Purification and characterization of proteinase inhibitors from adzuki beans (Phaseolus angularis).

Two proteinase inhibitors, designated as inhibitors I and II, were purified from adzuki beans (Phaseolus angularis) by chromatographies on DEAE- and CM-cellulose, and gel filtration on a Sephadex G-100 column. Each inhibitor shows unique inhibitory activities. Inhibitor I was a powerful inhibitor of trypsin [EC 3.4.21.4], but essentially not of chymotrypsin ]EC 3.4.21.1]. On the other hand, inhibitor II inhibited chymotrypsin more strongly than trypsin. The molecular weights estimated from the enzyme inhibition were 3,750 and 9,700 for inhibitors I and II, respectively, assuming that the inhibitions were stoichiometric and in 1 : 1 molar ratio. The amino acid compositions of both inhibitors closely resemble those of low molecular weight inhibitors of other leguminous seeds: they contain large amounts of half-cystine, aspartic acid and serine, and little or no hydrophobic and aromatic amino acids. Inhibitor I lacks both tyrosine and tryptophan residues. The molecular weights were calculated to be 7,894 and 8,620 for inhibitors I and II, respectively. The reliability of these molecular weights was confirmed by the sedimentation equilibrium and 6 M guanidine gel filtration methods. On comparison with the values obtained from enzyme inhibition, it was concluded that inhibitor I and two trypsin inhibitory sites on the molecule, whereas inhibitor II had one chymotrypsin and one trypsin inhibitory sites on the molecule.     

Characterization of the proteinase inhibitors from bull seminal plasma and spermatozoa.

Two acid stable proteinase inhibitors are present in bull seminal plasma and washed ejaculated bull spermatozoa. Inhibitor I with a molecular weight of about 8700 (estimated by gel filtration) is a very strong inhibitor of bull sperm acrosin but also inhibits bovine trypsin and chymotrypsin and porcine plasmin; inhibition of porcine pancreatic and urinary kallikrein was not observed. In this respect inhibitor I resembles the well known cow colostrum trypsin inhibitor. Inhibitor II with a molecular weight near 6800 (estimated by gel filtration) inhibits bovine trypsin and chymotrypsin, porcine plasmin and pancreatic and urinary kallikrein as well as bull acrosin. The inhibition specificity of inhibitor II is thus very similar to that of the basic inhibitor from bovine organs (Kunitz-type). In view of the inhibition strength and other characteristics, however, the acid stable bull seminal inhibitors are not identical with the inhibitor from cow colostrum or bovine lung (organs).     

Trypsin inhibitor in the hemolymph of a solitary ascidian, Halocynthia roretzi. Purification and characterization

A purified preparation of trypsin inhibitor was obtained from the hemolymph of a solitary ascidian, Halocynthia roretzi, by a procedure including trypsin-Sepharose chromatography, DEAE-cellulose chromatography, and Sephadex G-50 gel filtration. The product was a mixture of two isoinhibitors, inhibitors I and II. They were separated from each other by high-performance liquid chromatography on an anion exchanger column, and showed almost identical amino acid compositions. They were also indistinguishable in terms of apparent specific inhibitory activity against bovine trypsin when the activity was assayed with the inhibitors at rather high concentrations (greater than 50 nM). A large difference was observed between them, however, in the inhibition constants, which correspond to the dissociation constants of the inhibitor-trypsin complexes; the inhibition constant of inhibitor I was 90 pM, whereas that of inhibitor II was 4.7 nM. The molecular weights of inhibitors I and II were estimated to be 6,000 and 4,500, respectively, by SDS-polyacrylamide gel electrophoresis, while an almost identical value, 9,000, was obtained for both of them by gel filtration. The molecular weight calculated from the amino acid compositions was 5,929 for both. The isoelectric points were also identical, that is about 5.0. Both of the inhibitors were heat-stable. Ascidian inhibitor I also inhibited other trypsin-like enzymes of mammalian origin, as well as those of ascidian origin.     

Purification, partial characterization, and immunological relationships of multiple low molecular weight protease inhibitors of soybean.

Five protease inhibitors, I--V, in the molecular weight range 7000--8000 were purified from Tracy soybeans by ammonium sulfate precipitation, gel filtration on Sephadex G-100 and G-75, and column chromatography on DEAE-cellulose. In common with previously described trypsin inhibitors from legumes, I--V have a high content of half-cystine and lack tryptophan. By contrast with other legume inhibitors, inhibitor II contains 3 methionine residues. Isoelectric points range from 6.2 to 4.2 in order from inhibitor I to V. Molar ratios (inhibitor/enzyme) for 50% trypsin inhibition are I = 4.76, II = 1.32, III = 3.22, IV = 2.17, V = 0.97. Only V inhibit chymotrypsin significantly (molar ratio = 1.33 for 50% inhibition). The sequence of the first 16 N-terminal amino acid residued of inhibitor V is identical to that of the Bowman-Birk inhibitor; all other observations also indicate that inhibitor V and Bowman-Birk are identical. The first 20 N-terminal amino acid residues of inhibitor II show high homology to those of Bowman-Birk inhibitor, differing by 1 deletion and 5 substitutions. Immunological tests show that inhibitors I through IV are fully cross-reactive with each other but are distinct from inhibitor V.     

Trypsin and chymotrypsin inhibitors from viper venom

Inhibitors of trypsin and alpha-chymotrypsin with Mr of about 7000 Da and isoelectric points of greater than 10 and 9.9, respectively, were isolated from the venom of the common viper Vipera berus berus, using gel filtration and ion exchange chromatography. The inhibitor I prefers alpha-chymotrypsin (Ki = 4.6 X 10(-10) M) for the formation of an enzymeinhibitor complex at a molar ratio of 1:1. The inhibitor II prefers trypsin (Ki = 6.7 X 10(-11) M), forms an EI-complex at a molar ratio of 1:2, but also inhibits alpha-chymotrypsin (Ki = 1.4 X 10(-9) M) and hog pancreatic kallikrein (Ki = 1.6 X 10(-8) M). The inhibitor II contains no valine or methionine.     

Low molecular weight serine proteinase inhibitors of the human intervertebral disc

Human lumbar disc tissue when extracted with 4M GuHCl and subjected to dissociative CsCl density gradient ultracentrifugation yielded trypsin inhibitor activity in the low bouyant density fractions (rho less than or equal to 1.38 g/ml). Disc proteoglycans sedimented in the high bouyant density fractions (rho greater than or equal to 1.5 g/ml). Sephadex G75F gel filtration of the low bouyant density protein fractions afforded a major low molecular weight (Kav = 0.5) trypsin inhibitor pool which was further purified by trypsin affinity chromatography. This latter step facilitated separation of the trypsin inhibitors from neutral proteinase activity also present. The trypsin inhibitor fraction so isolated was shown to possess potent inhibitory activity against a range of human serine proteinases including leukocyte elastase and cathepsin G, urokinase, kallikrein, plasmin and thrombin. Significantly this serine proteinase inhibitor preparation effectively prevented degradation of proteoglycans by a neutral proteinase also isolated from the human intervertebral disc.     

Purification and characterization of trypsin inhibitor from seeds of faba bean (Vicia faba L.)

A trypsin inhibitor from seeds of faba bean (Vicia faba L.) was purified to near homogeneity as judged by native-PAGE with about 11 % recovery using ammonium sulphate fractionation, ion-exchange chromatography on DEAE-cellulose and gel filtration through Sephadex G-100. The inhibitor had a molecular weight of 18 kD as determined by SDS-PAGE and Sephadex G-100. The inhibitor inhibited trypsin and chymotrypsin to the extent of 48 and 12 %, respectively. The inhibtion was of non-competitive type with dissociation constant for the enzyme inhibitor complex in the region of 0.07 mg·ml⁻¹. The inhibtor was stable between pH 4 and 5. It completely lost its activity when heated at 125 °C for 1 h or at 100 °C for 2 h. The inhibitor also lost its activity on exposure to 2-mercaptoethanol. Based on these properties, it could be concluded that Vicia faba trypsin inhibitor belongs to Bowman-Birk type of inhibitors, as it has molecular weight lower than generally observed for Kunitz type inhibitors.     

Isolation, expression and characterization of a novel dual serine protease inhibitor, OH-TCI, from king cobra venom

Snake venom Kunitz/BPTI members are good tools for understanding of structure-functional relationship between serine proteases and their inhibitors. A novel dual Kunitz/BPTI serine proteinase inhibitor named OH-TCI (trypsin- and chymotrypsin-dual inhibitor from Ophiophagus hannah) was isolated from king cobra venom by three chromatographic steps of gel filtration, trypsin affinity and reverse phase HPLC. OH-TCI is composed of 58 amino acid residues with a molecular mass of 6339Da. Successful expression of OH-TCI was performed as the maltose-binding fusion protein in E. coli DH5alpha. Much different from Oh11-1, the purified native and recombinant OH-TCI both had strong inhibitory activities against trypsin and chymotrypsin although the sequence identity (74.1%) between them is very high. The inhibitor constants (K(i)) of recombinant OH-TCI were 3.91 x 10(-7) and 8.46 x10(-8)M for trypsin and chymotrypsin, respectively. To our knowledge, it was the first report of Kunitz/BPTI serine proteinase inhibitor from snake venom that had equivalent trypsin and chymotrypsin inhibitory activities.     

Proteolysis of the protein inhibitor of calcium-dependent proteases produces lower molecular weight fragments that retain inhibitory activity

The specific inhibitor of calcium-dependent proteases was purified from soluble extracts of bovine heart. The protein had a molecular weight of 125,000 on sodium dodecyl sulfate polyacrylamide gels and migrated on gel filtration chromatography with an apparent molecular weight of 250,000. The inhibitor specifically blocked the action of the two calcium-dependent proteases, CDP-I and CDP-II, but did not influence a variety of other proteases including trypsin, chymotrypsin, or Staphylococcus aureus V8 protease. These latter enzymes extensively degraded the inhibitor to discrete lower molecular weight peptides as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and by gel filtration chromatography. Under the conditions studied, proteolysis of the inhibitor had little or no effect on its inhibitory activity; isolated peptides with molecular weights as low as 17,000 retained inhibitory function. A number of various-sized inhibitor fragments were isolated by gel filtration chromatography and by SDS-PAGE. These fragments were compared with the intact inhibitor for their ability to inhibit CDPs. As suggested previously by us and others, one molecule of intact inhibitor appears to inhibit up to five molecules of calcium-dependent protease. The inhibitor fragments of decreasing size inhibited correspondingly fewer molecules of protease. These results suggest that the inhibitor protein contains multiple functional domains and may explain some of the discrepancies in reported molecular weights for this protein.     

Isolation and characterization of trypsin inhibitors from cowpea (Vigna unguiculata)

The trypsin inhibitor fraction from cowpea (Vigna unguiculata) has been purified and characterized. Although the total trypsin inhibitor as purified by affinity chromatography on immobilised trypsin was shown to be heterogeneous by gel electrophoresis and isoelectric focusing as well as by function, it was relatively homogeneous in MW (ca 17 000) on gel filtration. The total trypsin inhibitor was divided into inhibitors active against trypsin only and active against trypsin and chymotrypsin by affinity chromatography on immobilised chymotrypsin. The ‘trypsin-only’ inhibitor was the major component of the total trypsin inhibitor. It was shown by isoelectric focusing and gel electrophoresis to contain several isoinhibitors. Determination of the combining weight of this inhibitor and investigation of the complexes formed with trypsin by gel filtration indicated the presence of two protease binding sites per inhibitor molecule. The chymotrypsin/trypsin inhibitor was also shown to be composed of several isoinhibitors. On the basis of gel electrophoresis and gel filtration in dissociating and non-dissociating media both inhibitors were considered to be dimeric molecules with the subunits linked by disulphide bonds; this implies that the ‘trypsin-only’ inhibitor has one binding site per subunit.     

Proteinase inhibitors from the excretory gland cells of Stephanurus dentatus. Purification and properties of three secretory proteinase inhibitors

Three proteinase inhibitors designated as I, II, and III were isolated from the excretory gland cells of the swine kidney worm, Stephanurus dentatus. The inhibitors, which were trichloroacetic acid-soluble, were purified by affinity chromatography and ion exchange chromatography. The homogeneity of each inhibitor was shown by polyacrylamide gel electrophoresis and electrofocusing. The molecular weights of the inhibitors estimated by sodium dodecyl sulfate gel electrophoresis fell within a limited range of 9300 to 9700, and the isoelectric points were 6.45, 6.20, and 5.34 for Inhibitors I, II, and III, respectively. The inhibitors formed complexes with trypsin having apparent dissociation constants (Ki) of 2.9 X 10(-11), 7.6 X 10(-11), and 6.4 X 10(-11) M, respectively. Each inhibitor inhibits the esterolytic and proteolytic activities of both trypsin and chymotrypsin. A proteinase inhibitor present in the reproductive organs, intestines, body walls, and esophagi was identical with Inhibitor II found in the excretory gland cells. Culture medium collected after 24-h incubation with adult worms contained the same three inhibitors as the excretory gland cells. These data suggest that the gland cells may secrete the inhibitors internally and externally.     

Purification and characterization of a new trypsin inhibitor from Dimorphandra mollis seeds

A second trypsin inhibitor (DMTI-II) was purified from the seed of Dimorphandra mollis (Leguminosae-Mimosoideae) by ammonium sulfate precipitation (30–60%), gel filtration, and ion-exchange and affinity chromatography. A molecular weight of 23 kDa was estimated by gel filtration on a Superdex 75 column SDS-PAGE under reduced conditions showed that DMTI-II consisted of a single polypeptide chain, although isoelectric focusing revealed the presence of three isoforms. The dissociation constant of 1.7 × 10^–9 M with bovine trypsin indicated a high affinity between the inhibitor and this enzyme. The inhibitory activity was stable over a wide pH range and in the presence of DTT. The N-terminal sequence of DMTI-II showed a high degree of homology with other Kunitz-type inhibitors.     

Protease inhibitors from the parasitic worm Parascaris equorum

Two proteic inhibitors (I and II) of serine proteases have been purified from the parasitic worm Parascaris equorum by affinity chromatography on immobilized trypsin followed by preparative electrophoresis. They have an apparent relative molecular mass of 9000 and 7000 as determined by gel filtration, a slightly acid isoelectric point (5.5 and 6.1) and a similar amino acid composition. Both inhibitors lack serine, methionine and tyrosine. They bind bovine trypsin extremely strongly with an association constant, Ka, larger than 10(9) M-1, and form a 1:1 complex with this protease. The Ka values for the binding to bovine chymotrypsin are approximately 3.3 X 10(8) M-1 (inhibitor I) and approximately 2 X 10(6) M-1 (inhibitor II). Inhibitor I interacts also with porcine elastase (Ka approximately 5 X 10(7) M-1), while inhibitor II is inactive towards this enzyme.     

Purification and characterization of a trypsin inhibitor from seeds of Murraya koenigii

A protein with trypsin inhibitory activity was purified to homogeneity from the seeds of Murraya koenigii (curry leaf tree) by ion exchange chromatography and gel filtration chromatography on HPLC. The molecular mass of the protein was determined to be 27 kDa by SDS-PAGE analysis under reducing conditions. The solubility studies at different pH conditions showed that it is completely soluble at and above pH 7.5 and slowly precipitates below this pH at a protein concentration of 1 mg/ml. The purified protein inhibited bovine pancreatic trypsin completely in a molar ratio of 1:1.1. Maximum inhibition was observed at pH 8.0. Kinetic studies showed that Murraya koenigii trypsin inhibitor is a competitive inhibitor with an equilibrium dissociation constant of 7 x 10(-9) M. The N-terminal sequence of the first 15 amino acids showed no similarity with any of the known trypsin inhibitors, however, a short sequence search showed significant homology to a Kunitz-type chymotrypsin inhibitor from Erythrina variegata.     

Trypsin inhibitor from Dimorphandra mollis seeds: purification and properties

A trypsin inhibitor from Dimorphandra mollis seeds was isolated to apparent homogeneity by a combination of ammonium sulfate precipitation, gel filtration, ion-exchange and affinity chromatographic techniques. SDS-PAGE analysis gave an apparent molecular weight of 20 kDa, and isoelectric focusing analysis demonstrated the presence of three isoforms. The partial N-terminal amino acid sequence of the purified protein showed a high degree of homology with various members of the Kunitz family of inhibitors. This inhibitor, which inhibited trypsin activity with a Ki of 5.3 x 10(-10) M, is formed by a single polypeptide chain with an arginine residue in the reactive site.     

Demonstration of a new acrosin inhibitor in human seminal plasma

We have recently described the purification and characterization of a tumor-associated trypsin inhibitor (TATI). Studies on its N-terminal sequence suggested identity with the pancreatic secretory trypsin inhibitor (PSTI) (Huhtala, M.-L., Pesonen, K., Kalkkinen, N. & Stenman, U.-H. (1982) J. Biol. Chem. 257, 13713-13716). I report here the occurrence of a TATI-like activity in human seminal plasma. Concentrations of this inhibitor in seminal plasma varied considerably (4-500 ng/ml, n = 50). In radioimmunoassay the dose-response curves of the new seminal plasma inhibitor and purified TATI were parallel. The similarity between these two inhibitors was demonstrated by gel filtration, reverse phase liquid chromatography and ion-exchange chromatography. By ion exchange chromatography the new inhibitor could be separated from the main seminal plasma trypsin inhibitors. Purified TATI was shown to inhibit human acrosin effectively.     

A serine proteinase inhibitor from frog eggs with bacteriostatic activity

By Sephadex G-50 gel filtration, Resource Q anionic exchange and C4 reversed phase liquid high performance liquid chromatography, a proteinase inhibitor protein (Ranaserpin) was identified and purified from the eggs of the odour frog, Rana grahami. The protein displayed a single band adjacent to the molecular weight marker of 14.4 kDa analyzed by SDS-PAGE. The inhibitor protein homogeneity and its molecular weight were confirmed again by MALDI-TOF mass spectrometry analysis. The MALDI-TOF mass spectrum analysis gave this inhibitor protein an m/z of 14422.26 that was matched well with the result from SDS-PAGE. This protein is a serine proteinase inhibitor targeting multiple proteinases including trypsin, elastase, and subtilisin. Ranaserpin inhibited the proteolytic activities of trypsin, elastase, and subtilisin. It has an inhibitory constant (K(i)) of 6.2 x 10(-8) M, 2.7 x 10(-7) M and 2.2 x 10(-8) M for trypsin, elastase, and subtilisin, respectively. This serine proteinase inhibitor exhibited bacteriostatic effect on Gram-positive bacteria Bacillus subtilis (ATCC 6633). It was suggested that ranaserpin might act as a defensive role in resistance to invasion of pests or pathogens. This is the first report of serine proteinase inhibitor and its direct defensive role from amphibian eggs.     

A crystalline protein-proteinase inhibitor from pinto bean seeds.

A crystalline protein-proteinase inhibitor has been isolated from seeds of Pinto bean (Phaseolus vulgaris cultvar. Pinto). It has an average molecular weight of 19 000 as estimated by gel filtration. This crystalline inhibitor is highly active against both bovine pancreatic trypsin and alpha-chymotrypsin. Complexes of both trypsin-inhibitor and alpha-chymotrypsin-inhibitor have been isolated. The inhibitor which was derived from the dissociated trypsin-inhibitor complex was only 62% as effective as the original compound against either enzyme. In contrast, the inhibitor obtained from alpha-chymotrypsin-inhibitor complex retained its full original inhibitory activity for trypsin, but only 25% of its original activity against alpha-chymotrypsin. The dissociated inhibitor from alpha-chymotrypsin-inhibitor compex, despite its full inhibitory activity, had been modified to such an extent that it could no longer form any precipitable complex with trypsin. The crystalline protein-proteinase inhibitor is not homogeneous and has been resolved into two distinct inhibitors in terms of their physical and chemical properties. These two inhibitors are designated as Pinto bean proteinase inhibitor I and II and their respective minimum molecular weights are 9100 and 10 000. They differ most strikingly in their amino acid composition in that inhibitor II is void of both valine and methionine.     

Studies on a Doubleheaded Protease Inhibitor from Phaseolus mungo

A doubleheaded protease inhibitor showing inhibition of bovine pancreatic trypsin and α-chymotrypsin was isolated and purified from the seeds of Phaseolus mungo. The molecular weight of the protease inhibitor was found to be 14.2 kD by SDS-PAGE analysis and gel filtration. The native inhibitor inhibited trypsin and α-chymotrypsin stoichiometrically at the molar ratio 1:1 and 2:1 respectively. The Ki app for trypsin was found to be 0.35 nM and for α-chymotrypsin to be 2.4 nM. Bovine pepsin was not inhibited by the inhibitor. However, the pepsin treated inhibitor was still able to inhibit trypsin and α-chymotrypsin. The inhibitor was stable in 8M urea. Addition of 0.2 M mercaptoethanol resulted in significant loss of inhibitory activity. The inhibitor was extremely heat stable with only 50% loss of inhibitory activity after heating for 100°C for 20 min. Thus, the Phaseolus mungo trypsin/chymotrypsin inhibitor resembles other Bowman-Birk protease inhibitors.     

Acid-stable trypsin-plasmin inhibitors formed enzymatically from plasma precursor protein

Enzymatic formation of acid-stable trypsin-plasmin inhibitors (ASTPIs) in human plasma with several proteinases, particularly SH-proteinases, was demonstrated. The maximal activity obtained with bromelain was 40 U/ml plasma, which corresponded to about a 10-fold increase as compared to the untreated control plasma (4.2 U/ml). Gel filtration revealed at least two ASTPI activity peaks of molecular weight 16,000 (main peak) and 8000 (minor peak). The main ASTPI was further purified by trypsin-Sepharose affinity chromatography, isoelectric focusing and gel filtration on Sephadex G-75 superfine. The purified inhibitor was found to be identical to the active fragment of plasma ASTPI or urinary trypsin inhibitor (UTI) formed by bromelain treatment. It had an isoelectric point (pI) of 3.7, a molecular weight of 16,000 by SDS-polyacrylamide gel electrophoresis and was a glycine- and glutamic acid-rich protein lacking histidine. The NH2-terminal amino acid sequence was H2N-(Lys)-Glu-Asp-Ser-X-Gln-Leu-Gly-Tyr-Ser-Ala-Gly-Pro-X-Met-Gly-Met-Th r-X-Arg - Tyr-Phe-Tyr-... COOH, which was homologous to the Lys22-Met36 part (or Glu23-Met36 part; 30% of the total) of the plasma ASTPI or UTI molecule (molecular weight 70,000-80,000 by gel filtration). The purified ASTPI displayed the same antigenicity as UTI and exerted strong inhibitory effects on trypsin, chymotrypsin and plasmin amidolysis, but had a much lesser effect on plasmin fibrinolysis. It also strongly inhibited non-plasmic fibrinolysis with human leukocyte proteinase and earthworm proteinase.