Purification and characterization of a trypsin inhibitor from Solanum tuberosum.

A trypsin inhibitor isolated from a potato acetone powder has been purified by affinity chromatography. This protein inhibits trypsin mole per mole. To a lesser extent it combines also with chymotrypsin and elastase. For trypsin, K1 = 8 X 10(-7) M. The inhibitor has a single polypeptide chain of 207 amino acid residues. It contains no sugar or free sulfhydryl groups. Its extinction coefficient E2801% = 10.3 and its isoelectric point is 6.9. Its molecular weight is of the order of 21 000-22000, as determined by sedimentation equilbrium, by inhibition experiment or from its amino acid composition. These same techniques, taken together with the single band observed at different pH on polyacrylamide gel electrophoresis, indicate that the protein purified is monodisperse. However, the finding of two N-terminal amino acid residues, leucine and aspartic acid, and the different stoichometry observed during the interaction of the inhibitor, either with trypsin or with chymotrypsin and elastase, raises the possibility that our preparation is contaminated by a polyvalent inhibitor not detectable by physiochemical methods.     

Protein inhibitors of trypsin from the seeds of Cucurbitaceae plants

Seven trypsin inhibitors were isolated from the seeds of Cucurbitaceae plants: two from cucumber (Cucumis sativus) and red bryony (Bryonia diotica) and one from figleaf gourd (Cucurbita ficifolia), spaghetti squash (Cucurbita pepo var. (vegetable spaghetti) and water melon (Citrullus vulgaris). The inhibitors were purified by fractionation with ammonium sulphate, followed by ion-exchange chromatography and affinity chromatography using immobilized trypsin or anhydro-trypsin. The homogeneous inhibitors from cucumber and water melon are made up of 32 and 30 amino acid residues, respectively, whereas the remaining ones of 29 residues. All inhibitors contain three disulphide bridges and are free of threonine, phenylalanine and tryptophan. Inhibitors from spaghetti squash and CSTI IIb from cucumber are inactivated by acetylation of free amino groups whereas the remaining ones are inactivated by modification of arginine with 1,2-cyclohexanedione. Thus the P1 residues of the reactive sites of the inhibitors are lysine and arginine, respectively.     

On the activation of bovine chymotrypsinogen A. Preparation of alanine-neochymotrypsinogen and its activation to alpha-chymotrypsin

Alanine-neochymotrypsinogen was prepared by incubating 20 parts bovine pancreas chymotrypsinogen A with one part alpha-chymotrypsin in a solution containing 1 M (NH4)2SO4, 0.1 M sodium acetate, 0.05 M Tris buffer (pH 8.0) and 0.5 mg/ml soybean trypsin inhibitor. Optimal yields of NH2-terminal alanine were obtained after 60 h incubation at 4 degrees C. Ala-neochymotrypsinogen was isolated from the reaction mixture by affinity chromatography and ion-exchange chromatography on carboxymethyl-cellulose. As expected, the purified preparation was enzymatically inactive and, compared to chymotrypsinogen, had one additional NH2-terminal group identified as alanine. Ala-neochymotrypsinogen was activated by incubating with trypsin at a zymogen : trypsin ratio of 30 : 1 in 0.1 M phosphate buffer, pH 7.6 at 4 degrees C for 1 h. The fully active, stable species was identified as alpha-chymotrypsin.     

Purification and some properties of a low molecular weight trypsin inhibitor from acute pancreatitis urine

Urinary trypsin inhibitors (UTIs) from the urine of a patient with acute pancreatitis consisted of three forms with different molecular weights. These were highly purified by ammonium sulfate precipitation, Sephadex G-75, SP-Sephadex C-25 and trypsin-Sepharose 4B column chromatography. The lowest molecular weight of UTIs was estimated to be 6,200 daltons. Moreover, five residues of N-terminal amino acids and a C-terminal amino acid were the same as those of pancreatic secretory trypsin inhibitor.     

Isolation and characteristics of trypsin from pyloric ceca of the starfish Asterina pectinifera

Trypsin was purified from pyloric ceca of the starfish Asterina Pectinifera by ammonium sulfate precipitation, gel filtration, and cation-exchange chromatography. Final enzyme preparation was nearly homogeneous in sodium dodecyl sulfate-polyacrylamide gel electrophoresis and its molecular weight was estimated as approximately 28000. Optimum pH and temperature of A. pectinifera trypsin for hydrolysis of N(alpha)-p-Tosyl-L-arginine methyl ester hydrochloride were approximately pH 8.0 and 55 degrees C, respectively. A. pectinifera trypsin was unstable at above 50 degrees C and below pH 5.0, and was not activated by adding Ca(2+). The N-terminal amino acid sequence of A. pectinifera trypsin, IVGGHEF, was found.     

The isolation and partial characterization of trypsin from the pancreas of the ostrich Struthio camelus

Cationic trypsin was isolated and purified from the pancreas of the ostrich (Struthio camelus) by affinity chromatography on a Trasylol-Sepharose column. External activation of trypsinogen was required before trypsin could be isolated. The final preparation was homogeneous by SDS-PAGE and by sedimentation equilibrium centrifugation studies, resulting in Mr values of 24,547 and 22,091, respectively. The Mmin value obtained from amino acid analysis was 22,450. A mean sedimentation coefficient of 2S was obtained by sedimentation velocity centrifugation. Results obtained from N-terminal and amino acid analyses were similar to those from trypsins of other species. The effects of pH, temperature and inhibitors (LBTI, KBPTI and PMSF) on the tryptic activity were examined. The effect of calcium ions and enzyme concentration on the rate of self-digestion of ostrich trypsin was also investigated.     

Amino acid sequence of crayfish (Astacus fluviatilis) trypsin If

The complete amino acid sequence of trypsin from the crayfish Astacus fluviatilis has been determined. The protein was fragmented with cyanogen bromide after S-carboxymethylation of the reduced disulfide bonds and by trypsin after S-carboxymethylation as well as after succinylation of lysine residues and aminoethylation of the reduced disulfide bonds. Peptides were purified by gel filtration and by reversed-phase high-performance liquid chromatography. Stepwise degradation was performed in a spinning cup sequencer. The enzyme contains 237 amino acid residues and has a molecular weight of 25 030. In contrast to bovine trypsin, it contains three rather than six disulfide bonds which are paired in the same fashion as those in trypsin from Streptomyces griseus. The constituents of the active site of bovine trypsin are present in corresponding positions in the crayfish enzyme. Crayfish trypsin shows 43.6% sequence identity with the bovine enzyme as compared to 40.0% identity with the S. griseus enzyme. The present analysis affords the first detailed view into the evolution of trypsins at the invertebrate level.     

Kinetic and structural properties of two isoforms of trypsin isolated from the viscera of Japanese anchovy, Engraulis japonicus

Two isoforms of anchovy trypsin (aT-I and aT-II) were purified from the visceral extracts by (NH₄)₂SO₄ fractionation followed by affinity chromatography, gel filtration, and ion-exchange chromatography. The homogeneity of the purified preparation was evidenced by both native- and SDS-PAGE, and further by gelatin zymography. Identities of aT-I and aT-II as trypsins were established by N-terminal amino acid sequencing, which matched exactly to the corresponding stretches of their respective amino acid sequences obtained by molecular cloning [Ahsan et al. (2000), Marine Biotechnol., in press]. Both isoforms were completely inhibited by serine protease inhibitors as well as by specific trypsin inhibitors. The purified anchovy trypsins showed considerably higher catalytic efficiencies (k_cat/K_m) than bovine trypsin as measured toward benzoyl-arginine p-nitroanilide (BAPA) and benzoyl-arginine ethyl ester (BAEE) at 25°C; in particular, aT-II was 35 times more efficient than its mammalian counterpart against BAPA. This was due mainly to a dramatic decrease of K_m values for anchovy trypsins, which are indicative of an evolutionary response toward increased substrate binding at suboptimal temperatures in the marine environment.     

Isolation and partial characterization of the trypsin inhibitor from the seeds of Brassica oleracea var. sabellica

A trypsin inhibitor was extracted from the kale seeds with 0.01 M-HCl, precipitated with ammonium sulphate, and purified by affinity chromatography on immobilized trypsin and ion-exchange chromatography on QAE-Sephadex A-25. The inhibitor, of Mr 8 000, is composed of 64 amino acid residues and contains neither threonine nor methionine. Its isoelectric point is 8.9. In addition to trypsin, the inhibitor acts on subtilopeptidase A and shows a very weak antichymotrypsin activity. The factors modifying the arginine residues inactivate the inhibitor. A modified form of the inhibitor (with a broken reactive site peptide bond) has been isolated in pure form, and its properties were compared with those of the virgin form.     

Studies on trypsin inhibitors. Part IX. Synthesis and trypsin inhibitory activity of the duopentacontapeptide corresponding to the amino acid sequence of porcine pancreatic secretory trypsin inhibitor II (Kazal).

The synthesis of the protected duopentacontapeptide corresponding to the entire amino acid sequence I-52 of porcine pancreatic secretory trypsin inhibitor II (Kazal type) is described. The benzyloxycarbonyltetradecapeptide tert-butyloxycarbonylhydrazide (sequence 1-14) was selectively deblocked with trifluoroacetic acid and used to acylate, by the azide procedure, the peptide free base corresponding to the sequence 15-52. The isolated material was purified by ion exchange chromatography and the protecting groups were removed by successive treatments with anhydrous hydrogen fluoride, 1 M piperidine and mercuric acetate. F02M phosphate buffer, pH8. Determination of the inhibitory capacity indicated that the synthetic material is about 50% effective, at 30:1 inhibitor:trypsin molar ratio in inhibiting the tryptic hydrolysis of Nalpha-benzoyl-DL-arginine-4-nitroanilide. Full inhibition was achieved at a higher inhibitor:trypsin molar ratio. The stability constants and the standard free energy of binding of the complex between trypsin and the synthetic inhibitor have been determined.     

The Preparation of Trypsins and Chymotrypsins From Bovine and Porcine Residues After Insulin Extraction

Bovine and porcine pancreatic residue, remaining after the extraction of insulin, has been used to prepare a proteinase powder. This powder was used as a source of trypsin and chymo-trypsin. The individual enzymes were isolated and purified by chromatography on sulfopropyl (SP)-Sephadex C-25 and affinity chromatography on soybean trypsin inhibitor (STI)-Sepharose. The bovine proteinase powder contained a-chymotrypsin, trypsin and chymotrypsin B in the ratio 5:2:1. The porcine powder contained cationic trypsin, anionic trypsin and cationic chymotrypsin in the ratio 5 : 1. 4 : 3. The isolated enzymes were characterized and found to be identical with enzymes isolated from fresh tissue with the exception of porcine chymotrypsin. Porcine cationic chymotrypsin was isolated as two distinct forms, A-l and A-2, which appear to be different activation products of porcine chymotrypsinogen A. Both forms resemble bovine a-chymotrypsin, a three chain structure, rather than porcine chymo-trypsin A, a two chain structure. Furthermore, the B-chain appears to be cleaved, possibly at residues Phe₈₉-Lys₉₀.     

Purification, characterization, and cDNA cloning of a Bowman-Birk type trypsin inhibitor from Apios americana Medikus tubers

An Apios americana trypsin inhibitor, AATI, was purified from Apios tubers by chromatography on DEAE Cellulofine A-500 and Sephadex G-50. The molecular mass of AATI was determined to be 6,437 Da by matrix-assisted laser desorption and ionization time-of-flight mass spectrometer (MALDI-TOF-MS). It showed strong inhibitory activity toward serine proteases, and the inhibition constants toward trypsin and chymotrypsin were 3.0 x 10(-9) M and 1.0 x 10(-6) M respectively. The inhibitory activity was not affected by heating at 80 degrees C for 2 h or by incubation at a wide range of pH values, suggesting that AATI has remarkable heat-stability and pH-stability. AATI cDNA consists of 552 nucleotides, and includes an open reading frame encoding a protein of 116 amino acids. The results of N-terminal amino acid sequencing of AATI and MALDI-TOF-MS analysis suggested that the deduced amino acid sequence had 50 and seven extra amino acids at the N- and C-termini respectively. Thus the mature AATI protein consists of 59 amino acid residues. Comparison of the amino acid sequence with those of the trypsin inhibitors from plants suggests that AATI belongs to the Bowman-Birk family and that it contains two possible reactive sites toward trypsin at Lys62 and Arg88.     

Characterization of a trypsin inhibitor from equine urine.

A trypsin inhibitor was isolated from pregnant mares' urine by adsorption on bentonite and elution with aqueous pyridine followed by batch DEAE-cellulose treatment and column chromatography. Final purification to an electrophoretically homogenous glycoprotein was achieved by gel permeation chromatography. This equine urinary trypsin inhibitor (E-UTI) is acid- and heat-stable, has a molecular weight of 22 to 23 kDa, an isoelectric point of 4.55, forms a 1:1 molar complex with trypsin and has serine as its N-terminal amino acid. The N-terminal amino acid sequence of this protein is almost identical with that of EI-14, the inhibitor obtained from horse serum by tryptic treatment, except for two extra amino acid residues, Ser-Lys- on the N-terminal end of E-UTI. In its isoelectric point E-UTI differs from EI-14 and the inhibitor from human urine.     

Amino acid sequence of human D of the alternative complement pathway

The primary structure of human D, the serine protease activating the C3 convertase of the alternative complement pathway, has been deduced by sequencing peptides derived from various chemical (CNBr and o-iodosobenzoic acid) and enzymatic (trypsin, lysine protease, Staphylococcus aureus V8 protease, and chymotrypsin) cleavages. Carboxypeptidase A was also used to confirm the COOH-terminal sequence. The peptides were purified by high-pressure liquid chromatography. The proposed sequence of human D contains 222 amino acids and has a calculated molecular weight of 23 748. It exhibits a high degree of homology with other serine proteases, especially around the NH2-terminus as well as the three residues corresponding to the active-site His-57, Asp-102, and Ser-195 (chymotrypsinogen numbering). This sequence homology is highest (40%) with plasmin, intermediate (35%) with pancreatic serine proteases, such as elastase, trypsin, chymotrypsin, and kallikrein, and least (30%) with the serum enzymes thrombin and factor X. D, however, exhibits only minimal amino acid homology with the other sequenced complement serine proteases, Clr (25%) and Bb (20%). The substitution of a basic lysine for a neutral amino acid three residues NH2-terminal to the active-site serine as well as a small serine residue for a bulky aromatic amino acid at position 215 (chymotrypsinogen numbering) in the binding pocket may be important in determining the exquisite substrate specificity of D. The presence of His-40 which interacts with Asp-194 (chymotrypsinogen numbering) to stabilize other serine protease zymogens [Freer, S. T., Kraut, J., Robertus, J. D., Wright, H. T., & Xuong, N. H. (1970) Biochemistry 9, 1997] argues in favor of such a D precursor molecule.     

Photoreactive derivative of Kunitz's soybean trypsin inhibitor. Preparation by selective modification of a tryptophan residue and formation of a covalent complex of the modified inhibitor with trypsin

The photoreactive arylsulfenyl chloride 2-nitro-4-azidophenylsulfenyl chloride (2,4-NAPS-Cl) has been used for the selective modification of tryptophan in Kunitz's soybean trypsin inhibitor (SBTI). The ultraviolet absorption spectrum and amino acid analysis of 2,4-NAPS-SBTI indicated that only one of the two tryptophans (93 or 117) present in SBTI was modified. CNBr cleavage of 2,4-NAPS-SBTI resulted in two fragments 1-114 and 115-181. Amino acid analysis of the two separated fragments showed that only tryptophan 93 underwent modification. 2,4-NAPS-SBTI fully retained its inhibitory activity against trypsin. The photoaffinity labeling of trypsin with 2,4-NAPS-Cl was performed on tritiated trypsin prepared by reacting bovine trypsin with [3H]-succinimidyl propionate. The covalent attachment of 2,4-NAPS-SBTI to the tritiated trypsin after photolysis was demonstrated by exclusion chromatography on Sephadex G-50 in the presence of guanidine hydrochloride.     

Purification and characterization of two trypsin inhibitors from the hemolymph of Manduca sexta larvae

Trypsin inhibitory activity from the hemolymph of the tobacco hornworm (Manduca sexta) was purified by affinity chromatography on immobilized trypsin and resolved into two fractions with molecular weights of 14,000 (M. sexta hemolymph trypsin inhibitor (HLTI) A) and 8,000 (HLTI B) by molecular sieve chromatography on Sephadex G-75. Electrophoresis of these inhibitors under reducing conditions on polyacrylamide gels gave molecular weight estimates of 8,300 for HLTI A and 9,100 for HLTI B, suggesting that HLTI A is a dimer and HLTI B is a monomer. Isoelectrofocusing on polyacrylamide gels focused HLTI A as a single band with pI 5.7, whereas HLTI B was resolved into two components with pI values of 5.3 and 7.1. Both inhibitors were stable at 100 degrees C and pH 1.0 for at least 30 min. HLTIs A and B inhibited serine proteases such as trypsin, chymotrypsin, and plasmin, but did not inhibit elastase, papain, pepsin, subtilisin BPN', and thermolysin. In fact, subtilisin BPN' completely inactivated both inhibitors. Both inhibitors formed low-dissociation complexes with trypsin in a 1:1 molar ratio. The inhibition constant for trypsin inhibition by HLTI A was estimated to be 1.45 x 10(-8) M. The HLTI A-chymotrypsin complex did not inhibit trypsin; similarly, the HLTI A-trypsin complex did not inhibit chymotrypsin, indicating that HLTI A has a common binding site for both trypsin and chymotrypsin. The amino-terminal amino acid sequences of HLTIs A and B revealed that both these inhibitors are homologous to bovine pancreatic trypsin inhibitor (Kunitz).     

Isolation of a trypsin inhibitor with deletion of N-terminal pentapeptide from the seeds of Momordica cochinchinensis, the Chinese drug mubiezhi.

A trypsin inhibitor, MCCTI-1, with a molecular weight of 3479 Da as determined by mass spectrometry, was isolated from Momordica cochinchinensis seeds with a procedure involving extraction with 5% acetic acid, ammonium sulfate precipitation, ion exchange chromatography on CM-Sepharose and reverse-phase high performance liquid chromatography. The sequence of its first 13 N-terminal amino acid residues was ILKKCRRDSDCPG which was about 85% identical with the sequence of trypsin inhibitor MCTI-1 from Momordica charantia Linn. When compared with the sequences of most other squash family trypsin inhibitors, the sequence of MCCTI-1 was characterized by the deletion of a pentapeptide from the N-terminus. Trypsin inhibitors also existed in seeds of some hitherto uninvestigated Cucurbitaceae species.     

Isolation and partial amino acid sequence of the trypsin inhibitor from the seeds of Cucurbita maxima

Trypsin inhibitor from squash (Cucurbita maxima) seed was extracted with 0.1 M-acetate buffer, pH 4.5, purified on immobilized trypsin, and separated by SE-Sephadex C-50 chromatography into three active fractions. All of them inhibited trypsin to the same extent, showed no antichymotrypsin or antikallikrein activity, had a similar molecular weight (about 3300), and contained no tryptophan, phenylalanine or threonine. The partial amino acid sequence of tryptic and peptic peptides of fraction III was determined by Edman degradation procedure.     

Protein trypsin inhibitor from potato tubers

A protein of 22 kDa designated as PKTI-22 was isolated from potato tubers (Solanum tuberosum L., cv. Istrinskii) and purified to homogeneity using CM-Sepharose CL-6B ion-exchange chromatography. The protein efficiently suppressed the activity of trypsin, affected chymotrypsin less, and did not affect subtilisin Carlsberg. The N-terminal sequence of PKTI-22 (20 amino acid residues) was found to be highly homologous with the amino acid sequences of the potato Kunitz-type proteinase inhibitors of group B (PKPI-B) that were aligned from the corresponding gene sequences and was identical to the sequence (from the 2nd to the 20th residue) of the recombinant protein PKPI-B10. These data together with the observed similarity of the properties of two proteins indicate that the PKTI-22 protein is encoded by the PKPI-B10 gene.     

A Kazal-type trypsin inhibitor from the protochordate Ciona intestinalis.

A trypsin inhibitor from Ciona intestinalis, present throughout the animal, was purified by ion-exchange chromatography followed by four HPLC steps. By MS the molecular mass of the native form was determined to be 6675 Da. The N-terminal amino acid sequence was determined by protein sequencing, but appeared to be partial because the theoretical molecular mass of the protein was 1101 Da too low. Thermolysin treatment gave rise to several fragments each containing a single disulphide bridge. By sequence analysis and MS intramolecular disulphide bridges could unequivocally be assigned to connect the pairs Cys4-Cys37, Cys8-Cys30 and Cys16-Cys51. The structure of the inhibitor is homologous to Kazal-type trypsin inhibitors. The inhibitor constant, KI, for trypsin inhibition was 0.05 nM whereas chymotrypsin and elastase were not inhibited. To reveal the complete sequence the cDNA encoding the trypsin inhibitor was isolated. This cDNA of 454 bp predicts a protein of 82 amino acid residues including a 20 amino acid signal peptide. Moreover, the cDNA predicts a C-terminal extension of 11 amino acids compared to the part identified by protein sequencing. The molecular mass calculated for this predicted protein is in accordance with the measured value. This C-terminal sequence is unusual for Kazal-type trypsin inhibitors and has apparently been lost early in evolution. The high degree of conservation around the active site strongly supports the importance of the Kazal-type inhibitors.