.4 ,. II III III I III III IIII III II IIIII II III II III III L 3 1293 This is to certify that the thesis entitled Isolation and Characterization of Navy Bean Trypsin Inhibitor presented by Jose Carlos Gomes has been accepted towards fulfillment of the requirements for Ph.D. degree in Food Science and Human Nutrition W Major professor Damian—113” 044 1980 0-7639 OVERDUE FINES ARE 25¢ PER DAY PER ITEM Return to book drop to remove this checkout from your record. ISOLATION AND CHARACTERIZATION OF NAVY BEAN TRYPSIN INHIBITOR By Jose Carlos Gomes 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 1980 ABSTRACT ISOLATION AND CHARACTERIZATION OF NAVY BEAN TRYPSIN INHIBITOR By Jose Carlos Games In addition to the deficiency of sulfur-containing amino acids in legume seed protein, the presence of anti- physiological substances constitute a factor for concern. Among the biologically active factors are the proteinase inhibitors. A trypsin inhibitor fraction was isolated from navy beans (3. vulgaris L.), Sanilac cultivar, by affinity chro- matography on agarose-bound trypsin and characterized. -13 Ultracentrifugal measurements yielded 530 = 2-04x10 59C and 030 = 9.2xl0'7 cmZ/sec. Molecular weight values calcu- lated by the Svedberg equation and sedimentation equilibrium were l7,000 and ZOAJOO, respectively. This inhibitor preparation was heterogeneous when examined by discontinuous polyacrylamide gel electrophoresis. Important aspects of its amino acid composition were the high content of half- cystine, low content of methionine and absence of trypto- phan. There was no available thiol group detected. Jose Carlos Gomes The trypsin inhibitor is present in the albumin frac- tion of the navy bean proteins. Rabbit antibodies against the affinity isolated inhibitor indicated that the inhibitor was localized in the protein bodies of the navy beans. A fraction obtained from the water soluble proteins of navy bean by gel filtration on Sephadex G-75 was closely related in mobility (Disc-PAGE) to the affinity-isolated inhibitor. A molecular weight of l6,000 was estimated from its eluting position. Further separation by Disc-PAGE indicated that the trypsin inhibitory activity is limited to two protein zones. The affinity-isolated inhibitor is highly heat resistant. The decimal reduction time at 120°C was l45 min with a "z" value Of 22.3OC (72.l0F). An Arrhenius plot yield an activation energy of 30.9 Kcal/mol. Heat treatment of intact beans indicated that the indigenous inhibitor is easily inactivated by heat. ACKNOWLEDGMENTS I wish to express my gratitude tO my major professor, Dr. J.R. Brunner, for his advice, guidance and patience during this study and preparation of this thesis. I also appreciated many interesting discussions we had. Apprecia- tion is also extended to Dr. P. Markakis and Dr. L. Dugan of the Department of Food Science and Human Nutrition; to Dr. H.A. Lillevik and Dr. L.L. Bieber of the Department of Biochemistry for reviewing this manuscript and for serving on my graduate committee. I also thank John Euber for useful discussions we had during this study. I wish to acknowledge the technical assistance of Ursula Koch with the amino acid analyses and Ultracentri- fugal studies. I also feel grateful to the Departmento de Tecnologia de Alimentos at Universidade Federal de Vicosa (Brazil) and to the Programa de Ensino Agricola Superior (PEAS) for the opportunity and financial assistance granted to me. 11' TABLE OF CONTENTS Page LIST OF TABLES. . . . . . . . . . . . . . . . . . . . vi LIST OF FIGURES . . . . . .‘. . . . . . . . . . . . . vii INTRODUCTION. . . . . . . . . . . . . . . . . . . . . 1 LITERATURE REVIEW . . . . . . . . . . . . . . . . . . 3 Isolation and Characterization of Navy Bean Trypsin Inhibitors. . . . . . . . . . . . . . . 3 Nutritional Importance. . . . 7 EXPERIMENTAL. . . . . . . . . . . . . . . . . . . . . l7 Chemicals and Materials . . . . . . . . . . . l7 Equipment . . . . . . . . . . . . . . . . . . l7 Chemical Methods. . . . . . l9 Synthesis of ll -amino Undecanoic Methyl Ester . . . . . . . . . . . . . . l9 Nitrogen Determination. . . . . . . . . . . . l9 Amino Acid Analysis 20 TryptOphan determination. . . . . . . .'. 20 Amino acid composition. . . . . . . . Zl Methionine and cystine analyses . . . . . 22 Sulfhydryl determination. . . . . . . . . 23 Biuret Determination of Proteins. . . . . . . 24 Trypsin Inhibitor Assay . . . . . . . 24 Immobilization of Trypsin on Agarose. . . . . 25 Preparative Procedures. . . . . . . . . . . . . . 29 Protein Extraction. . . . . . . . . . 29 Separation of albumins and globulins. . . 29 Heat treated albumins . . . . . . . . . 3l Trypsin inhibitor enriched .fraction . . . 31 Extraction efficiency as a function of pH. . . . . . . . . . . 32 Extraction efficiency as a function of time. . . . . . . . . . 34 Extraction efficiency as a function of ionic strength. . . . . . . . . 34 Optimized extraction method . . . . . . . 34 Immobilized Trypsin Column. . . Affinity Separation of the Trypsin InhibitOr. Gel Filtration Column . . . . Fractionation Of Navy Bean Proteins on Gel Filtration Column . . . Trypsin Inhibitor- enriched fraction Heated albumin. Acid extracted proteins Separation of Protein Bodies. Immunological Analysis. . Preparation of Rabbit Anti- -sera against the Trypsin Inhibitor . . . . . . . . . . Immuno Double Diffusion Physical Methods. Discontinuous Polyacrylamide Gel Electro- phoresis. . . Disc- PAGE of 2S Fraction. . . Standardization of the Gel Filtration Column. Ultracentrifugal Characterization Sedimentation velocity. Diffusion coefficient . . Sedimentation equilibrium . Heat Inactivation In vitro heat inactivation. Indigenous heat inactivation. . Indigenous heat inactivation with inhibi- tor added . . . . . . . . . . Determination Of Bound Trypsin. . UV Spectrum of the Trypsin Inhibitor. Moisture Determination. . . RESULTS AND DISCUSSION. Protein Extraction. . Immobilization Of Trypsin . The Trypsin Inhibitor Assay . Separation of the Inhibitor Affinity Separation Gel Filtration. . . Calibration of the Sephadex G- 75 Column . . Discontinuous Polyacrylamide Gel Electrophoresis. The Ferguson Plot . Presence of Trypsin Inhibitor in the Protein Bodies. . . . Amino Acid Composition. Amino Acid Analysis . . . Sulfhydryl Determination. . Trend in the Amino Acid Composition of. Inhi- bitors. . . Ultracentrifugal Characterization Molecular Weight. iv Svedberg equation. Sedimentation equilibrium. Heat Inactivation. In vitro Measurements. In vivo Measurements . . UV Spectra of ZS and Inhibitor Fractions CONCLUSIONS. REFERENCES . APPENDICES 104 Table A1 A2 A3 A4 LIST OF TABLES Timetable for immunization procedure. Trypsin inhibitor activity of albumins and globulins . . . . . Amino acid composition of navy beans, 25 frac- tion and the affinity isolated inhibitor. Heat inactivation of the affinity-isolated trypsin inhibitor . Heat inactivation Of indigenous inhibitor Determination of molecular weight from inter- ference patterns. Specific volume of the affinity-isolated trypsin inhibitor . . . . . . . . . Data for calculation Of sedimentation and diffusion coefficients. List of chemicals used in this study. vi Page 40 51 76 88 91 108 109 110 111 LIST OF FIGURES Figure l Mode of action of trypsin inhibitors and regula- tion of trypsin levels in the intestine. 2 Immobilization of trypsin on agarose 3 Procedure for separation of albumins and globu- lins from navy beans . . 4 Procedure for separation of trypsin inhibitors from navy beans. . . . . . . . 5 Extraction of total protein 0 and trypsin inhibitorsEJ from navy beans . . 6 Affinity separation of navy bean trypsin inhibi- tors . . . . . . . . . . . . . . . . . 7 Gel filtration on Sephadex G—75 column 8 Gel filtration on Sephadex G-75 column 9 Gel filtration on Sephadex G-75 column of navy bean proteins Obtained by three different procedures . . . . . . . . lD Calibration of a Sephadex G-75 column. ll Discontinuous polyacrylamide gel electrophoretic patterns of affinity- isolated inhibitor (A), 25 fraction (8 and C) . . . . . . 12 Trypsin inhibitory activity of 23 fraction after separation by Disc- PAGE. . . . . l3 Trypsin inhibitory activity of 25 fraction after separation by Disc- PAGE. . . . . . . . . l4 Ferguson plot for the affinity isolated inhibi- tor. . . . . . . . . . . . . . . vii Page 14 3O 33 54 58 60 61 62 64 65 67 68 69 Figure 15 16 17 18 19 1A A - Protein bodies isolated from navy beans. B - Immuno double-diffusion pattern. A - Diffusion patterns of the affinity-isolated trypsin inhibitor. 8 - Sedimentation velocity patterns. C - Interference pattern of sedimen- tation equilibrium . A - Plot for calculation of diffusion coeffi- cients Of the affinity-isolated trypsin inhibi- tor. . . . . . . . . . . . . . . . . . . . . . B - Plot for calculation of sedimentation velocity coefficients. C - Dependence of sedimentation velocity C>and diffusion coefficientsZB on concentration. Heat inactivation of the affinity-isolated inhibitor. Arrhenius plot for the inactivation of the affinity-isolated inhibitor. Ultraviolet spectra of 25 fraction (A) and affinity-isolated trypsin inhibitor (B). viii Page 69 71 79 80 80 81 84 87 113 INTRODUCTION Legume grains are relatively high in protein content and constitute one Of the world's major sources Of protein. In addition to the well-documented deficiency of sulfur- containing amino acids in legume seed protein, the presence of antiphysiological substances constitute a factor for concern. Among these biologically active factors are the proteinase inhibitors. If not destroyed these inhibition has undesirable effects on the nutritive value of legumes Navy bean and soybeans represent the first legumes where the presence Of trypsin inhibitors was reported. While soybean trypsin inhibitors have been extensively studied, relatively little work has been devoted to the navy bean trypsin inhibitor. The latter inhibitor has been isolated by differential precipitation, ion exchange and gel chro- matography or by a combination of these techniques. Recently, it was isolated by affinity chromatography on agarose-bound trypsin. Conventional procedures of protein separation and puri- fication are generally based on small differences in physio- chemical properties of proteins. Affinity chromatography exploits the biological specificity of the protein-ligand interaction and, thus, yield high resolution and selectivi- ty. However, there is always a possibility that the substance being isolated maysuffer modification during the affinity separation step. The purpose of this study was to re-examine and extend the available information pertaining to selected physio- chemical parameters of the navy bean trypsin inhibitor. For this purpose, specimens isolated by affinity chromato- graphy and by gel filtration were employed. Because the trypsin inhibitor seems to be an undesirable nutritional factor if not inactivated, heat inactivation studies were conducted with the isolated inhibitor and with intact navy beans.~ Extraction procedures for the inhibitor were investigated in a wide range of pH, ionic strength and length of extraction period. Localization of the trypsin inhibitor in the seed was determined, using rabbit antibodies prepared against the inhibitor. LITERATURE REVIEW Isolation and Characterization of Navy Bean Trypsin Inhibi- Egg; Read and Haas (l928) 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) assayed navy beans, soybeans, wheat and corn aqueous extracts for trypsin inhibitory activity. He found that the extracts prepared from navy beans and soy- beans considerably retarded the digestion Of casein by trypsin. Comparable extracts from wheat and corn had little influence. He was the first to suggest that the presence of this heat labile protein, which inhibited trypsin, might account for the low nutritive value of raw legumes. The digestion-retarding fraction from navy beans could be con- centrated by precipitation with acetone or alcohol while satisfactory precipitation from soybean extract could be achieved with acetone only. Kunitz (1945, 1946) succeeded in crystallizing a tryp- sin inhibitor protein of globulin type from the extracts of soybean which later became the most extensively studied inhibitor of plant origin. Soybean and navy bean trypsin inhibitors were differentiated by Bowman (l948) in regard to their solubilities and activities. He found that crude navy bean inhibitor was considerably more active and water soluble than the crystalline, globulin type soybean trypsin inhibitor. He indicated that activity and solubility characteristics of the navy bean antitryptic factor suggest that it has a smaller molecular weight than the soybean inhibitor, more nearly resembling the plasma trypsin inhi- bitor. 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). The ethanol-washed ground seeds were extracted with a low concentration of hydrochloric acid followed by ammo- nium sulfate fractionation. The proteins in the 50-80% saturation of ammonium sulfare fraction were taken up in water, heated at 80°C for 5 min. and the supernatant applied to a Sephadex G-75 column. Fractions containing trypsin inhibitor activity were further fractionated by ion-exchange chromatography on DEAE-cellulose. Gel filtration of the oxidized inhibitor indicated that the native protein was a single polypeptide chain. 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 two moles of hexose per mole of protein and no thiol groups. Sedimentation equilibrium data yielded a molecular weight of 23,000. The stoichio- metry of the reaction between inhibitor and trypsin sugges- ted an inhibitor to enzyme ratio of 1:2. Bowman (l97l) chromatographed a protein fraction obtained from the aqueous extract of navy beans on a DEAE-cellulose column, utilizing a sodium chloride gradient. The seeds used were from Sanilac, Seaway and Gratiot varieties. Except for the first peak eluted without the aid of NaCl, the entire chromatographic elution pattern represented inhibiting material. One of the components further investigated was homogeneous on disc electrophore- sis and inhibited the proteolytic and esterolytic activities of both trypsin and a-chymotrypsin. The constant apparent specific activity of navy bean inhibitor when reacted with a mixture of active and inactive trypsin suggested that the inhibitor reacts with active as well as inactive trypsin. In a subsequent experiment, Whitley and Bowman (1975) reinvestigated the protein fraction Obtained by Bowman (l97l). 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 on 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 an inhibitor with an amino acid composition similar, on a relative basis, to the two compo- nents investigated. Gomes (1978) isolated the trypsin inhibitors from navy beans (Sanilac variety) by percolation of the albumin frac- tion through an affinity column with trypsin immobilized on Sepharose. An acidic solution was used to elute the inhi- bitor fraction off the column. This protein fraction inhibited strongly the proteinase activity of trypsin and a-chymotrypsin. Amino acid composition was similar to that reported by Wagner and Riehm (1967). Isoelectric focusing revealed a major band at pH 4.40 and a minor band at pH 4.45. Molecular weight calculated by SDS-PAGE was 16,600 for the major band. Molecular weight estimated from inhi- bition measurements was 11,900, and the minimum molecular weight, on the basis of methionine as limiting amino acid, was 12,200. Thus, there is a controversy surrounding the molecular weight of navy bean trypsin inhibitor(s). Apparently this anti-tryptic component contains at least two proteins with closely related characteristics, making resolution diffi- cult. The amino acid composition indicates some degree of hydrophobicipy, The high content of half-cystine and no free thiol groups indicate a highly compact molecule, since all the half-cystine are involved in disulfide bonds. Attempts to determine the molecular weight should make use of dissociating agents for the disulfide bonds (e.g. oxida- tion or reduction) as well as for the hydrophobic regions (e.g. sodium dodecyl sulfate). Oxidation of disulfide bonds as performed by Wagner and Riehm (1967) does not eliminate the possibility of association through other residues. Intermolecular association for the Bowman-Birk inhibitor from soybeans does not involve disulfide bonds (Birk, 1976). Reversible subunit self-assembly has been reported for nearly all inhibitors Obtained from legume seeds (Kassel, 1970). Black-eyed peas have double-headed inhibitors which form dimers even after boiling in the presence of sodium dodecyl sulfate and B-mercaptoethanol. This extremely stable dimer was dissociated only by complete reduction and carbamidoethylation, followed by heating in sodium dodecyl sulfate (Gennis and Cantor, 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 t l. (1917) fed navy bean meal as a source of protein and the animals failed to grow. These authors attributedthe failure of navy beans to promote growth to the presence of hemicullulose. 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 the destruction of toxic material or improve- ment in digestibility of the proteins. Waterman and Johns (1921) tested the later hypothesis. They found that heat treatment of navy bean protein increased the in 11353 diges- tion by trypsin and pepsin. Everson and Heckert (1944) studied the biological value of several legume seeds, including navy beans. They repor- ted 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 fractions were found to inhibit growth in rats. The growth inhibitory effect was attributed neither to the hemagglutinin nor the trypsin inhibitor but to a toxic material scattered throughout 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 support the growth of experimental animals. This growth inhibitor factor was later isolated and characterized (Evans _3 11., 1973) which was identified as a phytohemagglutinin with 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 _t_gl,, 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). Evans and Bandemer (1967) studied the nutritive value of several legume seeds, including navy beans Of Sanilac variety. They found that the protein nutritive values of most legume seeds were improved by heat. Navy and kidney beans promoted no growth unless heated before feeding. The protein nutritive value of a mixture of navy beans and. Gary oats was greater than that of either one alone. Except for dry peas, all of the raw legume seeds studied appeared to contain toxic or growth inhibitory substances, but in most cases these substances were destroyed by heat. 10 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 cystine of the navy beans. Since the navy bean trypsin inhibitor is poorly attacked by digestive enzymes unless modified by heat, Kakade at al. (1969a) suggested that this disproportionate distribution of cystine was the major factor involved in the low nutritive value of raw navy beans. Evans _£._l. (1974) investigated the bio-availability of methionine and cystine in dry bean seeds (Sanilac var.). Growing rats were fed diets containing 10% protein in which all of the protein was supplied as heated (121°C, 10 min) bean or soybean meal, or diets supplemented with methionine. Methionine and cystine contained in the bean seeds were more poorly utilized by growing rats than those in soy- beans. Nitrogen balance studies of methionine and cystine were also conducted. Approximately 49% of the methionine and 25% of the cystine Of the beans were excreted with the undigested proteins. They concluded that the methionine and cystine present in the undigested portion of the bean protein accounted for the apparent lack of utilization of these residues in navy beans; and, since methionine added to soybean meal was more fully utilized than that added to the bean, they postulate the presence of unknown substance(s) which interfere with the utilization Of methionine. 11 In a subsequent experiment Evans and Bauer (1978) presented similar results related to the poor utilization of methionine and cystine in heated navy beans. However, contrary to earlier findings (Evans st 11., 1974), the results indicated that the methionine supplemented to navy bean flour was completely absorbed, and that the poor utilization of methionine in dry beans is not caused by something which interferes with the absorption of methio- nine, but rather by the undigestability of the methionine- containing proteins. Presumably, this resistance to enzymatic attack is due to the stability of the molecule produced by a large number of disulfide bonds. Heat treatment causes an unfolding of the molecule resulting in the exposure of peptide bonds susceptible to enzymatic cleavage (Birk, 1968). In a rela- ted experiment Kakade gt 31, (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 growth to the same level obtained with anti- biotics. They explained the results on the basis of conver- sion 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. 12 Jayne-Williams (1976) reviewed a series of investiga- tions relating the intestinal microflora and the nature of the toxic components in raw navy and jack beans. This review can be summarized as follows: raw navy beans in the diet of Japanese quail caused enlargement of the pancreas due to the presence of trypsin inhibitors; combined activi- ties of coliform bacteria in the intestine and the phyto- haemagglutinins in the beans have lethal effects; the meta- bolic activity of intestinal bacteria might liberate toxins from innocuous precursors present in raw beans; the hypo- thermia observed in birds on raw navy bean diets seems to be a manifestation of coliform endotoxaemia; and, a syste- matic liver infection occurred with quail fed raw navy beans. The existence Of trypsin inhibitors in legume seeds seemed to Offer a reasonable explanation for the observa- tion that heat treatment improved the nutritional value of these seeds. From the results of a study on the physiologi- cal response of rats to raw or treated soybean meal diets, it was concluded that trypsin inhibitors are responsible for the pancreatic hypertrophy and from 30 to 50% of the growth-inhibiting effect (Steiner and Frattali, 1969). Liener (1979) reviewed the mode of action of trypsin inhibitors. The observation that trypsin inhibitors were capable of inhibiting growth even when predigested proteins were incorporated into diets ruled out inhibition of 13 proteolysis as the sole factor responsible for growth inhibition. Hypertrophy of the pancreas is accompanied by an increase in secretory activity. This led to the sugges- tion that growth depression caused by trypsin inhibitors might be the consequence of an endogenous loss Of essential amino acids being secreted by a hyperactive pancreas. Since pancreatic enzymes such as trypsin and chymotrypsin are particularly rich in sulfur-containing amino acids, pancreatic hypertrophy causes a drain on the body tissue of these particular amino acids which are required for the synthesis of these enzymes. The mechanism whereby the trypsin inhibitor induces pancreatic enlargement still is not fully understood. Pancreatic secretion is controlled by a mechanism of feedback inhibition which depends upon the level of trypsin and chymotrypsin in the small intes- tine. Levels Of these enzymes below a threshold value induce the pancreas to produce more enzyme. It is believed that the mediating agent between trypsin and the pancreas is the hormone cholecystokinin, which is released from the intestinal mucose when the level of trypsin in the intes- tine falls below its threshold level. If trypsin is complexed with the inhibitor this mechanism is seriously disturbed. The mechanism is illustrated in Figure l. Elias _t‘al. (1979) carried out studies to determine the possible relationship between color of the seed coat of beans (3. vulgaris L.) and the nutritive value of its 14 Trypsinogen Cholecystokinin (.—— (pancreas) (mucosa) Trypsin (intestine) Dietary Trypsin Protein Inhibitor Peptides Trypsin-TI Complex Figure 1. Mode of action Of trypsin inhibitors and regula- tion of trypsin levels in the intestine. 15 protein. Beans with white, red and black seed coats and a black coated bean and its white mutant were chosen for the experiments. Tannin content of the seed coat and inhibition of trypsin activity yielded a positive corre- lation. Protein digestability was lower for beans with colored seed coats. The addition of the cooking broth to the feed lowered the protein digestability for the colored coated beans but not for the white beans. Trypsin inhibi- tors were detected in the cotyledons as well as in the seed coat, being higher in the seed coat. In the case of white seed coat cultivars the inhibition of trypsin activity was higher in the cotyledons. After heat treatment (121°C - 20 min), inhibition Of trypsin was still detected in the whole seeds as well as in the cooking broth Of colored varieties while very little inhibition was found in the cooking liquor of white varieties. The authors pointed out that the polyphenols present in the seed coat could be responsible for the lower performance of the colored beans varieties when compared to white cultivars. They differen- tiated the trypsin inhibitory activity as a heat labile factor, more concentrated in the cotyledons, and a heat resistant factor, which predominates in the seed coat. The heat resistant factor seems to be directly related to the tannin content. However, it should be noted that the trypsin inhibitor assay employed by Elias gt_al, (1979) does not differentiate between protein and phenolic 16 substances. Because trypsin inhibitors can produce adverse physio- logical effects in animals, the question arises as to whether or not these are of physiological significance to man. The cationic form of human trypsin is only weakly inhibited by the soybean inhibitor, but the anionic form can be fully inactivated. Human trypsin, which fails to be inhibited by soybean inhibitors, represents the major part Of the trypsin activity of the digestive juice (Liener, 1976). EXPERIMENTAL Chemicals and Materials The principal chemicals used in this study and their source are listed in the Appendix, Table A4. All were reagent grade unless otherwise indicated. Navy beans, Sanilac cultivar (P. vulgaris), from 1977 and 1979 harvests were supplied by the Bean and Beet Research Farm (Saginaw) through the courtesy of Dr. M.W. Adams. These beans contained 23.0% protein (Nx6.25) in a free water basis and 10.9% moisture. Equipment Equipment used regularly during the course of this study is presented here. Instrumentation specific for certain experiment is referred to in the appropriate section. An Instrumentation Laboratory pH/mV Electrometer model 245, was used for all pH measurements. Protein samples were dried in a laboratory made 1yophilizer. Most weighings were performed on a top-load digital Sartorius model 3716 balance. Analytical weighings were performed in a Sartorius model 2433. A Bausch & Lomb Spectronic 21 was used for most spectrophotometric determinations in the visible region and a Beckman DK-2A spectrophotometer equipped with 1 cm path 17 18 way Silica cells was used for measurements in the UV region and when recording was necessary. Centrifugation was carried out in a Sorvall model RC2-B centrifuge using either the large capacity model GSA or the superspeed 55-34 rotors. Analytical ultracen- trifugation was performed in a Beckman (Spinco) model E ultracentrifuge equipped with Schlieren and Rayleigh interferometer optical systems. A Nikon digital microcom- parator was used to measure the patterns. Column eluates weremonitored at 254 or 280 nm with an ISCD Recording Ultraviolet Analyser, model UA-2 and/or fractions collected and absorbance measured at 280 nm. Where fractions were collected, a Scientific Glass Apparatus CO., Inc. or an ISCO fraction collector model 1100 was used. A channel alternator model 580 from ISCO was used when eluates from two columns were monitored simultaneously with the same recorder. An electrophoretic apparatus manufactured by Buchler Instruments and a Bio-Rad Laboratories model 400 power supply (voltage range 0-500 V; current 0-100 mA) were used for all polyacrylamide gel electrophoresis experiment. Amino acid analyses were conducted on a Beckman model 120C Amino Acid Analyser. 19 Chemical Methods Synthesis of ll-aminoundecanoate Methyl Ester Methylation of ll-aminoundecanoic acid was performed as described by McKay gt_al. (1958):50.0 g of ll-amino undecanoic acid was added to 1250 m1 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 by 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. Nitrogen Determination Duplicate samples ranging from 4 to 15 mg Of freeze- dried protein or about 100 mg of the 40 mesh powder were digested with 4 ml of the digestion mixture over a gas flame for 1 h. The digestion mixture consisted of 5.0 g of CuSO4'5 H20 and 5.0 g Of SeOZ in 500 m1 of concentrated sulfuric acid. After cooling the digestion mixture, 1 ml 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, and 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 20 five drops of indicator consisting Of 400 mg of bromocresol green and 40 mg of methyl red in 100 m1 of 95% ethanol. Distillation was continued until a final volume Of 50 to 60 m1 of distillate was collected. The resulting ammonium borate was titrated with 0.0193 N HCl. Hydrochloric acid was standardized with tris-hydroxymethyl amino methane (Sigma-Trizma base). Titrations were performed with a 10 m1 micro-burette. Recoveries of tryptophan standards performed in each run varied from 93.1 to 101% with an average of 97.9%. 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 m1 of phosphate buffer was added. These uncapped vials were placed into Erlenmeyer flasks containing 9.0 m1 of 21.2 N sulfuric acid and 30 mg of dimethylaminobenzaldehyde and 21 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 tryptophan content of pronase was subtracted from the total tryptophan 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 per- formed on 24 and 72 h or 48 h acid hydrolysates employing a Beckman Amino Acid Analyser, Model 120 C according to the procedures of Moore £3 11. (1958). Eight milligrams of navy bean trypsin inhibitor or 20 mg of navy bean 40 mesh powder was weighed into 10 m1 glass ampoules and 5 ml 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 or 48 h. 22 The ampoules were broken on tOp and 1 ml of norleucine 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, and 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 hydro- lysate Of each ampoule was transferred to 5 ml volumetric flask with citrate-HCl buffer, pH 2.2. An aliquot of 0.2 ml was applied to the analyser column. The chromatograms were quantitated by peak integration using a Spectra Physics Autolab System AA. Methionine and cystine analyses. The methods of Schram gt_al. (1954) and Lewis (1966) were used. These methods involve performic acid oxidation of methionine and cystine to methionine sulfone and cysteic acid, respecti- vely. Ten milligrams of navy bean trypsin inhibitor or 25 mg of navy bean 40 mesh powder was weighed into 25 m1 pear-shaped flask. The protein was oxidized for 15 h with 10 ml of performic acid at 4°C. After oxidation, 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. 23 Sulfhydryl Determination A method adapted from Glazer _t _l. (1973) was employed for the determination of sulfhydryl group content in the navy bean trypsin inhibitor and 25 fraction. Ten milli- grams of navy bean trypsin inhibitor or 5.0 mg of 25 frac- tion was dissolved in 3.0 m1 of Tris-SDS buffer, consisting of 0.1 M tris-hydroxymethyl amino methane which contained 0.01 M EDTA-Naz, 1% sodium dodecyl sulfate and adjusted to pH 8.0 with hydrochloric acid. Absorbances were read at 280 nm and converted to protein concentration. A 0.01 M solution of 5,5' dithiobis (2-nitrobenzoate) was prepared in 0.05 M phosphate buffer pH 7.0. For complete dissolution of the reagent, the pH was adjusted to 6.5 with 0.1 N sodium hydroxide. Two milliliter Of the tris-SDS buffer was transferred to each cell of a double bean spectrophotometer set at 412 nm. Then, 0.5 m1 of reagent was added to each cell and the instrument was calibrated. The sample cell was removed, washed and 2.0 ml of protein solution and 0.5 ml of reagent solution were added. Absorbance was recorded for 30 min and the values obtained at the plateau region were used for calculations according to the relation- ship: moles SH = A412 x _V__ MW X— mol 13,600 1000 m 24 where: A412 is the absorbance at 412 nm (1 cm); V, the total volume of the reaction mixture; m, the mass in grams of protein; and MW, the molecular weight of the protein fraction. Biuret Determination of Proteins When rapid, but not highly accurate, protein determi- nations were required, the Biuret method was employed as described by Cooper (1977). The reagent was prepared as follows: To a l l volumetric flask, 1.50 g of CuS04'5H20, 6.00 g of potassium sodium tartrate and 500 ml of distilled water were added. Under vigorous stirring, 300 m1 of 10% sodium hydroxide was added and the volume made to 1 1. This solution was stored in a plastic bottle and used until a black precipitate appeared at which time it was discarded. Protein samples were added to a test tube, the volume adjusted to 1.0 ml with distilled water and 4.0 ml of the Biuret reagent was added. Following incubation at 37°C for 20 min, absorbances were measured at 540 nm. Standard curves were prepared from the navy bean globulin and albu- min fractions. Plots of absorbance vs protein were linear in the range 1 to 10 mg. Trypsin Inhibitor Assay Trypsin inhibitor assay was performed by a modification of the method of Kunitz (1947) as proposed by Kakade t 1. (1969b). This method estimates the activity of trypsin 25 inhibitor by monitoring the hydrolysis Of casein by trypsin as indicated by the peptides soluble in trichloroacetic acid (TCA). Aliquots ranging from 5 to 500 ul of the samples being assayed were added to a triplicate set of tubes and adjusted to 2.0 ml with 0.1 M phosphate buffer pH 7.6. One milliliter of 50 ug/ml of trypsin solution in 0.001 M HCl was added to each tube; which were placed in a water bath at 37°C for 10 min. To one of the tubes (blank), 6.0 ml of 5% TCA was added. Then, 2.0 m1 Of 2% casein solution, previously adjusted to 37°C, was added to each tube. The casein solution consisted of 2 g of casein dissolved in 100 m1 of phosphate buffer. After exactly 20 min, the reaction was stopped by adding 6.0 m1 of 5% TCA to the sample tubes. After standing 1 h at room temperature, the suspension was filtered through Whatman no. 1, and absorbance of the filtrate was measured at 280 nm against the blank for each sample. In all cases, absorbances were transformed to A”2 (see Results and Discussion) as origi- nally proposed by Miller and Johnson (1951). One trypsin inhibitor unit (TIU) was arbitrarily defined as the decrease of 0.01 in absorbance after the transformation given above. Immobilization of Trypsin on Agarose Activated agarose was obtained by the method of Cuatre- cases and Anfinsen (1971). One hundred milliliters of decanted agarose was mixed with 100 m1 of distilled water. 26 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 tempera- ture was maintained at about 20°C by adding pieces of ice as needed. The reaction was completed in 10 min as indi- cated by the cessation of base uptake. A large amount of ice was added to the suspension which was transferred to a coarse sintered-glass funnel and washed under suction with 300-400 ml of a cold solution of NaHC03 (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 with the same solution of NaHCO3 and stirred overnight at 0-2°C. 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 m1 of water. After stirring for 20 min at O-2°C, 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 1&3303), Two and half grams of trypsin dissolved in 100 m1 of buffer pH = 4.0 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 = 4.0. Filtrate and washings were collected, volume adjusted to 500 ml with water and absorbance read at 280 nm. To the washed gel, 100 ml of buffer pH = 9.0 (0.1 M NH4C1, 0.1 M HN4OH, 0.1 M CaClZ) was added and the suspension stirred at 0°C for 4 h. The suspension was filtered and washed with a cold solution at pH = 3.8 (0.1 M CaCl 01001 M HCl). As before, filtrate 2. and washings were collected for absorbance readings. 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. 28 ‘ s0 nan-(Chla-C . o 1.2.19.2... .cm-H ‘°""' -c-N-and trypsin inhibi- tor(j from navy beans. pHs were adjusted to desired values with hydrochloric acid or sodium hydroxide. A - Extraction with water for 1 h; B - Extraction with water for 2 h; C - Extraction for 2 h with 0.25, 0.50 and 1.0 M NaCl; and 0 - Extraction with water at pH 2.0. 55 Immobilization of Trypsin Agarose beads both before and after activation and coupling of ligands exhibit very little nonspecific adsorp- tion of proteins provided the ionic strength of the buffer is 0.05 M or greater (Cuatrecasasand Anfinsen, 1971). This property, among other considerations (5rere and Uyeda, 1976), made agarose the supporting material of choice for the purpose Of this study. The amount of trypsin bound to agarose was measured by the difference between the initial amount added and that recovered in the washings. The concentration of trypsin in these washings was estimated by reading absorbance at 280 nm. Previous experience with this immobilization procedure (Gomes, 1978 and Gomes 11 11., 1979) indicated that about 18% of the enzymatic activity is retained after immobilization. A column prepared with this immobilized form of trypsin remains active for over two years, provided adequate storage conditions. 'These conditions include the presence of calcium ions, a low pH and low temperatures - but remained above freezing to stabilize the trypsin; and an antimicrobial agent. Care must be taken with extracts which might contain enzymes that hydrolyze polyssacharides. Percolation of an extract from Triticale hydrolized the agarose matrix of a column, decreasing the flow rate and the column volume. 56 The Trypsin Inhibitor Assay The rate of hydrolysis of casein by trypsin does not follow zero order kinetics under the conditions defined by Kunitz (1947). This aberration has been attributed to limited substrate concentration (Bundy and Mehl, 1958). The modification proposed by Kakade 11 11. (1969b) employs 2% casein solution as substrate, instead of 1% in the original 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 a linear relationship between A3/2 and enzyme concentration over a large range (Gomes, 1978). During the course of this study, over 1,000 individual determinations of trypsin inhibitory activity were per- formed. Usually the assays were carried out in batches of 10-12 samples. Each batch required the inclusion Of a "trypsin blank", i.e. measurement of the hydrolysis of casein by trypsin (absorbance at 280 nm of TCA soluble products) in absence of inhibitory activity. Absorbances (280 nm) for the "trypsin blank" of 27 batches, which included different preparations of trypsin and casein solutions, yielded a mean of A280 = 0.516 with a relative standard deviation of 9.5%. 57 Separation of the Inhibitor Affinity Separation Figure 6 shows the resulting chromatogram Obtained when the protein fraction obtained by the optimized extrac- tion method (page 34) was percolated through the affinity column. The albumin fraction yielded similar chromato- grams, but with lower ratios of inhibitor to non-inhibitor proteins. The buffering capacity of the solution used to percolate the proteins through the column should be kept high (i.e. 20.1 M Tris) or the protein concentration low (i.e. wpom acouwn_scw cwmaxeu was 0539:. .8338 o.~ .3 .8. z N... 3 eaten. 81-2.: ea... 2.22: 323...... 30cc< .:PE\—E P mo mums m pm me: cowuzpu .Aso m._ x om. can—cu cwmaxcu -mmoemme mzu op woven mm: o.m In edemaa _u:-mweh z P.o cm cowuapom cessapm RP mo mcmuwpeppwe auew. .Louwnwzcw :_ma>eu :mmn x>mc we co_umcmamm xu_:_ee< .m mezmwa .OZ cozogu om om Ilerl o. OIUHHW. .UnYAXOAVAyoAu . .IAYILUIIO H O? 0.. w... I. .m; .. / I. O D \. cam now. ,0 o < O O \ O . —. 59 Gel Filtration Solutions of navy bean trypsin inhibitor-enriched frac- tion (Figure 4), heat-treated albumins (page 31) and the proteins obtained by the optimized extraction procedure (page 34) were fractionated on;a Sephadex G-75 column. Sample volumes of 15 m1 resulted in chromatograms similar to the one shown in Figure 7. Because all the trypsin inhibitory activity is concentrated in a well resolved peak, the sample volume could be increased without over- lapping with non-inhibitor proteins peak. The resulting chromatogram from a 50 m1 sample is shown in Figure 8. Based upon its sedimentation velocity, this protein frac- tion with trypsin inhibitory activity is designated the 25 fraction. Chromatograms comparing the elution profiles of navy bean proteins obtained by the three extraction procedure employed herein are shown in Figure 9. All three methods yielded similar chromatographic profiles, differing in the inhibitor to non-inhibitor peak areas ratios only. The freeze—dried weight of 25 fraction represented 3.1% of the albumin fraction, on a protein basis (Nx6.25). Calibration of the Sephadex G-75 Column The Sephadex G-75 gel filtration column was calibrated on the basis of molecular weight distribution. Percolation of a solution containing Blue Dextran 2000, bovine serum albumin, ovalbumin, chymotrypsinogen and 1yzozyme resulted 60 .Ezemm .Hmzuwucm zap: cowuumme m>wo_moa mmumupocw A I--- . .cxpe oe mo mums zap» m on cwuumppou mew: mco_uomem emu_~wp_ws cm» .cE:_ou mga co nmwpaam mm: eoeeeaee edeeeeee-eee.e.;e_ e_aaaee age co eae......e came... ..5e e.~ x om. cszpou msiu xmcmgamm.co :owuomcw um;o_ecm couwnwgcw :Pmaxep eo :o_umeupwm Poo .N weaned .oZ cote—u...— ov on ON or . . - %.IQIOIOIOIQIO e88 . deeded '000000'-‘ '00.... I OQN< 5.3.3 : I k. 3. 61 .e\.e oe as meat zap; m on uwuumppou mew; meowuumee emumpwppms cop .csapou one :o nmwpaam we: ee.eeaee ee:e.eee-eoe_a.ee. eeaaxee age ea eae......e seeea ..50 e.~ x om. cszpoo mmuw xmcmsgmm co cowaomce umguwecm eoownwgcw cwmaaeu we cowumeuywe —mw .m mezmw. .02 .3208". .uN .OP . . 0 0000000 / _ a. . v. O V. ‘ Z o .mfim IO/V Q. . . e 53:00 z. n. Figure 9. 62 10 2‘0 30 do so Fraction No. Gel filtration on Sephadex G-75 column (90 x 2.6 cm) of Navy bean proteins obtained by three different procedures: A - Optimized extraction procedure; 8 - Heat-treated albumins; and C - Trypsin inhibitor enriched fraction (See text for details). Fifteen milliliter aliquots were applied on the column. Ten milliliter fractions were collected. Bars indicate fractions with trypsin inhibitory activity. 63 in the chromatogram shown in Figure 10a. The exact posi- tions of peak maxima were determined by measuring absorban- ces at 280 nm of the collected fractions. The distribution coefficients (Kd) were calculated by the relation (Ackers, 1970): Kd = (Ve-Vo)/(Vm-Vo) where: Va is the elution volume of standard protein; Vm, the total available volume as measured with B-mercaptoethanol; and V0 is the void volume as measured with Blue Dextran 2000. A plot of log MW 11 distribution coefficient is pre- sented in Figure 10b. The observation made by Tanford _1 _1. (1974) related to the chemical affinity of 1yzozyme, in aqueous buffer, for dextran polymers was not confirmed. The elution volume of 1yzozyme was correlated with its molecular weight, thus fitting well in the above mentioned plot. The difference in behavior for this protein might be due to different buffer systems used. The elution posi- tion for the peak with trypsin inhibitory activity (Figures 7, 8 and 9) indicated a molecular weight of 16,000. Discontinuous Polyacrylamide Gel Electrophoresis Disc-PAGE was used to verify whether or not the trypsin inhibitor isolated by affinity chromatography on agarose- trypsin differed from that inhibitor present in the 25 fraction, isolated by gel filtration. Figure 11 shows 64 3...? .434 A 205' <8 0' I °, 110 {30 100 240 300 Volumeiml) B 4.3- ° o 41% 3! I: 34.41 .4 4L2 0 1 2 .3 41 5 6 Kd Figure 10. Calibration of a Sephadex G-75 column (90x2.6 cm). Flow rate was at 40 ml/h with 0.1 M ammonium formate/formic acid pH 3.2. A - Elution profile of standard proteins: Blue Dextran 2000 (void volume), bovine serum albumin, ovalbumin, chymotrypsinogen and 1yzozyme in the elution order, respectively. Total available volume was measured with B-mercaptoethanol in another experiment. 8 - Logarithmic plot of molecular weight vs distribution coefficient of the proteins Tfidi- cated in A. 65 W W“ A B C Figure 11. Discontinous polyacrylamide gel electrophoretic patterns of affinity- isolated trypsin inhibitor (A), 2 S frac- tion separated from the trypsin inhibi- tor-enriched fraction (8) and 2 S frac» tion separated from heated albumins (C). Running gels were 12% T (5% C). One hundred micrograms of protein was appli- ed to each gel, and stained with Cooma- ssie Blue R-250-trichloroacetic acid after electrophoresis. 66 electropherograms of these fractions. The affinity iso- lated inhibitor showed different mobilities than the proteins present in the 25 fraction. The extent of the modification can not be derived from the Disc-PAGE patterns. Because the 25 fraction showed a larger number of protein bands than the affinity isolated inhibitor, under the staining conditions used, trypsin inhibitory activity was assayed in those gels. Figure 12 shows the anti-tryptic activity after electrophoresis of the 25 fraction. Trypsin inhibi- tory activity is concentrated in the two protein zones indicated in the gel. In order to verify whether both bands were trypsin inhibitors or overlapping of the gel slices, the 25 proteins were fractionated in longer running gels. The two protein bands were found to have trypsin inhibitory activity as seen in Figure 13. Other bands do not contain trypsin inhibitory activity or were in too low concentration to reveal any activity under the conditions of the assay used. The Ferguson Plot A Ferguson plot (Rodbard and Chrambach, 1971) was used to determine the optimum gel concentration for separation of two protein zones of the affinity isolated trypsin inhibitor with close mobilities. Electrophoresis was performed on gels at 7.5, 9.0, 12.0 and 15.0% total concen- tration. The Ferguson plot for the two protein zones 67 .xmmmm one c. E: cam on mestLOmnm :. Po.o mo mmemeumv on» muzmmmcam. ADHFV pee: eopwnwgcw :Pmaxce mco .mcop as oo mew: .8 am we em.. m.am aeeeeem ..ewuom coawnwgc_ cwmaxc» .NF mczmwm 68 .xmmmo osp cw e: omm an oucon.0mno c_ _o.o oo omoocuoc one mucomoeaoe ADHH. awe: couwnwgcw :wmaxeu oco .mcop as mop oeoz Au &m .H ympv m_om mcwccsm .mo.eea seee.a.ee. eemasee .m. ae:m.a 3H,... : LJDQI FInn 69 '.:Z' ,, . . . . 7.5 9.0 12. 15. Gel Concontratlon 1%) Figure 14. Ferguson plot for the affinity-isolated inhibitor. Gel concentrations were 7.5 (A), 9.0 (B), 12.0 (C) and 15.0- (0). Arrows indicate location of protein bands used in the plot. 70 referred to above is shown in Figure 14. The slope in this plot represents the retardation coefficient (Kr) for a protein under the electrophoretic conditions used; and the intercept yields the mobility at zero gel concentration (YO). The linear equations obtained were: 0.1946-0.0334X R2= 0.998 (band 1 - faster) log Rm 0.0661-0.0251X R2= 0.998 (band 2 - slower) and log Rm hence, Kr] 0.0334; Kr2 = 0.0251; Y0] = 1.565 and Y02 = 1.164. . These parameters allowed the calculation of a gel con- centration which affords optimum separation, Topt, according to the relationship (Chrambach 11 11., 1976; and Chrambach and Rodbard, 1971): 2 10 YO -K Y -K ! Topt. = °[‘ 1 r°)/(°§ r2) Krl'Krz This equation indicated that a total gel concentration of 45.4% (5% C) was necessary to achieve Optimum separation Of the two protein zones under the experimental conditions used. For practical reasons such high concentration of acrylamide can not be used. Presence Of Trypsin Inhibitor in the Protein Bodies Protein bodies isolated from navy beans (Figure 15a) were tested for the presence Of trypsin inhibitors using antibodies prepared against the affinity isolated inhibitor. These organelles yielded precipitin zones against the 71 A- Protein Bodies NBTI PB NBTI Alb. Figure 15. A; Protein bodies isolated from navy beans: reference bar, 3 pm. B; Immuno double-diffusion pattern using antibodies to the affinity-isolated tryp- sin inhibitor (NBTI): P8 = protein bodies; Alb = albumin fraction. I 72 antibodies as shown in Figure 15b. Albumins are generally thought to be enzymes and other metabolic proteins (Millerd, 1975) while classic studies show that globulins constitute storage proteins Of legumes and Oil seeds (Danielson, 1949; Derbyshire _1 11., 1976). Many seeds have a high content Of albumins which is not explained by assuming an entirely enzymatic role. Youle and Huang (1978) studied the albumins Of Castor beans (Ricinus communis L.) and found that approximately 40% of the total protein in the protein bodies was represented by a group of closely related albumins, localized in the matrix Of the organelle. This group of albumins possess a sedimentation value of 25 and is resolved into several proteins of molecular weight approximately 12,000 as deter- mined by SDS-PAGE. They have high contents of glutamic acid/glutamine, serine and half-cystine; a low content Of methionine and traces Of tryptophan. These proteins undergo degradation rapidly during germination. Based upon these findings they concluded that the 25 albumins are storage proteins. Although the effect Of germination on the trypsin inhi- bitor activity Of navy beans was not investigated in this study, Kakade and Evans (1966) found that trypsin inhibitor activity first decreased and then increased during germina- tion. These findings do not support the role Of trypsin inhibitor as a storage protein in navy beans in spite Of its presence in the protein bodies. 73 Amino Acid Composition Amino Acid Analysis The amino acid compositions of navy beans, 25 fraction and the affinity isolated trypsin inhibitor are shown in Table 3. Amino acids which are subject to degradation during hydrolysis were extrapolated to zero time of hydroly- sis. Assuming a first order decomposition rate, the relationship log A0 = [tZ/(tz/t])]1og A1-[t1/(t2-t1)]1og A2 was applied; where A0, A1.and A2 are the concentrations Of each amino acid after 0, 24, 48 or 72 hours of hydrolysis, respectively. Values Of 48 or 72 h were used for amino acid which are more resistant to hydrolysis. Important aspects Of the amino acid composition of the navy bean trypsin inhibitor were the absence of tryptophan, the low amount of methionine and the high content of half- cystine. Sulfhydryl Determination The determination of sulfhydryl groups in proteins by reaction with 5,5'dithiobis (2-nitrobenzoate)-DTNB- was 74 introduced by Ellman (1959). Sulfhydryl content is measured indirectly by absorption at 412 nm of 3-carboxylat0 4- nitrothiophenolate, a product Of the reaction of DTNB with sulfhydryl groups Of proteins. Since the anion 3-carboxy- late 4-nitrothiophenolate is reported to undergo hydrolysis, Janatova 11 11. (1968) and Glazer 11 11. (1973) recommend that absorbance at 412 nm be recorded with time, and after development Of maximum color the downward sloping line be extrapolated to zero time to determine the absorbance value to use in calculating the concentration of sulfhydryl groups. The E412 for 3-carboxylate 4-nitrothiophenolate is 13,600. Under the conditions employed to determine the sulfhydryl content Of navy bean trypsin inhibitor and 25 fraction the decrease in absorbance after reaching a maximum was not Observed. Rather, a plateau was Observed after approximately 5 min Of reaction. Therefore the absorbance at the plateau region was used to calculate the sulfhydryl content. This method yielded a value Of 0.37 moles of 5H per 20,000 daltons for the 25 fraction and no detectable 5H groups for the navy bean trypsin inhibitor. Trend in Amino Acid Composition of Inhibitors Systematic trends in the amino acid composition pointed out by Birk (1968) for other trypsin inhibitors of plant origin were verified with the navy bean trypsin inhibitor; namely, a remarkably high and constant content Of proline, 75 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 the small contribution Of methionine. Since the sulfhydryl determination indicated that the affinity isolated inhibitor had no free -5H groups, all the half-cystine determined by amino acid analysis was derived from indigenous disulfide bonds. Considering the high content of this amino acid, the inhibitor should exist in a highly compact structure. 76 Table 3. Amino acid composition Of navy beans, 25 fraction and the affinity isolated trypsin inhibitora Navy bean 25 Affinity inhibitor lysine 5.12 6.81 histidine 2.09 6.35 arginine 4.88 5.28 tryptophanb 0.98 0.17 0 aspartic acid 10.9 17.0 threonine 4.16 6.54 serine 4.95 14.3 glutamic acid 12.5 12.2 proline 3.79 7.07 glycine 3.30 1.43 alanine 3.60 2.76 half-cystinec 1.97 10.2 12.9 valine 5.91 2.29 methioninec 1.11 1.21 1.21 valine 4.66 5.60 methionineC 1.63 4.23 isoleucine 2.95 2.92 leucine 5.63 2.85 tyrosine phenylalanine ag Of residue/100 g protein. bDetermined by the procedure Of Spies (1967) and Spies and Chambers (1948). cDetermined as cysteic acid and methionine sulfone, respec- tively. 77 Ultracentrifugal Characterization U1tracentrifugally-derived diffusion patterns for the affinity isolated trypsin inhibitor are shown in Figure 16a. Apparent diffusion coefficients (Dapp) were calculated as the slopes Of (l/4ir)(A/Hm)2 _1 time (sec); where A is the area under the peaks and Hm, peak height (Figure 17a). Dapp obtained at three protein concentrations allowed to extrapolate the diffusion coefficient to zero protein con- centration (0°). The value obtained for 0° at 20°C in 0.1 M phosphate buffer pH 7.6 was 9.2x10‘7 cmZ/sec (Figure 17c). Sedimentation velocity patterns are shown in Figure 16b. Apparent sedimentation velocity coefficients (Sapp) were calculated from the slopes of log (x+xo) _1 time (min) - see Figure 17b -, where (x+xo) is a measure of boundary migration. Three protein concentrations were used to extrapolate Sapp to sedimentation velocity coefficient at zero concentration (5°). The value calculated for 5° at 20°C in phosphate buffer pH 7.6 was 2.04x10'7 sec (Figure 17c). Data used for plotting Figures 17a, b, c are shown in Appendix Table A3. 78 Molecular Weight Svedberg equation. The sedimentation coefficient Of a macromolecule provides a rough estimate of molecular weight; however it is influenced by molecular shape and dimensions. The ambiguity introduced by the frictional coefficient into sedimentation coefficient can be overcome by combining sedimentation and diffusion measurements (van Holde, 1971): where 5° and 0° are sedimentation and diffusion coefficients corrected for concentration dependence; v, specific volume of the macromolecule; 9 solution density 3 solvent density; R, gas constant; T, absolute temperature. The parameters used for the affinity isolated inhibitor were: S‘é’o=2.04x10"7 sec; 0(2)0=9.2x10"7 cmZ/sec; v=0.684 cm3/g (see Table A2); 0=l.006 g/cm3; R=8.314x10-7 erg/mol.°K; T=293°K. These values yielded a molecular weight of 17,000. Sedimentation eguilibrium. A pattern fOr a sedimenta- tion equilibrium run is shown in Figure 16c. The method detailed in Appendix C was used to analyse the data. Para- meters 1, p, R and T were the same as above. The molecular 0 w) was weight extrapolated to zero protein concentration (M 20,900. 79 in... m~o§ 63% L33. ...o3~.5=ou..ou 23:: mo 5523—3 e8 :3 Eggs 335...? u 5:. .3533 F332. coco—28-9 Aim... To 282223 .56 .Em 2N: 63% .33. .53an 57522.3 8335.589”. 4:3 .5269; EB... mé v.3 :Fou 3: ER... 2 8o: neon—552.8 .Ex 85.3 .3on .33. 6532:. 538—2. 523:0..53-.. 4.5.6: :3 .3269. 0...... .8 2.3.... m6 v.5 Tonga. :8 +0” 0...» Le FEE... 3 Eu: 2335523 fax 08; .3on L33. .og T... 32.323 56 5 533:...— 53?» 932373.53. it mo 2.33:. cgmetoi .2 v.53. SE h 9:. VN 80 .23... .0542; ED .2 o 8.63 28.: -m.u:mu=ou .m:.muum3 cm.mwpzum ms» cw :o_um.mws xmma .o w.:mmme m m_ on+xv umwxmn> mg» cm .mucmwuv$mmou zuwuo—m> cowumacma_umm mo cowumpaupmu .o. pop; . m .Pe\ms Nfinwucm op_u.m.o nvm.mz mcowum.u:mucou .m:.muum3 =m.m_p;um we acmwm; xmma mg» E: use mm.m mucmmm.am. < "mwxmn> mg» c“ ..ou_nwncm cmmax.u umpmpomwuaupcwmmm,mgu mo mucmwuwwwmou :o_m:mw_c mo :o_am_:u~mu .0. qu3 u < .5” w.=mwu .225 2.5... . .00. .5 ma ow MP 3 LT. owe“ com. a com: o3 ! D «I \ . .0. ) \O . OQOQ. WV O U 1. .fl m VA H + A w. 629x -.z W o; G . \ 6a; a; m < 81 '8 .. 8- 41.6 3 § A A g"3 N; 7. O $1.5 g 0. no A (g '2 6- «1.4 a. \ 3 O ‘103 a 5 . . . A 1 5 , 1O Concentration (mg/ml) Figure 17c. Dependence of sedimentation veiocity o and diffusion coefficients A on concentration. 81 5 , ‘ ‘ A ‘ 1o Concentration (mg/ml) Figure 17c. Dependence of sedimentation veiocity o and diffusion coefficients A on concentration. '8 - 8- «1.6 3 0 (9 s: A A '2, N; 7- O '1.5 v: No A g '3 a. 41.4 m v: \ 55 o -1.3 82 Heat Inactivation In-vitro Measurements The data related to heat inactivation of the navy bean trypsin inhibitor were anaiysed using a first-order rate mode], i.e., the rate of inactivation at any given tempera- ture depends oniy on the first power of the concentration of a singie reacting species, in this case, active inhibitor moiecuies. Therefore: 0. O a kc at constant temperature (1) Q. t where c is the concentration of native (active) species; t, time and k, the rate constant with the units of the reciprocai of time. Integration from initial concentration Co, at t=0, to a concentration c at some iater time, gives: - gig = dt (2) C and 83 According equation 3 if the ratio c/co is known, quantities c and CO need not to be measured separately, and can be substituted for: C = constant x a C o constant X a0 Thus, the measurement of any quantity a that is proportional to the concentration c and co can be used (Barrow, 1973). From equation 3: _ k 0 2.303 log 0. = log a t (4) In this study a0 represents the initial trypsin inhibitor units, equated to 100; and 6 represents the units remaining after heating at a given temperature for an interval of time. A plot of activity 1; heating time for the temperature range 100 to l50°C is shown in Figure 18. The rate con- stants, therein designated inactivation rate constants, are shown in Table 4. The dependence of the inactivation rate constant on temperature is described by the Anhenius equation (Marshall, 1978): Ea/RT where k is the rate constant; Ea, activation energys T, 84 .mpcms_.wqu mam—3:50 03» we ounce; PS 3:25 .ooomF‘ucm ozflémplémpflé: O 530 no.3; 3.3232: £32.25 umampomw$t5tm 93 we 5.53.505... new: 4: 9:3: 1:2:v0::h 00 on O? on ON or < 4 .omx I 4 V a m. m .M 4 U Q Q / /._ u/ 4 won ab” 2: 84 mumpasou 0:» mo mumcm>m m.m mucwoa no.m3 mm.:um.maemh .mucmsw.maxm .uoomp‘ucm o: flog-6306: 0 .oo— 0 ..o._a.;=. omen—om_-»..:...a as. .o :o_ua>.uoae_ paw: 3.2.5 «EC. om Om ov on am e 4. AV .ON 1 I 4 V AV Mw w. “M 4. D Q Q on wuumcmn .m.:um.ma§ma gunman Acwsv mswuux_u:m >a_>wuum an» “tongue m.m:cm ammmgm omm.o m. _-o_xwm._ x~-o_xom.o-m¢o.~u> no. om. aom.o .m N-o_on.o x~-o.x~..~-.mo.mu> moo ca. mqm.o o. N-o_x~o.m x -o_x_m._-mmo.mu> no; om. m.m.o me_ ~-o_xmm._ xm-o_xmm.©-m_o.mu> mo; ON_ oem.o mmm m-o.x~m.¢ xm-o_x.m._-mma._u> no; OF. mmm.o comm ¢-o_xoo.m x¢-o_xo..m-.oo.~u> mo; oo_ «a oficwsv o nApncwva meowumzcm Auov m.:uw.masm» .o._a.;=_ =.m.».u embaPOW.-»8_:_..a an. .o =o_oa>_.ua=. “am: .a a_am. 89 TZ-T] Z = 109 (kZ/k1) (9) Values for D in the range l00 to lSOOC are shown in Table 4. The value for 2 calculated from equation 9 was 22.300 or 72.1°F. In-vivo Measurements Because affinity-isolated trypsin inhibitor showed such high resistance to inactivation by heat, navy beans were submitted to moist heat to determine if behavior in-vitgg and 1 -vivo was similar in respect to heat stability. The results are reported in Table 5. The inhibitor was completely inactivated when beans were heated for a few minutes at tem- peratures where the inhibitor resisted inactivation during in-vitrg_treatment. To determine whether the inhibitor was being inactivated during the i -vivo heat treatment or the extraction procedure failed to extract the inhibitor fraction after heat treat- ment of the beans, navy beans were treated by moist heat after soaking in affinity-isolated inhibitor solution. There was complete inactivation of the inhibitor (see Table 5), thus favoring the first hypothesis. Some components which are present in navy beans were tested for possible interaction with the inhibitor; none of these substances changed the profile of in-vitgg heat inac- tivation (Table 5). 90 Atmospheric or enzyme-catalysed oxidation of phenols in seeds results in quinoidal production and the formation of hydrogen peroxide. Both products degrade labile amino acids, denature proteins and inhibit enzymes. Once formed, o-qui- nones react non-enzymatically to polymerize and are reduced or bind covalently to amino, thiol and methylene groups. The e-amino groups of lysine and the thioether group of methio- nine are commonly attacked. Tannins bind to enzymes and other proteins by hydrogen bonding to amide groups to form insoluble complexes. The combined reaction of quinones, polyphenols and tannins on the e-amino groups of lysine and subsequent polymerization into tannin-protein complexes will form large blocks of modified amino acids. Thus, a low level of oxidation can result in a substantial modifi- cation of proteins (Sokulski, l979). Thus, in view of the possible interactions between proteins and polyphenols and their oxidation products, it was anticipated that heat treatment of the inhibitor in a system containing tannic acid would lead readily to its inactivation. Therefore the drastic difference between the heat-resistance observed 1n-v1vo and in-vitro could be attributed to polyphenols present in the seed coat of navy beans. Because this was not observed experimentally, the rapid heat inactivation that the trypsin inhibitor undergoes in-vivo should be attributed to components of beans other than those tested. 91 = = = = = om o» o_ ONF uwom u_=:mu + .opwn_;:H = = = = = on op o_ omp mmooz—m + .ou_n_;cH . = = = = co a» 0. OF. m=._=no_m + .oo.a_;=_ mpw.o.3 cowpm>wuumcw :_ mmcmgo o: on on o_ o__ cu.mum + .oawnwgcm = = mp o» m c__ .ou_n_;:_ + comm = = mm ow m om_ mcmmm = = om cu m om. mcamm cowpm>wpumcw muopasoo ow ca o_ op— mcmmm mapsmmm A.:PEV wave Aoov .QEm» mucmcoaeou .0pwaw;:_ mzocmmwvcw ms“ .0 :owum>_uomcw “mm: .m mpamp 92 UV Spectra of 25 and Inhibitor Fractions The ultraviolet absorption spectra of proteins, between 250 and 300 um are due almost entirely to the indole side chains of tryptophan and the phenolic side chains of tyro- sine. The phenyl side chains of phenylalanine also absorb in this region, but their molar absorbance is much lower (Tanford, 1961). UV spectra of the affinity-isolated trypsin inhibitor and ZS fraction are shown in Figure 1A (Appendix). Absor- 280 nm bance values at 280 um were used to calculate E1% The values of Efigo nm were 4.2 and 4.6 for the affinity-isolated inhibitor and ZS fraction, respectively. Amino acid analyses show that the 25 fraction has a low content of tryptophan, while the affinity-isolated inhibitor contains no tryptophan. The slightly higher value of E320 nm for the 25 fraction reflects the presence of tryptophan in these proteins. CONCLUSIONS A trypsin inhibitor fraction was isolated by affinity- chromatography on agarose-bound trypsin and characterized. Ultracentrifugal measurements yielded a sedimentation velocity and diffusion coefficients of 2.04x10"13 sec and 9.2x10'7 cmz/sec, respectively. Molecular weight values calculared by the Svedberg equation and sedimentation equilibrium were 17,000 and 18,600, respectively. This inhibitor preparation was heterogeneous when examined by discontinuous polyacrylamide gel electrophoresis. Molecular weights reported for the navy bean trypsin inhibitor were 23,000 obtained by sedimentation equilibrium method, 16,600 and 17,650 by SDS-PAGE, and 7,900, 11,500 and 11,900 by the stoichoimetry of the reaction with tryp- sin. A value of 16,000 was obtained by gel filtration. Thus, it seems that methods for estimation of molecular weight which depends on shape of the molecule yield lower values. The values obtained by the interaction inhibitor- trypsin suggest that the inhibitor has two sites against trypsin. Important aspects of its amino acid composition were the high content of half-cystine, low content of methionine and absence of tryptophan. Since there was no available 93 94 thiol group detected, a large number of disulphide bonds is indicated. The trypsin inhibitor is present in the albumin fraction of the navy bean proteins. Extraction of the navy bean meal with water, adjusted to pH 2.0 with hydrochloric acid and held for one hour yielded optimum results, i.e., a maximum extraction of inhibitor with a minimum extraction of pro- tein. Rabbit antibodies against the affinity-isolated inhibitor indicated that the inhibitor was localized in the protein bodies of the navy beans. Fractionation of water soluble proteins of navy beans by gel filtration yielded a fraction which contained nearly all the trypsin inhibitory activity of navy beans. A molecular weight of 16,000 was estimated from its eluting position. Further separation by discontinuous polyacryla- mide gel electrophoresis indicated that the trypsin inhibi- tory activity is limited to two protein zones. Other minor zones had no inhibitory activity under the assay employed. These two protein zones are closely related in mobility to the affinity-isolated inhibitor. Heat inactivation studies indicated that the affinity- isolated inhibitor is highly heat resistant. The decimal reduction time at 120°C was 145 min. with a "2" value of 22.300 (72.10F). An Arrhenius plot for the inactivation rate constants in the range loo-150°C yielded an activation energy of 30.9 Kcal/mol. Heat treatment of intact navy 95 beans indicated that the indigenous inhibitor is easily inactivated by heat. REFERENCES REFERENCES Acker, G.K. 1970. Analytical gel chromatography of pro- teins. In: Advanced Protein Chemistry. Vol. 44:349. Barrow, G.M. 1973. Physical Chemistry. Third edition. McGraw-Hill Book Co. New York. Beckman Instruction Manual. 1966. Instruction manual E-IM-3. Spinco Division of Beckman Instruments, Inc. Palo Alto, California. 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. 45:695. L. Lorand ed. Academic Press Inc. Publishers, New York. Bowman, D.E. 1944. Fractions derived from navy beans and soybeans which retard tryptic digestion of casein. Proc. Soc. Exp. Biol. Med. 57:139. Bowman, D.E. 1948. Further differentiation of bean trypsin inhibiting factors. Arch. Biochem. Biophys. 16:109. Bowman, D.E. 1971. Isolation and properties of a proteinase inhibitor of navy beans. Arch. Biochem. Biophys. 144:541. Bundy, H.F. and Mehl, J.N. 1958. Trypsin inhibitors of human serum: I. Standardization, mechanism of reaction and normal values. J. Clin. Inv. 37:947. Chrambach, A., Reisfeld, R.A., Wyckoff, M., and Zaccari, J. 1967. A procedure for rapid and sensitive staining of protein fractionated by polyacrylamide gel electrophore- sis. Anal. Biochem. 20:150. Chrambach, A. and Rodbard, 0. 1971. Polyacrylamide gel electrophoresis. Science 172:440. Chrambach, A., Jovin, T.M., Svendensen, P.J., and Rodbard, D. 1976. Analytical and preparative polyacrylamide gel electrophoresis. In: Methods of Protein Separation. Vol. 2, p. 27. N Catsimpoolas, ed. Plenum Press, New York. 96 97 Cohn, E.J. and Edsall, J.T. 1950. Density and apparent specific volume of proteins. In: Proteins, Amino Acid and Peptides as Ion and Dipolar Ions. E.D. Cohn and J.T. Edsall, ed. 4th ed. Reinhold Publishing Corp. New York. Cooper, 1.6. 1977. The Tools of Biochemistry. John Wiley and Sons. New York. Cuatrecacas, P. and Anfinsen, C.B. 1971. Affinity Chro- matography. In: Methods in Enzymology. Vol. 22:345, v.3. Jacoby ed. Academic Press Inc. Publishers, New York. Dahlgren, K., Porath, J., and Lindahl-Kiessling, K. 1970. On the purification of phytohemagglutinins from Phaseo- ‘ lus vulgaris seeds. Arch. Biochem. Biophys. 137:306. Danielson, C.E. 1949. Seed globulins of the Graminae and Leguminosae. Biochem. J. 44:387. Danielson, C.E. 1950. An electrophoretic investigation of vicilin and legumin from seeds of peas. Acta Chem. Scand. 4:762. Derbyshire, E., Wright, D.J. and Boulter, D. 1976. Review. Legumin and vicilin, storage proteins of legume seeds. Phytochemistry 15:3. Elias, L.G., DeFernandez, 0.0., and Bressani, R. 1979. Pos- sible effects of seed coat polyphenolics on the nutri- tional quality of bean protein. J. Food Sci. 44:524. Ellman, G.L. 1959. Tissue sulfhydryl groups. Arch. Bio- chem. Bi0phys. 82:70. Evans, R.J. and Bandemer, S.L. 1967. Nutritive value of legume seeds proteins. J. Agric. Food Chem. 15:439. Evans, R.J. and Bauer, D.H. 1978. Studies of the poor uti- lization by the rat of methionine and cystine in heated dry bean seed (Phaseolus vulgaris). J. Agric. Food Chem. 26:799. Evans, R.J., Bauer, D.H., Kathleen, A.S. and Ryan, P.A. 1974. The availability for the rat of methionine and cystine contained in dry bean seed (Phaseolus vulgaris). J. Agr. Food Chem. 22:130. 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 303:175. 98 Everson, G., and Heckert, A. 1944. The biological value of some leguminous sources of protein. J. Am. Diet. Assoc. 20:81. Gennis, L.S., and Cantor, C.R. 1976. Double-headed pro- tease inhibitors from black-eyed peas. III. Subunit interactions of the native and half-site chemically modified proteins. J. Biol. Chem. 251:747. Glazer, A.N., Delange, R.J., and Sigman, 0.5. 1973. Titra- tion of thiols with 5,5'-dithiobis (2-nitrobenzoate). In: Chemical Modification of Proteins. p. 113. North- Holland/American Elsevier. New York. Goa, J. and Strid, L. 1969. Amino acid content of legu- minous proteins as affected by genetic and nutritional factors. III. Arch. Microbiol. 33:253. Gomes, J.C. 1978. Isolation of trypsin inhibitor from navy beans (3. vulgaris L.) by affinity chromatography. M.S. thesis. Michigan State University. Gomes, J.C., Koch, U., and Brunner, J.R. 1979. Isolation of a trypsin inhibitor from navy beans by affinity chromatography. Cereal Chem. 56:525. Hermansson, A.M. 1979. Methods of studying functional characteristics of vegetable proteins. In: Proceedings of the world conference on vegetable food proteins, held Oct. 29, 1978. Amsterdam. J. Am. 0il Chem. Soc. 56:121. Holbrook, I.B. and Leaver, A.G. 1976. A procedure to increase the sensitivity of staining by Coomassie brilliant blue G-250-perchloric acid solution. Anal. Biochem. 75:634. Ishino, K. and Ortega, D.M.L. 1975. Fractionation and characterization of major reserve proteins from seeds of Phaseolus vulgaris. J. Agric. Food Chem. 23:529. Janatova, J., Fuller, J.K., and Hunter, M.J. 1968. The heterogeneity of bovine albumin with respect to sulfhy- dryl and dimer content. J. Biol. Chem. 243:3612. Jayne-Williams, D.J. 1976. The significance of the intes- tinal microflora in relation to the oral toxicity of raw navy beans and jack beans for japanese quail. In: Plant Proteins p. 141. G. Norton ed. Butterworths, London. 99 Johns, C.0. and Finks, A.J. 1920. Studies in nutrition. II. The role of cystine in nutrition as exemplified by nutrition experiments with the proteins of navy beans, Phaseolus vulgaris. J. Biol. Chem. 41:349. Joly, M. 1965. A Physico-chemical Approach to the dena- turation of Proteins. Academic Press. London. 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. 13:450. Kakade, M.L. and Evans, R.J. 1965b. Nutritive value of navy beans (Phaseolus vulgaris). Brit. J. Nutr. 19: 269. Kakade, M.L. and Evans, R.J. 1966. Effect of soaking and germinating on the nutritive value of navy beans. J. Food Sci. 31:781. Kakade, M.L., Arnold, R.L., Liener, I.E., and Naibel, P.E. 1969a.' Unavailability of cystine from trypsin inhibi- tors as a factor contributing to the poor nutritive value of navy beans. J. Nutr. 99:34. Kakade, M.L., Simons, N. and Liener, I.E. 1969b. An evaluation of natural vs synthetic substrates for measuring the antitryptic activity of soybean samples. Cereal Chem. 46:518. 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. Kassel, B. 1970. Trypsin inhibitors from other legumes. In: Methods in Enzymology. Vol. 19:862, G.E. Perlmann and L. Lorand ed. Academic Press Inc. Publishers. New York. Kunitz, M. 1945. Crystallization of a trypsin inhibitor from soybean. Science 101:668. Kunitz, M. 1946. Crystalline soybean trypsin inhibitor. J. Gen. Physiol. 29:149. Kunitz, M. 1948. Crystalline soybean trypsin inhibitor. II. General properties. J. Gen. Physiol. 30:291. Lansing, w.o. and Kraemer, E.0. 1935. Molecular weight analysis of mixtures by sedimentation equilibrium in the Svedberg ultracentrifuge. J. Am. Chem. Soc 57:1369. 100 Loeffler, L.J. and Pierce, J.V. 1973. Acyl azyde deriva- tives in affinity chromatography immobilization of enzymatically active trypsin on beaded agarose and porous glass. Biochim. Biophys. Acta 317:20. Liener, I. 1979. Significance for humans of biologically active factors in soybeans and other food legumes. In: Proceedings of the world conference on vegetable food proteins, held Oct. 29, 1978. Amsterdam, J. Am. 0il Chem. Soc. 56:121. Liener, I.E. 1976. Proteast inhibitors and other toxic factors in seeds. In: Plant Proteins, p. 117. G. Norton ed. Butterworths, London. Lewis, 0.A.M. 1966. Short ion-exchange column method for the estimation of cystine and methionine. Nature 209: 1239. Lund, D. 1975. Heat processing. In: Physical principles of food preservation. M. Karel, D. Fennema, and D. Lund, ed. Marcel Dekker, Inc. New York. Marshall, 1978. Biophysical Chemistry Principles, Techniques and Applications. John Wiley and Sons. New York. McCollum, E.V., Simonds, N., and Aitz, W. 1917. The dietary deficiencies of the white bean Phaseolus vul- garis. J. Biol. Chem. 29:521. McKay, A.F., Skulski, M., and Garmaise, D.L. 1958. Reac- tion of amino alcohols with carbon disulphide. Can. J. Chem. 36:147. McMeekin, T.L. and Marshall, K. 1952. Specific volumes of proteins and their relationship to their amino acid content. Science 116:142. Melachouris, N. 1969. Discontinuous gel electrophoresis of whey proteins, casein, and clotting enzymes. J. Dairy Sci. 52:456. Miller, B.S. and Johnson, J.A. 1951. A simple linear relationship and definition of a unit for proteinase activity.w Arch. Biochem. Biophys. 32:200. Millerd, A. 1975. Biochemistry of legume seed proteins. Annual Rev. Plant Physiol. 26:53. Mitchell, H.L., Parrish, D.B., Cormey, U., and Wassom, C.E. 1976. Effect of corn trypsin inhibitor on growth of rats. J. Agric. Food Chem. 24:1254. Moore, S., Spackman, D.H., and Stein, W.H. 1958; Chro- matography of amino acids on sulfonated polysterene resin. Anal. Chem. 30:1185. 101 Mosolov, V.V. and Fedurkina, N.V. 1974. Isolation of trypsin inhibitor from beans by means of specific binding on trypsin-agarose. Biokhimiya 39:624. Osborn, T.B., and Mendel, L.B. 1912. Beobachtungen Uber wachstum bei futterungsversuchen mit isolierten nahurgssubstazen. Z. Physiol. Chem. 80:307. Ouchterlony, 0. 1949. Antigen-antibody reactions in gels. Acta Pathol. Microbiol. Scand. 26:507. Pharmacia Fine Chemicals. 1976. Sephadex-gel filtration in theory and practice. September 1976-16. Uppsala, Sweden. , Porath, J. and Axen, R. 1976. Immobilization of enzymes to agar, agarose, and sephadex supports. In: Methods in Enzymology. Vol. 44:19. K. Mosbach ed. Academic Press Inc. Publishers, New York. Read, J.W., and Haas, L.W. 1938. Studies on the baking quality of flour as affected by certain enzymes actions. Cereal Chem. 15:59. Reisner, A.H., Nemes, P., Buchalty, C. 1975. The use of Coomassie brilliant blue G-250 perchloric acid solution for staining in electrophoresis and isoelectric focusing on polyacrylamide gels. Anal. Biochem. 64:509. Rodbard, D. and Chrambach, A. 1971. Estimation of molecu- lar radius, free mobility, and valence using polyacryla- mide gel electrophoresis. Anal. Biochem. 40:95. Scarbieri, V.C., Antunes, P.L. and Almeida, L.D. 1979. Nutritional evaluation of four varieties of dry beans. J. Food Sci. 44:1306. Schachman, H.K. 1957. Ultracentrifugation, diffusion and viscometry. In: Methods in Enzymology: Vol. 4:32. S.P. Colowick and N.0. Kaplan, ed. Academic Press, Inc. Publishers. Schram, E., Moore, 5., and Bigwood, E.J. 1954. Chromato- graphic determination of cystine as cysteic acid. Biochem. J. 57:33. Seidl, 0., Jaffe, M., and Jaffe, W. 1969.Digestabi1ity and proteinase inhibiting action of a kidney bean globulin. J. Agr. Food Chem. 17: 1318. 102 Sokulski, F. 1979. Organoleptic and nutritional effects of phenolic compounds on oilseed protein products: A review. J. Am. Oil Chem. Soc. 56:711. Spies, J.R., and Chambers, D.C. 1948. Chemical determi- nation of tryptophan. Anal. Chem. 20:30. Spies, J.R. 1967. Determination of tryptophan in proteins. Anal. Chem. 39:1412. Srere, P. and Uyeda, K. 1976. Functional groups on enzymes suitable for binding for matrices. In: Methods in Enzymology. Vol. 44:11. K. Mosbach ed. Academic Press Inc. Publishers, New York. Steiner, R.F., and Frattali, V. 1969. Purification and properties of soybean protein inhibitors of proteolytic enzymes. J. Agric. Food Chem 17:513. Tanford, C. 1961. Physical Chemistry of Macromolecules John Wiley & Sons, Inc. New York. Tanford, C., Nozaki, Y., Reynolds, J.A., and Makino, S. 1974. Molecular characterization of proteins in deter- gent solutions. Biochemistry 13:2369. VanHolde, K.E. 1967. Sedimentation equilibrium. Reprint SR-lOl by Spinco Division of Beckman Instruments, Inc. Palo Alto, California. VanHolde, K.E. 1971. Physical Biochemistry. Prentice- Hall, Inc. Englewood Cliffs, New Jersey. Waterman, H.C., and Johns, C.0. 1921. Studies on the digestability of proteins in vitro. I. The effect of cooking on the digestability of phaseolin. J. Biol. Chem. 46:9. 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. Whitaker, J.R. 1972. Principles of Enzymology for the Food Sciences. Marcell Dekker, Inc. New York. Whitley, E.J. Jr., and Bowman, D.E. 1975. Isolation and properties of navy bean proteinase inhibitor component I. Arch. Biochem. Biophys. 169:42. Wolf, W.J. 1977. Legumes: seed composition and structure, processing into protein products and protein properties. In: Food Proteins. J.R. Whitaker ed. AVI Publishing Co. Inc. Westport, Connecticut. 103 Yatsu, L.Y. and Jacks, T.J. 1968. Association of lysosomal activity with aleurone grains in plant seeds. Arch. Biophys. Biochem. 124:466. Youle, R.J. and Huang, A.H.C. 1978. Albumin storage pro- teins in the protein bodies of castor beans. Plant Physiol. 61:13. APPENDICES 104 APPENDIX StainingrProcedures A. Method of Chrambach g£_al, The method of Chrambach 33 11. (1967) represents an improvement over previous methods using Coomassie Blue R-250. After electrophoresis, the polyacrylamide cylinder is immersed in 12.5% TCA for 30 to 60 min. The gels are transferred to the staining solution freshly prepared by a 1:20 dilution in 12.5% TCA of a 1% aqueous stock solution of Coomassie Blue R-250. After 30 to 60 min of staining, the gels are transferred into 10% TCA. The color intensity of the zones increase during the succeeding 48 h of storage. If more sensitivity is needed, the same gel may be subjected to a second immersion in staining solution prepared by 1:10 dilution in 12.5% TCA of the aqueous stock and by doubling the staining periods. 8. Method with Coomassie Blue G-250/Perchloric Acid This procedure exploits a change of color exhibited by Coomassie Blue G-250 in perchloric acid solution (Reisner gt 31., 1975). No destaining is necessary since blue pro- tein zones appear against a light background. A 0.04% solution of Coomassie Blue G-250 is prepared in 3.5% 105 perchloric acid; the solution is stirred for l h at room temperature, filtered through Whatman no. 1 and passed through 0.45 um Millipore filter. The gels are immersed in this solution for 2-10 h at room temperature. The gels are removed from the staining solution and soaked in 5% acetic acid (Holbrook and Leaver, 1976) to increase the intensity of the stained zones. Gels can be stored in this solution. Molecular Weight by Sedimentation Equilibrium The most satisfactory method developed for the deter- mination of molecular weight of macromolecular materials is by means of sedimentation equilibrium in an analytical ultracentrifuge. The method has the advantage of a satis- factory theoretical background and a small experimental error. Calculation of molecular weight from the schlieren pat- terns was performed according to Lansing and Kraemer (1935) and Schachman (1957). The approach used yields molecular weight average, i.e. Mw=2wiM1/Zwi, where "i is the total weight of molecular species with molecular weight Mi’ The following example is based on the schlieren patterns obtained for the affinity-isolated navy bean trypsin inhibi- tor at concentrations of 5, 6.7 and 10 mg/ml in 0.1 M phos- phate buffer pH 7.6. Speed was 11,272 RPM (nominal) and a volume of about 0.12 ml of protein solution was used for 106 concentrations of 5 and 6.7 mg/ml. Apparent molecular weight for concentration of 10 mg/ml was calculated from a short column experiment using 0.06 ml of solution. Calculation from schlieren patterns Distance from reference wire to center of rotation (r 5.62 cm 0): Distance from the wire to the memiscus: Xm/2.118; where Xm is the microcomparator reading, and 2.118 is the magnification factor Distance from center of rotation to the memiscus (rm): where Xb is the microcomparator reading from wire to the cell bottom. Solution column length: rb - rm Integration of the schlieren pattern is carried out using 25 increments (AX), i.e. (rb - rm)/25. In terms of the microcomparator scale this becomes: Ax = [(rb - rm)/25] x 2.118 107 Thus, values in the Y-axis (Y1) are computed from the memis- cus to the bottom of the cell for each AX increment. The concentration difference between the bottom of the cell (Cb) and the air-solution memiscus (Cm) is obtained from: The initial concentration (Co) is obtained, in units of area, from a diffusion experiment using a synthetic boundary cell. Calculation of the area under the schlieren peak is performed in the same way as for calculation of diffusion coefficients. The area obtained in the microcomparator scale is divided by the magnification factor. Apparent molecular weight for each concentration is obtained from the relationship: 2RT . Cb-Cm 1 Mwapp - (l-Vo)w2 Co rb ' rm where R = 8.314x10'7 erg/mol. 0K; T is the absolute tempera- ture (OK); 9, the specific volume of the protein;p , solution density and w is the angular velocity. Apparent molecular weights were calculated for three concentrations of the affinity-isolated trypsin inhibitor and extrapolated to yield molecular weight at zero protein concen- tration (Mg). Table A1 shows the calculations in detail. 108 .Apmaouev 2am NmN.__uz mm\msu amo.o"» ”gammu u h oom.op . mNo_.o quo.o .mmo.~ qu.m N.__.m ommm.~ o_ oom.o~ ocm.m_ qmmo.o ammo.o one.“ mm~.o o¢o_.m N__¢.~ L.o oo¢.m_ mooo.o moqo.o Lmo.~ N¢~.o _mo_.m Numm.m o.m a: z: oo 28-38 AEUVQL Agave; ax Ex A_s\mev .ocou .nozums azwgawppzcm cowumucmewvmm mg» An ucmwmz gmpaow—os mo cowumpzupmu .F< wpamh 109 Table A2. Specific Volumea of the affinity isolated tryp- sin inhibitorb Residue % W Residue M.W. Sp. Vol. Wx(Sp. Vol.) Lysine 6.80 128.08 0.82 5.58 Histidine 6.35 137.08 0.67 4.25 Arginine 5.28 156.08 0.70 3.70 TryptOphan 0.0 186.08 0.74 0 Aspartic acid 17.03 115.08 0.60 10.22 Threonine 6.54 101.10 0.70 4.58 Serine 14.31 87.08 0.63 9.02 Glutamic acid 12.30 129.08 0.66 9.12 Proline 7.07 97.08 0.76 5.37 Glycine 1.43 57.03 0.64 0.92 Alanine 2.76 71.04 0.74 2.04 % Cystine 15.30 121.09 0.61 9.33 Valine 2.29 99.08 0.86 1.97 Methionine 1.21 131.18 0.75 0.91 Isoleucine 5.60 113.08 0.90 5.04 Leucine 5.60 113.08 0.90 3.81 Tyrosine 2.92 163.08 0.71 2.07 Phenylalanine 2.85 147.08 0.77 2.19 aCohn and Edsall, 1950; and McMeekin and Marshall, 1952. t’v =(ZWxSP.Vol.)/Z%W = 0.684 cm3/q 1'10 po~.— comp —ppw.o o~ 888.. 888. 88.8.8 88 8.8 8.88.8 888. 8.8. 88.. . 8888.8 8. 8.8. 8888.8 88.. 8888.8 8. 8.88.8 888 8888.8 8. 88... 888. 88.8.8 88 8888.8 888. 8888.8 .8 8.8 8888.8, 888. 8.8. 88.. 8888.8 8. 8.8. 8888.8 88. 8888.8 8. 8888.8 888 8888.8 8 888.. 888. 88.8.8 88 8888.8 888. 8..8 8 88 8.. 88.8.8 888. 88.8 88.. 8..8.8 8. 88.8 8888.8 888. 88.8.8 8. 8888.8 888 8888.8 8. Auomxu xsco—Vaaaa Auauwacpvwaazx_uomamw. .Pox :..o :. fimo.o 8:8 a.~o.o 8883 888.88.8888889 .A 88888.:88 :_8ngu cues—88.nza_:_wmm 8:8 A888 .85 59.26283 own 0mm omu 0mm 4 1 8 q d 14 _ q a q u — O 8. .8 .8. 8888.8 IQ 8 aouquosqv _L L "1111111111111111111