem. gg:518. Kakade, M.L., Simons, N., and Liener, I.E. 1970. Nutri- tional effects induced in rats by feeding natural and synthetic trypsin inhibitors. J. Nutr. 100:1003. Kawasaki, T. and Ashwell, G. 1976. Chemical and physical properties of an hepatic membrane protein that speci- fically binds asialoglycoproteins. J. Biol. Chem. 251: 1296. 62 Kogen, H.M., Crombie, G.D., Jordon, R.E., Lewis, N., and Franzblau, C. 1972. Proteolysis ofelastin-ligand com- plexes. Stimulation of elastase digestion of insoluble elastin by sodium dodecyl sulfate. Biochemistry tt: 3412. Kunitz, M. 1945. Crystallization of a trypsin inhibitor from soybean. Science 101:668. Kunitz, M. 1946. Crystalline soybean trypsin inhibitor. J. Gen. Physiol. pg 149. Kunitz, M. 1947. Crystalline soybean trypsin inhibitor. 11. General properties. J. Gen. Physiol. §Q;291. Lewis, 0.A.M. 1966. Short ion-exchange column method for the estimation of cystine and methionine. Nature 209: 1239. Loeffler, L.J., and Pierce, J.V. 1973. Acyl azide deriva- tives in affinity chromatography immobilization of enzymatically active trypsin of beaded agarose and porous glass. Biochim. Biophys. Acta. §t1;20. Lyman, R L., and Lepkovsky. S. 1957. The effect of raw soy- bean and trypsin inhibitor diets on pancreatic enzyme secretion in rat. J. Nutr. 15:445. Malik, N. and Berrie. A. 1972. New staining fixative for proteins separated by gel isoelectric focusing based on coomassie brilliant blue. Anal. Biochem. 12:173. McCollum, E.V., Simonds, N. and Pitz, W. 1917. The dietary deficiencies of the white bean Phaseolus vulgaris. J. Biol. Chem. gg:521. McKay, A.F., Skulski, M., and Garmaise, D.L. 1958. Reaction of amino alcohols with carbon disulphide. Can. J. Chem. gg:147. Messing, R.A. 1976. Absorption and inorganic bridge forma- tions. In: Methods in Enzymolqu. Vol. ggzl48. K. Mosbach ed. Academic Press Inc. Publishers, New York. Miller, B.S. and Johnson, J.A. 1951. A simple linear rela- tionship and definition of a unit for proteinase acti- vity. Arch. Biochem. Biophys. gt:200. Mitchell, H.L., Parrish, 0.8., Cormey, U. and Wassom, C.E. 1976. Effect of corn trypsin inhibitor on growth of rats. J. Agric. Food Chem. gg 1254. 63 Moore, 5., Spackman, D.H., and Stein, W.H. 1958. Chromato- graphy of amino acids on sulfonated polysterene resin. Anal. Chem. gQ:1185. Mosolov, V.V., and Fedurkina, N.Y. 1974. Isolation of trypsin inhibitor from beans by means of specific binding on trypsin-agarose. Biokhimiya gg:624. Osborn, T.B. 1894. The proteins of the kidney bean. J. Am. Chem. Soc. tt:633, 703, 757. Osborn, T.B., and Mendel, L.B. 1912. Beobachtungenuber wachstum bei futterungsversuchen mit isolierten nah- rungssubstazen. Z. Physiol. Chem. 82:307. Porath, J. and Axen, R. 1976. Immobilization of enzymes to agar, agarose, and sephadex supports. In: Methods in Enzymology, Vol. 33:19. K. Mosbach ed. Academic Press Inc. Publishers. New York. Puztai, A. 1967. Trypsin inhibitors of plant origin, their chemistry and potential role in animal nutrition. Nutr. Abstr. Rev. girl. Read, J.W., and Haas, L.W. 1938. Studies on the baking quality of flour as affected by certain enzyme actions. Cereal Chem. t§z59. Rick, W. 1974. Chymotrypsin. In: Methods of Enzymatic Analysis. Vol. 2:1006. H.U. Bergmeyer ed. Academic Press Inc. Publishers, New York. Robard, D. 1976. Estimation of molecular weight by gel filtration and gel electrophoresis. I. Mathematical principles. In: Methods of Protein Separation. Vol. 2. N. Catsimpoolas ed. Plenum Press, New York. Schram, E., Moore, 5., and Bigwood, E.J. 1954. Chromato- graphic determination of cystine as cysteic acid. Biochem. J. gtz33. Seidl, D., Jaffe, M. and Jaffe, H. 1969. Digestability and proteinase inhibiting action of a kidney bean globulin. J. Agr. Food Chem. ttzl318. Spies, J.R. 1967. Determination of tryptophan in proteins. Anal. Chem. gg:1412. Spies, J.R. and Chambers, 0.0. 1948. Chemical determination of tryptophan. Anal. Chem. gp:30. 64 Srere, P. and Uyeda, K. 1976. Functional groups on enzymes suitable for binding for matrices. In: Methods in Enzymology. Vol. 55:11. K. Mosbach ed. Academic Press Inc. Publishers, New York. Steffans, R., Fox, F.R., Hassel, B. 1978. Effect of trypsin inhibitors on growth and metamorphosis of borer corn larvae Ostinia nubilalis (Hubner). J. Agric. Food Chem. 26:170. Steiner, R.F., and Frattali, V. 1969. Purification and properties of soybean protein inhibitors of proteolytic enzymes. J. Agric. Food Chem. tt:513. Svasti, J., and Parrijpan, B. 1977. SOS-polyacrylamide gel electrophoresis. J. Chem. Educ. 55:560. Ugel, A.R., Chrambach, A., and Rodbard, D. 1971. Fractiona- tion and characterization of an oligomeric series of bovine ketatohyalin by polyacrylamide gel electrophore- sis. Anal. Biochem. 55:410. Wagner, L.P., and Riehm, P.J. 1967. Purification and partial characterization of a trypsin inhibitor isolated from the navy bean. Arch. Biochem. Biophys. 121:672. Walsh, K.A., and Wilcox, P.E. 1970. Serine proteases. In. Methods in Enzymolpgy, Vol. 15:31 G.E. Perlmann ed. Academic Press Inc. Publishers, New York. Waterman, H.C. and Johns, C.0. 1921. Studies on the diges- tability of proteins tg vitro. 1. The effect of cooking on the digestibility of phaseolin. J. Biol. Chem. 55:9. Weber, K., and Kuter, D.J. 1971. Reversible denaturation of enzymes by sodium dodecyl sulfate. J. Biol. Chem. 246: 4504. Weber, K., and Osborn, M. 1969. The reliability of molecular weight determinations by dodecyl sulfate-polyacrylamide gel electrophoresis. J. Biol. Chem. 244:4406. Weetall, H.H. 1975. Immobilized enzymes and their applica- tion in the food and beverage industry. Proc. Biochem. tg_(July/August):3. Whitley, E.J. Jr., and Bowman, P.E. 1975. Isolation and properties of navy bean proteinase inhibitor component 1. Arch. Biochem. Biophys. 169:42. 65 Wrigley, C.W. 1971. Gel electrofocusing. In: Methods in Engymology. Vol. 55:559. W.B. Jakoby ed. Academic Press Inc. Publishers, New York. Wu, Y.V., and Scheraga, H.A. 1962. Studies of soybean trypsin inhibitor. 1. Physicochemical properties. Biochemistry t:698. APPENDIX 66 Table I. Inhibition of Trypsin Activity by Navy Bean Tryp- sin Inhibitor NBTI (ug) A280* A3/2 % Activity 0 0.552 0.410 100 2.97 0.450 0.302- 73.6 4.45 0.405 0.258 62.9 5.94 0.357 0.213 51.9 7.43 0.281 0.149 36.3 8.91 0.220 0.103 25.1 10.40 0.197 0.087 21.2 11.88 0.152 0.059 14.4 13.36 0.112 0.037 9.0 14.85 0.067 0.017 4.1 16.33 0.060 0.015 3.6 17.82 0.060 0.015 3.6 19.30 0.047 0.010 2.4 20.79 0.045 0.009 2.2 *Average of two duplicate determinations. 67 Table II. Inhibition of Trypsin Activity by Soybean Tryp- sin Inhibitor SBTI (ug) A280* A3/2 % Activity 0 0.552 0.410 100 3.97 0.492 0.345 84.1 7.94 0.430 0.282 68.8 11.94 0.325 0.185 45.1 15.88 0.260 0.133 32.4 23.82 0.040 0.008 1.9 27.79 0.017 0.002 0.5 *Average of two duplicate determinations Table III. Inhibition of oChymotrypsin Activity by Navy Bean Trypsin Inhibitor NBTI A280* A3/2 % Activity 0 0.912 0.871 100 2.97 0.805 0.722 82.9 5.94 0.735 0.630 72.3 8.91 0.620 0.488 56.0 11.88 0.530 0.386 44.3 14.85 0.487 0.340 39.0 17.82 0.422 0.274 31.5 20.79 0.350 0.207 23.8 23.76 0.280 0.148 17.0 *Average of two duplicate determinations 68 .0>wm 50g$ mp gown: mcowu -mggcmucoo Pom mm.op quoxw mpcmsrgmaxm manorpasc 030 $0 mmmgm>m 000 mucwoa .op 0020*; cm czozm m? popa 05h .Axm.mp 0:0 w.cp .m.u.mv mcowgmgucmucoo pmm 0:0» 500% 00>Icmoa .AINIFI IcaaeaI Ia euccaeec m_ NIII-mIm tee papa IsmsmcaI ea easeaee_a>a 0N._ mNo.o mumo.c m~o.o+xm~0.0rum r IoawnNIIN cpmaxeu ammo x>cz NF.F opo.o NNwa.o ch.o+cho.onum oomep mEXNOmxg FN.P mNo.o wmwm.o mwo.o+xmmo.oium oompm Louwnw;:w :wmaagu :mmaxom wd.F mvo.o opmm.o Pup.o+xm¢o.c|um commm cQGOCPmahsuoE%;u nm.~ nmc.c emmm.o oom.o+xnwo.cium ooome :wE:nFm>o mo.m ¢cp.c cmmm.o Npm.o+x¢cF.ciu% ooowo :rsanpw Eszm mcw>om 0> NI NI 000p; comzmsmm mcowumscu 3: cwmuoga mmuopa comzmsmm soc» 00>Igwo mcowumacm .>H mpamh MICHIGAN STATE UNIV. 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