Binding of the bovine and porcine pancreatic secretory trypsin inhibitor (Kazal) to human leukocyte elastase: a thermodynamic study.

The effect of pH and temperature on the apparent association equilibrium constant (Ka) for the binding of the bovine and porcine pancreatic secretory trypsin inhibitor (Kazal-type inhibitor, PSTI) to human leukocyte elastase has been investigated. At pH 8.0, values of the apparent thermodynamic parameters for human leukocyte elastase: Kazal-type inhibitor complex formation are: bovine PSTI--Ka = 6.3 x 10(4) M-1, delta G degree = -26.9 kJ/mol, delta H degree = +11.7 kJ/mol, and delta S degree = +1.3 x 10(2) entropy units; porcine PSTI--Ka = 7.0 x 10(3) M-1, delta G degree = -21.5 kJ/mol, delta H degree = +13.0 kJ/mol, and delta S degree = +1.2 x 10(2) entropy units (values of Ka, delta G degree and delta S degree were obtained at 21.0 degrees C; values of delta H degree were temperature independent over the range (between 5.0 degrees C and 45.0 degrees C) explored). On increasing the pH from 4.5 to 9.5, values of Ka for bovine and porcine PSTI binding to human leukocyte elastase increase thus reflecting the acidic pK-shift of the His57 catalytic residue from congruent to 7.0, in the free enzyme, to congruent to 5.1, in the serine proteinase: inhibitor complexes. Thermodynamics of bovine and porcine PSTI binding to human leukocyte elastase has been analyzed in parallel with that of related serine (pro)enzyme/Kazal-type inhibitor systems. Considering the known molecular models, the observed binding behaviour of bovine and porcine PSTI to human leukocyte elastase was related to the inferred stereochemistry of the serine proteinase/inhibitor contact region(s).     

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.     

Studies on trypsin inhibitors. Part I. Synthesis of protected peptides related to sequence 1-10 of porcine pancreatic secretory trypsin inhibitor II (Kazal).

The general strategy for the synthesis, by conventional procedures, of the entire sequence of porcine pancreatic secretory trypsin inhibitor II (Kazal) is discussed. The synthesis of two protected peptides corresponding to positions 2-10 and 1-10 of the proposed primary structure of the inhibitor is described. The heptapeptide free base threonyl-S-acetamidomethylcysteinylthreonylseryl-gamma-tert-butylglutamylvalylserine tert-butyloxycarbonylhydrazide (sequence 4-10) was acylated, by the azide procedure, with either the dipeptide benzyloxycarbonyl-gamma-tert-butylglutamylalanine hydrazide (sequence 2-3) or the tripeptide Nalpha-benzyloxycarbonyl-Nomega-nitroarginylglutamylala-nine hydrazide (sequence 1-3). The stereochemical homogeneity of the resulting peptides, benzyloxycarbonyl-gamma-tert-butylglutamylalanylthreonyl-S-acetamidomethyl-cysteinylthreonylseryl-gamma-tert-butylglutamylvalylserine tert-butyloxycarbonylhydrazide and Nalpha-benzyloxycarbonyl-Nomega-nitroarginylglutamylalanylthreonyl-S-acetamido-methylcysteinylthreonylseryl-gamma-tert-butylglutamylvalylserine tert-butyloxycarbonyl-hydrazide, was assessed, after partial deprotection with liquid hydrogen fluoride, by digestion with aminopeptidase M followed by quantitative amino acid analysis.     

Immunochemical studies of human urinary trypsin inhibitor

Antisera against purified urinary trypsin inhibitor (UTI-I, molecular weight 67,000) and UTI-III (molecular weight 23,000) were first produced in rabbits. Both anti-UTI-I and anti-UTI-III sera formed a single immunoprecipitin line with human plasma inter-alpha-trypsin inhibitor (I alpha TI), whereas two immunoprecipitin lines were formed with crude urine. It was speculated that both UTI-I and UTI-II might be present in normal human urine. In the present study, the inhibitory effects of anti-UTI sera on UTI activity were examined by three different assay methods. The results indicated that the inhibitory effect was almost immediate. Although the inhibitory effect of anti-UTI-III serum on UTI-III was almost of the same degree of completeness for the three assay methods. UTI-I was partially inhibited by the anti-UTI-I serum when residual trypsin activity was measured by the caseinolytic or fibrinolytic assay method. This discrepancy was considered to be due to the difference in conformational change between UTI-I and UTI-III by antigen-antibody reaction.     

Purification of human tissue plasminogen activator with Erythrina trypsin inhibitor

Seeds of the legume Erythrina latissima contain a 20,000-dalton, single-chain protein that has been shown to inhibit the amidolytic activity of trypsin and tissue plasminogen activator. It had no comparable effect on urokinase. IC50 values of 1.1 X 10(-7) M for tissue plasminogen activator and 6.9 X 10(-10) M for trypsin were determined by titration. When coupled to agarose, the Erythrina inhibitor provided an effective reagent for affinity purification of tissue plasminogen activator from melanoma cell-conditioned tissue culture medium. Using this as a single-step procedure, 270-fold purified enzyme was reproducibly obtained with yields of 90% or greater. Both one- and two-chain forms of tissue plasminogen activator were purified. The enzyme migrated, in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, as a predominant 72,000-dalton doublet with lesser amounts of immunochemically similar, 115,000- and 68,000-dalton components.     

Studies on trypsin inhibitors. Part V. Synthesis of the protected octapeptide (sequence 36-43) of porcine pancreatic secretory trypsin inhibitor II (Kazal).

Synthesis is described of the partially protected octapeptide tert-butyloxycarbonyl-gamma-tert-butylglutamylasparaginyl-N-trifluoroacetyllysyl-N-trifluoracetyllysylarginyl-Ngamma-4,4'-dimethoxybenzhydryglutaminylthreonylproline corresponding to positions 36-43 of the amino acid sequence of porcine pancreatic secretory trypsin inhibitor II. The tetrapeptide free base arginyl-Ngamma-4,4'-dimethoxybenzhydrylglutaminylthreonylproline was acylated, by the azide proceedure, with the tripeptide benzyloxycarbonyl-asparaginyl-N-trifluoroacetyllsyl-N-trifluoroacetyllysine hydrazide. The resulting protected heptapeptide was partially deblocked by catalytic hydrogenation and reacted with alpha-1-succinimidyl-gamma-tert-butyl tert-butyloxycarbonylglutamate. The stereochemical homogeneity of the ensuing octapeptide was assessed, after partial deprotection with aqueous 90% trifluoroacetic acid, by digestion with papain and aminopeptidase M followed by quantitative amino acid analysis.     

Studies on trypsin inhibitors. Part III. Synthesis of the protected decapeptide (sequence 15-24) of porcine pancreatic secretory trypsin inhibitor II (Kazal).

The synthesis of the amino-protected decapeptide tert-butyloxycarbonylhydrazide corresponding to positions 15-24 of the amino acid sequence of porcine pancreatic secretory trypsin inhibitor II (Kazal inhibitor) is described. The tripeptide free base threonyl-beta-tert-butylaspartylglycine tert-butyloxycarbonylhydrazide (sequence 22-24) was acylated with 1-succinimidyl o-nitrophenylsulfenylvalyl-S-acetamidomethylcysteinylglycinate (sequence 19-21). Removal of the amino protecting group from the resulting hexapeptide followed by acylation of the free base with either benzyloxycarbonylisoleucyl-O-tert-butyltyrosylasparaginylproline or O-nitrophenylsulfenylisoleucyl-O-tert-butyltyrosylasparaginylproline, via the pyrazoline active ester method, yielded the decapeptide tert-butyloxycarbonylhydrazide (sequence 15-24) in the form of Nalpha-benzyloxycarbonyl or Nalpha-O-nitrophenylsulfenyl derivative. The stereochemical homogeneity of the two decapeptides was assessed, after partial deprotection with liquid hydrogen fluoride, or thioacetamide and aqueous 90% trifluoroacetic acid, by digestion with papain and aminopeptidase M followed by quantitative amino acid analysis.     

Studies on trypsin inhibitors. Part VI. Synthesis of the protected nonapeptide (sequence 44-52) of porcine pancreatic secretory trypsin inhibitor II (Kazal).

Synthesis is described of the partially protected nonapeptide tert-butyloxycarbonyl-valylleucylisoleucylglutaminyl-N-trifluoroacetyllsylserylglycylprolyl-S-acetamido-methylcysteine corresponding to positions 44-52 of the amino acid sequence of porcine pancreatic secretory trypsin inhibitor II. The hexapeptide free base glutaminyl-N-trifluoroacetyllsylserylglycylprolyl-S-acetamidomethylcysteine, prepared by two alternative routes, was acylated by the azide procedure, with the tripeptide tert-butyloxycarbonylvalylleucylisoleucine hydrazide. The stereochemical homogeneity of the resulting nonapeptide was assessed, after partial deprotection by treatment with aqueous 90% trifluoroacetic acid, by digestion with papain and aminopeptidase M followed by quantitative amino acid analysis.     

Studies on trypsin inhbitors. Part II. Synthesis of the protected tetrapeptide (sequence 11-14) of porcine pancreatic secretory trypsin inhibitor II (Kazal).

Synthesis is described of the protected tetrapeptide corresponding to positions 11-14 of the primary structure of the porcine pancreatic secretory trypsin inhibitor II (Kazal), in the form of free acid as well as protected hydrazide. The tetrapeptide tert-butyloxycarbonylglycyl-S-acetamidomethylcysteinylprolyl-Nepsilon-trifluoroacetyl-lysine was prepared by stepwise elongation from the C-terminal Nepsilon-trifluoroacetyllysine using successively 1-succinimidyl benzyloxycarbonylprolinate, p-nitrophenyl N-tert-butyloxycarbonyl-S-acetamidomethylcysteinate and 1-phenyl-3-methyl-4-(tert-butyloxycarbonylglycyl)-oximinyl-5-(benzyloxycarbonylglycyl)-imino-2-pyrazoline as acylating agents. Alternately, the dipeptide benzyloxycarbonylprolyl-Nepsilon-trifluoroacetyllsine was transformed into the corresponding tert-butyloxycarbonylhydrazide which was reacted, after catalytic hydrogenolysis, with tritylglycyl-S-acetamido-methylcysteine to give the tetrapeptide tritylglycyl-S-acetamidomethylcysteinylprolyl-Nepsilon-trifluoroacetyllsine tert-butyloxycarbonylhydrazide. The stereochemical homogeneity of the final products was assessed, after partial deprotection with aqueous 90% trifluoroacetic acid, by digestion with papain and aminopeptidase M, followed by quantitative amino acid analysis.     

Enzymatic properties of alpha 2-macroglobulin-proteinase complexes. Apparent discrimination between covalently and noncovalently bound trypsin by reaction with soybean trypsin inhibitor

The binding of trypsin to alpha 2-macroglobulin, the appearance of free beta-cysteinyl thiol groups of the formed complexes, the steady-state kinetics of their enzymic hydrolysis of carbobenzoxy-L-valyl-glycyl-L-arginyl-4-nitroanilide and finally their reactions with soybean trypsin inhibitor leading to the formation of ternary alpha 2-macroglobulin-trypsin-soybean trypsin inhibitor complexes were investigated. Each alpha 2-macroglobulin molecule binds two trypsin tightly; the dissociation constants were found to be unmeasureably small, but the extent of formation of 1:1 and 1:2 complexes at different molar ratios of alpha 2-macroglobulin to trypsin as determined from the appearance of thiol groups clearly indicated that binding of trypsin to alpha 2-macroglobulin shows negative cooperativity. Binding of the first trypsin makes the access of the second less easy. The kinetic results showed a decrease of the kc/Km value of hydrolysis of the tripeptide substrate by approx. 4-fold compared to that of free trypsin for each alpha 2-macroglobulin-bound trypsin. Here no differences were seen between the bound trypsins. The analysis of the reactions between the alpha 2-macroglobulin-trypsin complexes and soybean trypsin inhibitor shows that ternary complexes do form, although slowly, and that two processes occur, not only when 1:2 complexes but also when 1:1 complexes react with soybean trypsin inhibitor. Soybean trypsin inhibitor apparently discriminates between two distinct binding modes of trypsin to alpha 2-macroglobulin, the covalently and the noncovalently alpha 2-macroglobulin-bound trypsins.     

Studies on trypsin inhibitors. Part VIII. Synthesis of the protected octatriacontapeptide corresponding to the sequence 15-52 of porcine pancreatic secretory trypsin inhibitor II (Kazal)

The synthesis by fragment condensation of protected peptides corresponding to the amino acid sequences 15-35, 25-52 and 15-52 of porcine pancreatic secretory trypsin inhibitor II (Kazal type) is described. The Rudinger modification of the azide procedure was used in the fragment coupling steps. The tert-butyloxycarbonylheptapeptide hydrazide (sequence 22-28) was reacted with the heptapeptide methyl ester free base (sequence 29-35) and the resulting tert-butyloxycarbonyltetradecapeptide methyl ester after selective deprotection, coupled with the benzyloxycarbonylheptapeptide hydrazide (sequence 15-21) to give the protected peptide methyl ester corresponding to the 15-35 sequence which was then converted to the corresponding hydrazide. The synthesis of the 25-52 sequence was achieved by assembling the protected peptide hydrazide corresponding to the amino acid residues 25-35, with the C-terminal heptadecapeptide 36-52. The resulting protected octaeicosapeptide (sequence 25-52) was selectively deblocked with trifluoroacetic acid and acylated with the benzyloxycarbonyldecapeptide hydrazide 15-24 to give the desired octatriacontapeptide corresponding to sequence 15-52 of the inhibitor. An attempt to prepare the 15-52 sequence through the condensation of fragments corresponding to 15-35 and 36-52 sequences was unsuccessful. The identity and purity of the synthetized peptide derivatives wre established by elemental analysis (in some cases), amino acid analysis, optical rotation, and thin-layer chromatography in two solvent systems. The final products were also evaluated, after partial deprotection with anhydrous hydrogen fluoride or aqueous 90% trifluoroacetic acid, by paper electrophoresis at different pH values.     

Equilibrium of Bowman-Birk inhibitor association with trypsin and alpha-chymotrypsin.

Association constants, enthalpies, and stoichiometries of Bowman-Birk soybean inhibitor for trypsin and alpha-chymotrypsin were measured in the pH range 4-8 at 25 degrees, 0.01 M Ca2+. The results are quoted in terms of moles of protease active sites, from active site titration. Enthalpies were obtained from calorimetry. The inhibitor was modified by carboxyl group modification, and by tryptic and chymotryptic attack. Association thermodynamics and stoichiometries of the modified inhibitors with both proteases were also determined. There is one independent site for each protease on the inhibitor protein. Modification decreases association to some extent, but does not appear to change stoichiometry or protease binding site independency. In the pH 4 region the association enthalpies are endothermic, of the order 6 kcal/mol for both trypsin and chymotrypsin. With increasing pH, the enthalpies decrease and become exothermic at pH 8 for chymotrypsin. Positive entropies, 50 cal mol-1 deg-1, occur at pH 4-5. They decrease as pH increases, but are always positive in sign. The observed to accompany the overall reaction, such as H+ transfer steps. The enthalpies and entropies probably compensate over the pH range 4-8, with a characteristic temperature of 390 plus or minus 30 degrees K. Estimates were made of the macromolecular Coulomb charge products in inhibitor-protease interaction. These range from about +5 to -60, over pH range 4-8, depending on the protease. Although intermolecular Coulombic forces cannot be easily delineated at the specific side chain level, they may operate at the macromolecule level.     

Hydrogen exchange kinetics of bovine pancreatic trypsin inhibitor beta-sheet protons in trypsin-bovine pancreatic trypsin inhibitor, trypsinogen-bovine pancreatic trypsin inhibitor, and trypsinogen-isoleucylvaline-bovine pancreatic trypsin inhibitor

Hydrogen exchange rates of six beta-sheet peptide amide protons in bovine pancreatic trypsin inhibitor (BPTI) have been measured in free BPTI and in the complexes trypsinogen-BPTI, trypsinogen-Ile-Val-BPTI, bovine trypsin-BPTI, and porcine trypsin-BPTI. Exchange rates in the complexes are slower for Ile-18, Arg-20, Gln-31, Phe-33, Tyr-35, and Phe-45 NH, but the magnitude of the effect is highly variable. The ratio of the exchange rate constant in free BPTI to the exchange rate constant in the complex, k/kcpIx, ranges from 3 to much greater than 10(3). Gln-31, Phe-45, and Phe-33 NH exchange rate constants are the same in each of the complexes. For Ile-18 and Tyr-35, k/kcpIx is much greater than 10(3) for the trypsin complexes but is in the range 14-43 for the trypsinogen complexes. Only the Arg-20 NH exchange rate shows significant differences between trypsinogen-BPTI and trypsinogen-Ile-Val-BPTI and between porcine and bovine trypsin-BPTI.     

The multiplicity of human pancreatic secretory trypsin inhibitor

Four forms of pancreatic secretory trypsin inhibitor (PSTI; A1, A2, B, and C) were purified from human pancreatic juice. According to sequence results, the primary structure of B was different from that reported earlier (Greene, L.J., et al. (1976) Method Enzymol. 45, 813-825) at two positions, i.e. Asn21----Asp21, Asp29----Asn29. A1 and A2 were deamidated forms of B judging from peptide mappings with Staphylococcus aureus V8 protease. Gln45 in B was replaced by Glu in A1 and Gln51 in B was replaced by Glu in A2. C was an inhibitor lacking five amino acid residues from the amino terminal of B. B and C inhibited human cationic trypsin activity stoichiometrically with similar dissociation constants, but A1 and A2 showed poorer trypsin inhibitory activity than B and C.