Antitryptic Activity Research Articles

Anti-tryptic activity of a synthetic bicyclic fragment of soybean bowman-birk inhibitor

A bicyclic hexadecapeptide corresponding to the sequence 9–24 of soybean Bowman-Birk inhibitor has been synthesized. This peptide has two disulfide linkages, between residues 9 and 24 and between 14 and 22. A nonapeptide loop formed by the latter disulfide bridge contains a Lys-16-Ser-17 bond, which is the reactive site for trypsin inhibition. The bicyclic peptide showed strong anti-tryptic activity with an inhibition constant ( K i) of 1.5·10 −7 M. Comparison with other hexadecapeptides which have the same sequence but lack one or both disulfides suggested that a compact ring system increases the activity and protects the Lys-Ser bond from tryptic hydrolysis.

Natural plant enzyme inhibitors. Protease inhibitors in millets

AbstractProtease inhibitory activities were screened in 12 varieties of pearl millet (Pennisetum typhoideum), 12 varieties of echinocloa (Echinocloa colona), 12 varieties of setaria (Setaria italica), 11 varieties of kodo (Paspalum scorbiculatum), 13 varieties of proso (Panicium miliaceum), 11 varieties of miliare (Panicium miliare), 29 varieties of sorghum (Sorghum bicolor) and four varieties of ragi (Eleusine coracana). Proso, miliare and kodo had no detectable inhibitory activity. Pearl millet, setaria and echinocloa millets displayed only antitryptic activity. Ragi had both antitryptic and antichymotryptic activity. Two varieties of sorghum had neither antitryptic nor antichymotryptic activity. In most other strains of sorghum, the antichymotryptic activity was more than the antitryptic activity. Pearl millet, setaria, sorghum and echinocloa extracts inhibited the proteolytic activity of both human and bovine pancreatic preparations.

Urinary-proteinase inhibitor in premature infants and its comparison with amniotic-fluid acid-stable proteinase inhibitor

Urinary-antitryptic activity was found to be higher in neonates (2.25 ± 0.75 units, mean ± SD) than in infants (0.65 ± 0.50, 2–12 months old) or in children of 1–10 years old (0.63 ± 0.53). It was indicated in follow-up studies that a gradual decline of activity occurred in the first month of life, and stabilized by 30 days. The antitryptic activity in urine was higher in premature infants (5.97 ± 0.98) than in fullterm ones. The inhibitor was purified to homogeneity from the urine of premature infants by chromatography on DEAE-cellulose, concanavalin A-Sepharose 4B, and Sephadex G-100. The inhibitor which is a glycoprotein was found to have a molecular weight of 33,000 based upon SDS electrophoresis. The purified inhibitor appears to be made up of two identical polypeptide chains as suggested by electrophoresis in the presence of mercaptoethanol. The inhibitor was stable to exposure of a wide range of pH (1.0–11.8). Modification of the guanidino groups resulted in the loss of the antitryptic activity but not the antichymotryptic activity. The inhibitor was found to be similar to the proteinase inhibitor isolated from amniotic fluid (5) in terms of its molecular size, dimeric nature, carbohydrate content, and heat and acid stability and lability to pepsin treatment. However, the urinary inhibitor did not have mutually exclusive binding sites for trypsin and chymotrypsin, unlike the factor from amniotic fluid; the former inhibitor was more active on trypsin than on chymotrypsin.

Serum antipapain activity during normal pregnancy.

Antitryptic, antichymotryptic and antipapain activities in maternal serum were measured in the course of normal pregnancy. An increase in inhibitory capacity toward the three examined proteases was demonstrated. The level of antipapain activity was highest between the 13th and 24th weeks of gestation in contrast to antitrypsin and antichymotrypsin activities which have shown the highest values between the 25th and 36th weeks of pregnancy.

Turkey serum trypsin inhibitors: Description and characterization

1. 1. Turkey serum trypsin inhibitors were studied on whole and chromatographically fractionated normal turkey serum using both quantitative (trypsin inhibitory capacity measurement) and qualitative (antitryptic activity detection methods) determinations, coupled to electrophoretic and isoelectrophoretic studies. 2. 2. Five proteins with trypsin inhibitory activity were described, the most important ones being α 2 and β-globulins with a multibanded pattern revealed by isoelectric focusing. 3. 3. Trypsin inhibitory capacity assays, performed on individual sera, as well as isoelectric focusing studies, failed to find any quantitative and/or qualitative deficiency of these antiproteases. 4. 4. Evidence is given that round heart disease in turkeys is not related to serum trypsin inhibitor deficiency.

Optimum tryptic activation of inactive renin in human plasma is independent of endogenous anti-tryptic activity.

1. Plasma samples from 31 normal subjects were treated (at 4 degrees C, pH 7.0, for 2 min) with different concentrations of trypsin (500, 1000, 2000, 3000 and 4000 microgram/ml) in order to assess which concentration yielded the maximum activation of inactive renin. 2. Endogenous antitryptic activity was also measured in all samples; the mean value +/- SD (in microgram of trypsin inhibited by 1 ml of plasma) was 953 +/- 550 microgram/ml (range 34-1800 microgram/ml). 3. In the entire group of subjects the values of trypsin-activated renin measured with trypsin at 2000 microgram/ml were significantly higher than those obtained with lower or higher trypsin concentrations. 4. With subjects divided into subgroups according to their endogenous anti-tryptic activity, the maximum yield of activation was reached with trypsin at 2000 microgram/ml. 5. No significant correlations were found between single values of active, inactive or trypsin-activated renin and the corresponding levels of endogenous anti-tryptic activity. However, a weak but significant correlation (r = 0.39, P less than 0.05) was found between single values of anti-tryptic activity and the corresponding percentage of activation of inactive renin. 6. Thus the maximum activation of inactive renin at 4 degrees C for 2 min is obtained with trypsin at 2000 microgram/ml independently of the corresponding endogenous anti-tryptic activity. It is not excluded that the content of protease inhibitors in human plasma might affect the proportion in vivo of circulating active and inactive renin.

Trypsin inhibitor activity in vicia faba beans

Extracts of mature green, dry and germinated Vicia faba beans depressed the trypsin activity of casein. Germination of Vicia faba beans (for 60 h) lowered the trypsin inhibitor (TI) activity. 0.171 M Saline was the most efficient extractant for the TI. Minimal amounts of the TI were extracted in the pH range 4 to 5. The TI of Vicia faba beans was undialysable. The inhibitor activity originated in the seeds at the beginning of pod formation and increased with development of maturity. TI was active only towards trypsin and inactive towards papain, rennin and pepsin. Chromatographing Vicia faba bean proteins, possessing antitryptic activity, on a column of DEAE-cellulose yielded six peaks, all of which possessed antitryptic activity.

Natural plant enzyme inhibitors. Characterization of an unusual alpha-amylase/trypsin inhibitor from ragi (Eleusine coracana Geartn.).

An inhibitor I-1, capable of acting on both alpha-amylase and trypsin, was purified to homogeneity from ragi (finger-millet) grains. The factor was found to be stable to heat treatment at 100 degrees C for 1 h in the presence of NaCl and also was stable over the wide pH range 1-10. Pepsin and Pronase treatment of inhibitor I-1 resulted in gradual loss of both the inhibitory activities. Formation of trypsin-inhibitor I-1 complex, amylase-inhibitor I-1 complex and trypsin-inhibitor I-1-amylase trimer complex was demonstrated by chromatography on a Bio-Gel P-200 column. This indicated that the inhibitor is 'double-headed' in nature. The inhibitor was retained on a trypsin-Sepharose 4B column at pH 7.0. Elution at acidic pH resulted in almost complete recovery of amylase-inhibitory and trypsin-inhibitory activities. alpha-Amylase was retained on a trypsin-Sepharose column to which inhibitor I-1 was bound, but not on trypsin-Sepharose alone. Modification of amino groups of the inhibitor with 2,4,6-trinitrobenzenesulphonic acid resulted in complete loss of amylase-inhibitory activity but only 40% loss in antitryptic activity. Modification of arginine residues by cyclohexane-1,2-dione led to 85% loss of antitryptic activity after 5 h, but no effect on amylase-inhibitory activity. The results show that a single bifunctional protein factor is responsible for both amylase-inhibitory and trypsin-inhibitory activities with two different reactive sites.

Kunitz-type proteinase inhibitors derived by limited proteolysis of the inter-alpha-trypsin inhibitor, IV. The amino acid sequence of the human urinary trypsin inhibitor isolated by affinity chromatography.

An acid-resistant trypsin inhibitor from human urine and serum is released in vivo by limited proteolysis from the high molecular acid-labile inter-alpha-trypsin inhibitor. The inhibitor shows an apparent molecular mass of 30 000 Da and is composed of two Kunitz-type domains. The domains are released in vitro by prolonged tryptic hydrolysis. The C-terminal domain is responsible for antitryptic activity. For the other domain no inhibitory activity towards proteinases, i.e. chymotrypsin, trypsin, pancreatic and leucocytic elastase has been demonstrated so far. The polypeptide chain comprising both domains consists of 122 residues and has a molecular mass of only 13 400 Da. In this work we have found that both, the N-terminal extension peptide with 21 residues and the "inactive" domain are linked O-glycosidically and N-glycosidically, respectively, with large carbohydrate moieties. The N-terminal amino acid sequence of the human urinary trypsin inhibitor was determined by solid-phase Edman degradation of a single peptide. The molecular mass calculated for the total polypeptide chain of 143 residues should be 15 340 Da; from the difference to the measured value (30 000 Da) it is concluded that the glycopeptide contains a considerable carbohydrate moiety.

Natural plant enzyme inhibitors. Isolation and characterisation of a trypsin/chymotrypsin inhibitor from indian red wood (Adenanthera pavonia) seeds

AbstractA trypsin/chymotrypsin inhibitor was isolated from Indian red wood seeds by extraction with 0.01m HCl, chromatography on diethyl amino ethyl‐cellulose, ammonium sulphate fractionation and gel chromatography on Sephadex G‐100. The homogeneity of the final product was ascertained by affinity chromatography on trypsin sepharose and chromatography on phenyl sepharose CL‐4B columns. During all stages of purification and characterisation the ratio of activities against trypsin and chymotrypsin remained constant at about 1.1:1 indicating that the same factor is responsible for both activities. The size of the inhibitor was found to be 24 000 daltons based on gel chromatographic studies on Sephadex G‐100 and by sodium dodecyl sulphate‐polyacrylamide gel electrophoresis. The molar ratio of interaction between the inhibitor and bovine trypsin for complete inactivation of the enzyme was found to be 1.04:1. Electrophoretic and gel chromatographic studies indicated that the purified inhibitor is capable of undergoing aggregation to form dimers and trimers. Even in the presence of sodium dodecyl sulphate and sodium dodecyl sulphateurea, this phenomenon was discernible. The binding sites on the inhibitor for trypsin and chymotrypsin were not mutually exclusive, based on the data from mixed enzyme studies and on analysis of the inhibitor‐enzyme complexes by gel chromatography on Sephadex G‐100. Modification of the arginyl residues of the inhibitor resulted in the loss of more of the antitryptic activity than of antichymotryptic activity. Conversely, modification of amino groups resulted in the loss of more of the antichymotryptic activity. The inhibitor was stable to exposure to a wide range of pH (1.0–12.0), but it was completely inactivated on heat‐treatment at 100°C for 15 min. The mode of inhibition of trypsin as well as chymotrypsin was non‐competitive and Ki values for the inhibitor were 2.92 × 10‐10M and 4.46 × 10‐10M, respectively, for the two enzymes.

EFFECT OF COOKING ON THE CHEMICAL COMPOSITION OF WINGED BEANS (Psophocarpus tetragonolobus)

ABSTRACTThe effect of soaking and cooking methods on the chemical composition, digestibility and antitryptic activity of winged beans was investigated. Water imbibition was rather slow with winged bean seeds. To obtain beans sufficiently tender for human consumption, prolonged soaking prior to cooking for 5 hr was found to be suitable. Short‐cooking time was ineffective as the beans were very hard. Prolonged cooking after a prolonged soaking period improved the flavor, increased tenderness, and increased the weight of cooked beans. Also, increases in percent protein and in vitro digestibility and reduction in the activity of trypsin inhibitor were observed. Cooking influenced the mineral content of the beans leading to significant losses in potassium and magnesium.

PROTEASE INHIBITORS IN SERUM AND AMNIOTIC FLUID DURING PREGNANCY

Antitryptic and antichymotryptic activities in amniotic fluid and maternal serum were measured at various stages of pregnancy using the caseinolytic assay method for proteases. The inhibitory activities increased up to 20 weeks in amniotic fluid and remained in the same range up to 30 weeks; a sharp fall was observed at term. In serum, there was a gradual rise of activities with peak values around 30 weeks. The decrease with further advance of pregnancy was not as sharp as in amniotic fluid. The ratios of antitryptic to antichymotryptic activities remained fairly constant (1.54 +/- 0.07) in amniotic fluid throughout pregnancy. In maternal serum the ratio varied over a range of 1.28 to 2.67 and the increase in antichymotryptic activity was relatively greater between 16 and 30 weeks resulting in a lower ratio between the two activities. Amniotic fluid was found to contain a higher proportion of a heat stable inhibitor compared to serum and its contribution to total antitryptic activity varied from 8.7 to 15.6 per cent.

Natural plant enzyme inhibitors. VI. Studies on trypsin inhibitors of Colocasia antiquorum tubers

A trypsin inhibitor was purified from the tubers of Colocasia antiquorum. The inhibitor acted on bovine trypsin, human trypsin and weakly on bovine chymotrypsin. The inhibitor, which had a molecular weight of 40 000, contained trace amounts of carbohydrates. The purified inhibitor was stable over a pH range of 2.0–12.0 and was more thermostable than the crude preparations. Trinitrobenzene sulphonate treatment resulted in the inactivation of the inhibitor. Chymotrypsin, pepsin and pronase digested the inhibitor. Pretreatment with trypsin at neutral pH resulted in the partial loss of antitryptic activity, whereas treatment at pH 3.7 led to complete inactivation. Evidence for the formation of a trypsin-inhibitor complex at pH 7.6 is provided. During the plant growth, in the early phase (0–40 days) there was a gradual increase in protein content and in antitryptic activity. The middle phase (40–55 days) was characterized by a rapid fall and abolition of the antitryptic activity and a diminution in protein content in the tubers. The immature tubers had low antitryptic activity compared to the mature ones. Mild heat treatment caused a sharp rise in antitryptic activity in the extracts of immature tubers but not with the mature tuber preparations.

Kunitz-Type Proteinase Inhibitors Derived by Limited Proteolysis of the Inter-α-Trypsin Inhibitor, III. Sequence of the two Kunitz-Type Domains inside the Native Inter-α-Trypsin Inhibitor, Its Biological Aspects and also of Its Cleavage Products

The human inhibitor HI-14 consists of two Kunitz-type domains covalently connected. They are liberated from the human ITI by limited tryptic proteolysis. The inhibitor HI-14 is formed via a trypsin inhibitor complex. We have reported the amino acid sequences of the domain with antitryptic activity and the homologous domain without activity. Here we present the sequence of the domains as present in ITI. The domain lacking antitryptic activity is the N-terminal part of the inhibitor HI-14, whereas the domain with antitryptic activity represents the C-terminal part of HI-14 and probably the C-terminus of the ITI-molecule, too.

Effect of Modification of Reactive Lysine on Antitryptic and Antichymotryptic Activity of Proteinase Inhibitor from Cow Colostrum

40% of the primary structure of the cow colostrum proteinase inhibitor (CTI) is homologous with the structure of the trypsin kallikrein inhibitor (TKI) from bovine organs; the positions of the reactive lysine residues are also the same in both inhibitors. Both CTI and TKI were modified by carbamoylation and the fully labeled derivatives were isolated by ion-exchange chromatography. The effect of the modification on the antitryptic and antichymotryptic activity of both inhibitors was investigated. The antichymotryptic activity of both inhibitors is not decreased after the modification. The antitryptic activity of modified TKI is retained, yet the dissociation constant of the complex of the modified inhibitor with trypsin is considerably increased; nevertheless, modified TKI is a good trypsin inhibitor. The antitryptic activity of modified CTI is hardly detectable. We explain this difference in the behaviour of both inhibitors by a replacement of basic residues Arg-17 and Arg-39 in TKI by neutral amino acids Ala-20 and Gln-42 in CTI.

Trypsin reactivity site of the Vicia angustifolia proteinase inhibitor.

Trypsin [EC 3.4.21.4] modified (reactive site cleaved) Vicia angustifolia proteinase inhibitor was prepared at pH 3 with a catalytic amount of trypsin and purified using columns of Sephadex G-50 and DEAE-Sephadex A-25. The modified inhibitor, which still retained antitryptic activity, lost its activity upon treatment with carboxypeptidase B or citraconic anhydride. End-group analyses revealed that the carboxyl-terminal Arg and the amino-terminal Ser residues were newly exposed end-groups in the modified inhibitor. It takes a much longer incubation time (about 1 h) to exhibit the maximal inhibitory activity against trypsin. Reduction and carboxymethylation of the modified inhibitor produced two fragments on Sephadex G-50 chromatography. The smaller fragment consisted of about 32 amino acid residues and possessed a new carboxyl-terminal Arg residue. The larger fragment consisted of about 80 residues and possessed a Ser residue at its amino-terminus. These results indicate that the small fragment was derived from the amino-terminal portion of the modified inhibitor and the large fragment from the carboxyl-terminal. It is also concluded that an Arg-Ser bond is the reactive site as well as the inhibitory site of the V. angustifolia inhibitor against trypsin. The sequence around the antitryptic site exhibits high degrees of homology with other double-headed inhibitors of legume origin, such as the Bowman-Birk inhibitor, lima beam inhibitor, and the major inhibitor in chick-peas.

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

Measurement of antitryptic activity of serum with a centrifugal analyzer.

The Selected Method [Clin. Chem. 20, 396 (1974)] for the enzymatic assay of alpha1-antitrypsin in serum has been adapted for use with the E.N.I.-GEMSAEC. With alpha-N-benzoyl-D,L-arginine-p-nitroanilide as substrate, the difference between the tryptic activity measured with and without addition of serum in the same run has been used to calculate the trypsin-inhibitory capacity. The rate of increase in absorbance at 400 nm of the p-nitroanilide formed, has been evaluated during a reaction time of 140 s. Results correlated well (r = 0.986) for 54 human sera analyzed as described here and by the Selected Method. The adaptation on GEMSAEC can be used in detecting alpha1-antitrypsin deficiency in the newborn.

Human chorionic gonadotropin: Doubtful role as an inhibitor of cell-mediated immunity

HCG preparations from several commercial sources were tested for their ability to inhibit mitogen-induced lymphocyte blastogenesis. Proven biologically active preparations were found to vary widely in their ability to suppress PHA response, in their toxicity to lymphocytes, and in their anti-tryptic activity. Since high concentrations of a low toxicity, low trypsin inhibitor-containing preparation did not suppress blastogenesis, we conclude that previous reports of HCG being a potent inhibitor of CMI are erroneous and the effect observed by others probably due to contaminants of the HCG preparations used in their assay systems. Our findings cast doubt on the validity of the hypothesis that HCG is a potent inhibitor of CMI and therefore of importance in the role of maternal tolerance to the fetal allograft and in escape of HCG-secreting tumors from the host immune system.

75] Acid-stable proteinse inhibitors from human seminal plasma

Antitryptic activity in human seminal plasma was first detected by Rasmussen and Albrechtson. Haendle et al. described the occurrence of acid-stable trypsin inhibitors in testes, epididymis, and seminal vesicles as well as in the seminal plasma of many mammals, including man. Fink et al. and Suominen and Niemi showed that the antitryptic activity in human seminal plasma is due to two different trypsin inhibitots, the human seminal plasma inhibitors (HUSI) I and II. HUSI-I, trypsin-chymotrypsin inhibitor, and HUSI-II, a trypsin-acrosin inhibitot, were further characterized in laboratory. α-Antitrypsin, an acid-unstable glycoprotein, is also present in human seminal plasma. Trypsin inhibitors isolated from human spermatozoa show very similar characteristics to those of HUSI-I and HUSI-II. This chapter discusses the assay methods like: Trypsin Inhibition, Chymotrypsin Inhibition and Acrosin Inhibition. It also discusses the purification procedure and properties.