Soybean Protease Inhibitor Research Articles

Soybean Protease Inhibitors in Foods

ABSTRACTThe major types of soybean protease inhibitors are the Kunitz inhibitor (KSTI) and the Bowman‐Birk inhibitor (BBI). Enzymatic analysis and rocket Immunoelectrophoresis were used to specifically quantitate the amounts of KSTI and BBI in various soy foods. Many soy products such as soy flours, soy concentrates and soy isolates contained significant amounts of KSTI and BBI. Some soy foods contained small but still detectable levels of KSTI and BBI.

Effect of partially or totally replacing fish meal protein by soybean meal protein on growth, food utilization and proteolytic enzyme activities in rainbow trout ( Salmo gairdneri). New in vivo test for exocrine pancreatic secretion

This study was undertaken to determine whether the partial or total replacement of fish meal protein by protein from extracted, commercially available soybean meal would provoke a response in pancreatic proteolytic enzyme secretion in rainbow trout fry. At the end of the growth trial (177 days) the activity of chymotrypsin was determined by an in vivo method based on the oral introduction of N-benzoyl- l-tyrosyl- p-aminobenzoic acid (bt-PABA). Comparative trials were run with fish force-fed with PABA. The excretion index (bt-PABA/PABA) reflected the decreasing rate of pancreatic chymotrypsin activity with increasing amounts of soybean protease inhibitors introduced into the diet. This finding is in contrast to the several-fold increase of trypsin activity of faeces collected from fish fed a soybean-meal-supplemented diet. The growth rate of fish was reduced significantly when 50% of the fish meal was replaced by soybean meal, and 100% replacement resulted in growth arrestment and mortality. Amino acid absorption, particularly of methionine, leucine and threonine, decreased even when 25% of the fish meal protein had been replaced by soybean meal protein, and decreased further at the 50 and 100% substitution levels. It is suggested that the increase in secretion of pancreatic proteases caused by the soybean protease inhibitors does not compensate for the decrease in the efficiency of dietary protein digestion. The apparent contradiction between the in vivo assay of chymotrypsin activity and enzyme activity present in the faeces can be explained by inactivation of the protease inhibitors during digesta passage in the gut and by regeneration of inhibitor-bound trypsin in the posterior part of the intestine.

Heat inactivation of the Kunitz and Bowman-Birk soybean protease inhibitors

Etude de l'effet de differents parametres (duree, temperature, activite de l'eau) sur la vitesse et l'intensite de l'inactivation des deux principaux inhibiteurs proteasiques du soja

Isolation and sequence of cDNA encoding the soybean protease inhibitors PI IV and C-II

Isolation and sequence of cDNA encoding the soybean protease inhibitors PI IV and C-II

Kinetic studies on the inhibitions of mast cell chymase by natural serine protease inhibitors: indications for potential biological functions of these inhibitors.

We have found that degranulation from mast cells is specifically inhibited by the inhibitors of chymase (10). Among the natural serine protease inhibitors tested, Bowman-Birk soybean protease inhibitor, Eglin C, and human alpha 1-antichymotrypsin inhibited chymase more strongly than did chymostatin, Kunitz soybean protease inhibitor, and phosphatidylserine. Of the inhibitors tested, Bowman-Birk soybean protease inhibitor was the strongest inhibitor of chymase, its Ki value being 13.2 X 10(-9) M. Kinetic studies showed that these inhibitors were all noncompetitive inhibitors of chymase. Bowman-Birk and Kunitz soybean protease inhibitors inhibited both chymotrypsin-type and trypsin-type serine proteases but Eglin C specifically inhibited chymotrypsin-type proteases.

Chemical replacement of P1' serine residue at the second reactive site of soybean protease inhibitor C-II.

The P1' Ser(50) at the second reactive site of soybean protease inhibitor C-II was replaced with arginine to confirm the contribution of this residue to the inhibition. The Arg derivative had less trypsin inhibitory activity (Ki = 1 X 10(-7) M) than the Ser derivative (Ki = 2 X 10(-8) M), in contrast to the results obtained from studies on peanut protease inhibitor B-III reported in the previous paper (J. Biochem. 101, 723-728 (1987)). These results suggest that each Bowman-Birk type inhibitor has an amino acid at the P1' position inherently best suited to maintaining its inhibitory activity, and that serine is not unique for the P1' amino acid in Bowman-Birk type inhibitors.

Isolation and sequence of cDNA encoding the soybean protease inhibitors PI IV and C-II

Two full-length (or nearly so) cDNA clones containing information for the protease inhibitors PI IV and C-II from soybean seeds were identified by means of a synthetic probe. DNA sequencing revealed that the two protease inhibitors are synthesized as precursors with a short peptide leader. The coding regions of the two clones show 80% homology, wheraes the 5' non-coding regions are 90% homologous. Homology of 75% is found in the region extending beyond the stop codons.

Nanomolar concentrations of Bowman-Birk soybean protease inhibitor suppress x-ray-induced transformation in vitro.

Experiments reported here indicate a crude soybean extract, if defatted with acetone, effectively blocks cell transformation in vitro. An active component of this crude extract is the Bowman-Birk trypsin and chymotrypsin inhibitor. The chymotrypsin-inhibitory region of the Bowman-Birk inhibitor is responsible for suppressing in vitro transformation. Another low molecular weight soybean trypsin inhibitor does not significantly suppress transformation. The Bowman-Birk inhibitor (i) has an irreversible effect on the transformation process, (ii) can suppress radiation-induced transformation even when added to cultures many days after the carcinogen exposure, and (iii) is effective in its ability to suppress transformation when present in the medium at a concentration as low as 0.125 nM.

Selective DNA-amplification induced by carcinogens (initiators): evidence for a role of proteases and DNA polymerase alpha.

Inhibitors of DNA polymerase alpha (aphidicolin, phosphonoacetic acid, phosphonoformic acid) efficiently inhibit initiator-induced amplification of SV40 DNA sequences in the SV40-transformed Chinese hamster cell line CO631. Amplification is also inhibited by various protease inhibitors (antipain, leupeptin, aprotinin, alpha-I-antitrypsin, epsilon-amino-caproic acid, soy-bean protease inhibitor), by the non-initiating but DNA-damaging agent caffeine, and by sodium butyrate, which inhibits DNA synthesis by histone modification. In contrast, an inhibitor of topoisomerase II, nalidixic acid, enhances amplification when applied simultaneously with initiating treatment. This latter compound does not induce amplification when applied without initiator. Cycloheximide induces DNA amplification in the same way as chemical and physical carcinogens. This amplification can still be observed when protein synthesis is completely blocked. The data suggest a complex mechanism of selective DNA amplification. The possible involvement of proteases leading to a functional modification of DNA polymerase alpha is discussed.

Differential Expression of Kunitz and Bowman-Birk Soybean Proteinase Inhibitors in Plant and Callus Tissue

Bowman-Birk soybean trypsin inhibitor (BBSTI) but not Kunitz soybean trypsin inhibitor (KSTI) was found in samples of undifferentiated and partially differentiated Amsoy 71 tissue culture callus. This suggests the differential metabolism of these two classes of proteinase inhibitors, whether the difference be in synthesis, in rates of degradation, or both. The differential metabolism of the proteinase inhibitors is also seen in the plant. Both BBSTI and KSTI were found in the hypocotyl, root, and epicotyl of the Amsoy 71 soybean seedling in addition to their expected presence in the cotyledons. Whereas the ratio of KSTI to BBSTI in the cotyledon was higher, the ratio of BBSTI to KSTI was higher in the extracotyledonary tissues of the seedling. The levels of both classes of proteinase inhibitors declined during seedling growth, except in the epicotyl and the proximal root. In both of these tissues, an increase in BBSTI, but not in KSTI content, expressed as milligrams inhibitor per plant part, occurred.

Different Rates of Metabolism of Soybean Proteinase Inhibitors during Germination

During germination, the content of the major Bowman-Birk proteinase inhibitor (BB-E) in the cotyledons of soybean (Glycine max [L.] Merrill cv. Fiskeby V) seeds decreases, becoming a minor form by the sixth day of germination. One of the three other minor species (BB-D) of this inhibitor in the dry seed increases to become the major form in six-day cotyledons. The other two minor species (BB-C and BB-F) also appear to decrease during germination, but at a slower rate compared to the original major inhibitor form BB-E. By 13 days of germination, no distinct Bowman-Birk inhibitor species can be discerned in the cotyledons. The content of the major Kunitz proteinase inhibitor (K-B) also decreases during germination, but much more slowly. One new form of the Kunitz inhibitor (K-A) becomes apparent by the sixth day. By the 13th day, the proportion of the main isoinhibitor to the late-appearing form is approximately two to one. This difference in the rates of disappearance and appearance of isoinhibitor species in the Bowman-Birk and Kunitz proteinase inhibitor classes suggests a differential metabolism of these two types of proteins and a possible difference in function in the soybean plant.

In vitro synthesis of the Bowman-Birk and related soybean protease inhibitors

Poly (A)(+) RNAs from immature soybean seeds were size fractionated in denaturing sucrose gradients to identify mRNA that directs the cell-free synthesis of the Bowman-Birk protease inhibitor and the related inhibitors PI I-IV. Polypeptides synthesized in vitro were labeled with ((35)S)-cysteine and ((3)H)-serine and detected by immunoprecipitation with anti Bowman-Birk and anti PI I-IV sera. Immunoprecipitates of the translation products comigrated on SDS-polyacrylamide gels with the dimeric or trimeric aggregates of the authentic inhibitor proteins, which self-associate under certain conditions. Further evidence that these immunoprecipitates contained authentic polypeptides corresponding to the Bowman-Birk or PI IV inhibitor was shown by sequential amino acid analyses of peptides generated by cleavage with cyanogen bromide.

Soybean Proteinase Inhibitors and Human Proteolytic Enzymes: Selective Inactivation of Inhibitors by Treatment with Human Gastric Juice

The stability of soybean proteinase inhibitors to 0.1 M HCl and to human gastric juice was investigated. Purified Kunitz soybean inhibitor (SBTI), purified Lima bean inhibitor (LBI), and crude, aqueous extract (1:5 w/v) of three varieties of soybeans were incubated for 24 hours at 37°. The SBTI, the LBI, and the soybean extracts were mixed with gastric juice or 0.1 M HCl in amounts corresponding to 4.0 g SBTI, 2.4 g LBI, and 130 g soybean meal per liter. The trypsin and chymotrypsin inhibitor activities of the incubation mixtures were measured after 0.0, 0.5, 1.0, 2.0, 4.0, 8.0, and 24.0 hours of incubation. Human pancreatic juice was used as enzyme source. SBTI was rapidly inactivated by the incubation with gastric juice. LBI was hardly affected by the incubation. The trypsin and chymotrypsin inhibitor activities of the soybean extracts were reduced to 60–70%, indicating that SBTI contributed ⅓ of the inhibitor activity of these varieties. Thus, the compact, rigid inhibitors, such as the Bowman-Birk, were responsible for the remainder. The latter group of inhibitors, when ingested with the soybeans, will not be inactivated by passage through the stomach. They probably reach the duodenum in an active form.soybean inhibitors gastric juice pancreatic proteinase

Affinity chromatographic interactions of proteases with low-molecular-weight soybean protease inhibitors

One of the five low-molecular-weight soybean protease inhibitors, protease inhibitor V (PI-V, Bowman-Birk inhibitor) with a molecular weight of 8000 daltons, is used for the affinity chromatographic purification of trypsin, chymotrypsin and kallikrein. The PI-V covalently bound to Sepharose 4B adsorbs bovine trypsin, chymotrypsin or porcine kallikrein at neutral pH. The adsorbed enzyme is eluted from the column with buffer containing a higher salt concentration and a lower pH. This low-molecular-weight PI-V affinity gel can separate trypsin and chymotrypsin from mixtures of these crude enzymes and can purify both enzymes to a homogeneous degree.

Lima bean proteinase inhibitor. Origins of circular dichroism bands and modification by Br2- and (CNS)2-.

Origins of CD bands in lima bean proteinase inhibitor were deduced from an acetylation-deacetylation study of the sole tyrosyl residue in the protein (Tyr 69), and by analogy with Bowman-Birk soybean proteinase inhibitor, a homologous protein with similar spectral properties. Tyr 69 is relatively inaccessible to N-acetylimidazole; 100-fold molar excess of the reagent in the presence of 6 M guanidine hydrochloride elicited about 70 to 80% O-acetylation. A broad negative CD band centered around 280 nm arises mainly from the longest wavelength transition of cystinyl side chains (epsilon L--epsilon R approximately equal to -0.8 M-1 cm-1 per disulfide). The second cystinyl transition gives rise to a positive CD band of a comparable intensity at 247 nm. The Lb vibronic transition of Tyr 69 has negative CD around 280 nm, contributing approximately 10% of the total CD intensity at 278 nm (epsilon L--epsilon R approximately equal to -0.5 M-1 cm-1). The 232 nm positive shoulder is from the La vibronic transition of Tyr 69. Radical anions, Br2- and (CNS)2-, generated by the irradiation of N2O-saturated inhibitor solutions containing KBr or KCNS, reduced tyrosyl CD without affecting disulfide CD bands, indicating that the radical anions damaged Tyr 69 without altering protein conformation. The inhibitor modified at Tyr 69 by Br2- and (CNS)2- retained full activity toward trypsin and chymotrypsin. The irradiation of the inhibitor in the air-saturated solution led to loss in tyrosyl as well as cystinyl CD bands and decline in both antiproteinase activities.

Preliminary crystallographic data for Bowman-Birk inhibitor from soybean seeds.

A well characterized soybean protease inhibitor, the Bowman-Birk inhibitor, has been crystallized at room temperature in the presence of polyethylene glycol 4000 by vapor diffusion against an ammonium sulfate solution containing 2-methyl-2,4-pentanediol. An x-ray diffraction study reveals that the inhibitor crystallizes in a hexagonal unit cell of symmetry P6122 (or P6522) and dimensions a = b = 91.36(2) A and c = 63.92(2) A. Each of the 12 asymmetric units contains 2 molecules of molecular weight 8000. The crystal, which diffracts barely to 3-A spacings, is fairly stable to x-irradiation and has a solvent content of approximately 52% by volume.

Inhibition of Human and Rat Pancreatic Proteinases by Crude and Purified Soybean Proteinase Inhibitors

Effects of proteinase inhibitors on total proteolytic activity and trypsin and chymotrypsin activity in human pancreatic juice were determined separately. Purified inhibitors as well as crude extracts of raw soybeans completely inhibited trypsin and chymotrypsin activity while 40 to 50% of the total proteolytic activity remained. Inhibition experiments with 1,10-o-phenanthroline showed that this residual proteolytic activity was due mainly to carboxypeptidase A and B. Comparative studies with rat pancreatic enzymes demonstrated certain similarities between the corresponding enzymes from rat and man. However, differences were revealed which indicate that the rat enzymes must be used with great caution when applied as models for the human proteinases when studying effects of soybean inhibitors.

Rapid Release of Protease Inhibitors from Soybeans

Specific antisera were prepared against the Bowman-Birk trypsin inhibitor and four other trypsin inhibitors of low molecular weight isolated from soybeans (Glycine max L. cv. Tracy). These antisera were used to detect the presence and amount of the inhibitors in: (a) seeds and protein extracts of soybean meal; (b) seedlings; and (c) the water surrounding the seeds and roots of seedlings. Lectin activities in seeds, seedlings, and water were also determined at the same time as the protease inhibitor activities. By competitive inhibition of immunoprecipitation, the combined five low molecular weight protease inhibitors were found to constitute the following percentages of proteins (w/w): 6.3% in defatted soybean meal; 8.1% of the protein extracted from the meal by a buffer of pH 8.6; 8.3, 14.7, 15.2, 16.1, 17.2, and 18.9% of the protein in a lyophilisate of water in which seeds were incubated for 4, 8, 12, 16, 20, and 24 hours, respectively; 8.2% in a lyophilisate of water in which roots of seedlings grew for 20 days; 1.5% in cotyledons; and less than 0.1% in epicotyls, hypocotyls, and roots of 12-day-old seedlings. Hemagglutination activities, expressed as the lowest amount of protein required to give a positive agglutination of 0.2 ml of 2% rabbit red blood cells, were as follows: purified soybean lectin, 0.08 mug; lyophilisate of water in which seeds were incubated for 4, 8, 12, 16, 20, and 24 hours, 10, 2.5, 5, 5, and 2.5 mug, respectively; lyophilisate of water in which roots grew for 20 days, 5 mug; 12-day-old cotyledons, roots, epicotyls, and hypocotyls, 12.5, 100, >1,000, and >500 mug, respectively. The results indicate that a large amount of protease inhibitors as well as lectins are released from seeds during the first 8 hours of imbibition. Neither lima bean trypsin inhibitor (mol wt, 10,000) nor Kunitz soybean trypsin inhibitor (mol wt, 21,500) showed competitive inhibition in tests with antisera against low molecular weight soybean protease inhibitors.

γ Irradiation of Bowman-Birk Soybean Proteinase Inhibitor

WANDELL, J. L., AND KAY, E. 7 Irradiation of Bowman-Birk Soybean Proteinase Inhibitor. Radiat. Res. 72, 414-426 (1977). Radiation damage to Bowman-Birk soybean proteinase inhibitor was studied in dilute aqueous solutions containing: (a) air, (b) N20 plus KBr, and (c) NO20 plus KCNS. The degradation of tyrosines, monitored by changes in ultraviolet absorption and circular dichroism (CD) spectra of irradiated inhibitor, led to the loss of chymotrypsininhibitory activity without affecting antitrypsin activity. Of two tyrosyl residues in the inhibitor, Tyr 45, located next to the antichymotrypsin site, was shown to be essential to chymotrypsin-inhibitory activity. Radiation damage to Tyr 59 had no effect on either of the antiproteinase activities. The loss of trypsin-inhibitory activity paralleled decrease in disulfide CD. The breakage of disulfide bonds was confirmed by the p-chloromercuribenzoate assay for sulfhydryl groups. However, the exact role of disulfide bonds in antitryptic activity, other than the maintenance of conformational integrity, is not apparent. It seems more likely that the loss of trypsin-inhibitory activity is due to damage in other amino acid (s), although the degradation of tyrosine (2 residues) and histidine (1 residue) did not affect anti

Studies on soybean trypsin inhibitors. 3. Amino-acid sequences of the carboxyl-terminal region and the complete amino-acid sequence of soybean trypsin inhibitor (Kunitz).

For the elucidation of the amino‐acid sequence of the carboxyl‐terminal region of soybean trypsin inhibitor (Kunitz), fragments C and D were digested with trypsin, and the resulting peptides were separated by ion‐exchange chromatography on Dowex 50X2 or by gel nitration on Bio‐Gel P‐4.Further fractionation and purification of the peptides were performed by ion‐exchange chromatography on Dowex 1X2, by gel filtration on Bio‐Gel P‐2 or by high‐voltage paper electrophoresis at pH 1.9 and 3.6.Three peptides were obtained in pure form from fragment C and ten peptides from fragment D, and their amino‐acid sequences were determined by the direct Edman method and by carboxy‐peptidase digestion technique.Overlapping peptides necessary for the alignment of the tryptic peptides from fragment D were obtained from a chymotryptic hydrolysate of fragment D.Nine main peptides and nine minor peptides were obtained. The amino acid composition and the partial amino‐acid sequence of the 18 chymotryptic peptides of fragment D made it possible to establish the amino‐acid sequence of the carboxyl‐terminal region of the inhibitor.The complete amino‐acid sequence of soybean trypsin inhibitor (Kunitz) deduced here was compared with that of Bowman‐Birk inhibitor, another well‐known soybean proteinase inhibitor.