Characterization of -trypsin at acid pH by differential scanning calorimetry.

Trypsin is a serino-protease with a polypeptide chain of 223 amino acid residues and contains six disulfide bridges. It is a globular protein with a predominance of antiparallel -sheet and helix in its secondary structure and has two domains with similar structures. We assessed the stability of -trypsin in the acid pH range using microcalorimetric (differential scanning calorimetry) techniques. Protein concentrations varied in the range of 0.05 to 2.30 mg/ml. Buffer solutions of 50.0 mM -alanine and 20.0 mM CaCl2 at different pH values (from 2.0 to 4.2) and concentrations of sorbitol (1.0 and 2.0 M), urea (0.5 M) or guanidinium hydrochloride (0.5 and 1.0 M) were used. The data suggest that we are studying the same conformational transition of the protein in all experimental situations using pH, sorbitol, urea and guanidinium hydrochloride as perturbing agents. The observed van't Hoff ratios (deltaHcal/deltaHvH) of 1.0 to 0.5 in the pH range of 3.2 to 4.2 suggest protein aggregation. In contrast, deltaHcal/deltaHvH ratios equal to one in the pH range of 2.0 to 3.2 suggest that the protein unfolds as a monomer. At pH 3.00, -trypsin unfolded with Tm = 54 C and deltaH = 101.8 kcal/mol, and the change in heat capacity between the native and unfolded forms of the protein (deltaCp) was estimated to be 2.50 0.07 kcal mol-1 K-1. The stability of -trypsin calculated at 298 K was deltaG D = 5.7 kcal/mol at pH 3.00 and deltaG D = 15.2 kcal/mol at pH 7.00, values in the range expected for a small globular protein.

Characterization of á-trypsin at acid pH by differential scanning calorimetry

Trypsin is a serino-protease with a polypeptide chain of 223 amino acid residues and contains six disulfide bridges. It is a globular protein with a predominance of antiparallel á-sheet and helix in its secondary structure and has two domains with similar structures. We assessed the stability of á-trypsin in the acid pH range using microcalorimetric (differential scanning calorimetry) techniques. Protein concentrations varied in the range of 0.05 to 2.30 mg/ml. Buffer solutions of 50.0 mM á-alanine and 20.0 mM CaCl2 at different pH values (from 2.0 to 4.2) and concentrations of sorbitol (1.0 and 2.0 M), urea (0.5 M) or guanidinium hydrochloride (0.5 and 1.0 M) were used. The data suggest that we are studying the same conformational transition of the protein in all experimental situations using pH, sorbitol, urea and guanidinium hydrochloride as perturbing agents. The observed van't Hoff ratios (deltaHcal/deltaHvH) of 1.0 to 0.5 in the pH range of 3.2 to 4.2 suggest protein aggregation. In contrast, deltaHcal/deltaHvH ratios equal to one in the pH range of 2.0 to 3.2 suggest that the protein unfolds as a monomer. At pH 3.00, á-trypsin unfolded with Tm = 54ºC and deltaH = 101.8 kcal/mol, and the change in heat capacity between the native and unfolded forms of the protein (deltaCp) was estimated to be 2.50 ± 0.07 kcal mol-1 K-1. The stability of á-trypsin calculated at 298 K was deltaG D = 5.7 kcal/mol at pH 3.00 and deltaG D = 15.2 kcal/mol at pH 7.00, values in the range expected for a small globular protein.

Effect of angiotensin-(1-7) on jejunal absorption of water in rats.

The effect of angiotensin-(1-7) on jejunal water absorption in rats was investigated. The jejunal sac of anesthetized rats was filled with two ml of tyrode solution containing 3.7 MBq of tritiated water. A femoral vein was cannulated for administration of peptides and drugs. Infusion of Ang-(1-7) at the dose of 0.7 ng/kg.min produced a significant increase in jejunal water absorption compared to control (32% increase). The Ang-(1-7) antagonist A-779 abolished the effect of Ang-(1-7) on water absorption. A reduction of the Ang-(1-7) effect was also produced by treatment with the AT(1) receptor antagonist, losartan or the AT(2) receptor antagonist, PD123.177. The increase in jejunal water absorption produced by Ang-(1-7) was blocked by the nitric oxide synthase inhibitor, L-NAME and by indomethacin. These data suggest that the effect of Ang-(1-7) on the jejunal loop is mediated by activation of a multiple angiotensin receptors and/or by an atypical angiotensin receptor. Furthermore, the effect of Ang-(1-7) on jejunal water absorption is mediated by nitric oxide and by a cyclooxygenase-dependent mechanism.

Thermodynamics of unfolding of beta-trypsin at pH 2.8.

The unfolding equilibrium of beta-trypsin induced by thermal and chemical denaturation was thermodynamically characterized. Thermal unfolding equilibria were monitored using UV absorption and both far- and near-UV CD spectroscopy, while fluorescence was used to monitor urea-induced transitions. Thermal and urea transition curves are reversible and cooperative and both sets of data can be reasonably fitted using a two-state model for the unfolding of this protein. Plots of the fraction denatured, calculated from thermal denaturation curves at different wavelengths, versus temperature are coincident. In addition, the ratio of the enthalpy of denaturation obtained by scanning calorimetry to the van't Hoff enthalpy is close to unity, which supports the two-state model. Considering the differences in experimental approaches, the value for the stability of beta-trypsin estimated from spectroscopic data (deltaGu = 6.0 +/- 0.2 kcal/mol) is in reasonable agreement with the value calculated from urea titration curves (deltaGUH2O = 5.5 +/- 0.3 kcal/mol) at pH 2.8 and 300 degrees K.

Ph titration of native and unfolded beta-trypsin: evaluation of the delta delta G§ titration and the carboxyl pK values

The stabilizing free energy of beta-trypsin was determined by hydrogen ion titration. In the pH range from 3.0 to 7.0, the change in free energy difference for the stabilization of the native protein relative to the unfolded one (delta delta G° titration) was 9.51 + 0.06 kcal/mol. An isoelectric point of 10.0 was determined, allowing us to calculate the Tanford and Kirkwood electrostatic factor w. This factor presented a nonlinear behavior and indicated more than one type of titratable carboxyl groups in beta-trypsin. In fact, one class of carboxyl group with a pK= 3.91 + 0.01 and another one with a pK= 4.63 + 0.03 were also found by hydrogen in titration of the protein in the folded state.

Thrombin-like enzyme from Lachesis muta muta venom: isolation and topographical analysis of its active site structure by means of the binding of amidines and guanidines as competitive inhibitors.

A serine protease enzyme was purified from Lachesis muta muta venom, with 40% yield, by gel filtration on Sephadex G-100 and affinity chromatography on Sepharose-agmatin. Homogeneity of the enzyme preparation was demonstrated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and the enzyme had a relative mol. wt of 45,000. The molar extinction coefficient at 280 nm was 62,127 (M x cm)-1. The enzyme hydrolysed Bz-Arg-Nan with Ks = 0.233 +/- 0.08 mM and kcat = 2.80 +/- 0.07 sec-1. All the amidines and guanidines tested for their inhibitory effect on thrombin-like enzyme behaved as competitive inhibitors of the enzyme with Ki values in the range 6.2 microM to 42.3 mM for amidines and 0.19 mM to 9.31 mM for guanidines. Dissociation constant values were analyzed in terms of the binding of the inhibitors with the subsite S1, the specificity pocket of the enzyme, Ki values were discussed in accordance with those for trypsin inhibition. beta-Naphthamidine was the strongest inhibitor, while guanidine was the weakest. The differences among the Ki values were interpreted in terms of the shape of the enzyme active site. For meta- and para-substituted benzamidinium ions a good correlation was found between log l/Ki and sigma Hammett values of the substituents. The substituent effects in the pi-electrons of the benzamidine ring were considered in the frame of Hückel molecular orbital theory. A model for the binding of p-benzamidine derivatives with the primary specificity S1 subsite of the enzyme active site was proposed.

pH titration of native and unfolded beta-trypsin: evaluation of the delta delta G0 titration and the carboxyl pK values.

The stabilizing free energy of beta-trypsin was determined by hydrogen ion titration. In the pH range from 3.0 to 7.0, the change in free energy difference for the stabilization of the native protein relative to the unfolded one (delta delta G0 titration) was 9.51 +/- 0.06 kcal/mol. An isoelectric point of 10.0 was determined, allowing us to calculate the Tanford and Kirkwood electrostatic factor w. This factor presented a nonlinear behavior and indicated more than one type of titratable carboxyl groups in beta-trypsin. In fact, one class of carboxyl group with a pK = 3.91 +/- 0.01 and another one with a pK = 4.63 +/- 0.03 were also found by hydrogen ion titration of the protein in the folded state.

Kinetic mechanism of the inhibition of human urinary kallikrein by basic pancreatic trypsin inhibitor.

Hydrolysis of D-valyl-L-leucyl-L-arginine p-nitroanilide (D-Val-Leu-Arg-Nan) at five different concentrations (10-20 microM) by human urinary kallikrein was studied in the absence and in the presence of increasing concentrations of basic pancreatic trypsin inhibitor (BPTI) (1.35-9.15 nM). The data indicate that the inhibition of human urinary kallikrein by BPTI is not a simple competitive inhibition as reported by others, but that it is a competitive inhibition of the parabolic type, with two inhibitor molecules binding to one enzyme molecule, with the formation of a ternary enzymatic complex. Statistical analysis of the experimental data supports the kinetic model proposed. The calculated values of the constants Ki and Kii were 16.20 nM and 1.10 nM, respectively. It is noteworthy that the Kii < Ki, i.e., the second BPTI molecule binds to the enzyme with a larger affinity suggesting that this second binding site was probably created or positively modulated as a consequence of the binding of the first BPTI molecule.

Kinetic mechanism of the inhibition of human urinary kallikrein by basic pancreatic trypsin inhibitor

Hydrolysis of D-valvyl-L-leucyl-L-arginine p-nitroanilide (D-Val-Leu-Arg-Nan) at five different concentration (10-20µM) by human urinary kallikrein was studied in the absence and in the presence of increasing concentrations of basic pancreatic trypsin inhibitor (BPTI) (1.35-9.15nM). The data indicate that the inhbition of human urinary kallikrein by BPTI is not a simple competitive inhibition as reported by others, but that it is a competitive inhibition of the parabolic type, with two inhibitor molecules binding to one enzyme molecule, with the formetion of a ternary enzymatic complex. Statistical analysis of the experimental data supports the kinetic model proposed. The calculated values of the constants Ki and Kii were 16.20 nM and 1.10 nM, repectively. It is noteworthy that the Kii < Ki, i.e., the second BPTI molecule binds to the enzyme with a larger affinity suggesting that this second binding site was probably created or positively modulated as a consequence of the binding of the first BPTI molecule

Tyrosine 151 is part of the substrate activation binding site of bovine trypsin. Identification by covalent labeling with p-diazoniumbenzamidine and kinetic characterization of Tyr-151-(p-benzamidino)-azo-beta-trypsin.

Identification of the substrate activation site of beta-trypsin by a 1:1 reaction with p-diazoniumbenzamidine chloride was confirmed by spectral analysis. Proteolysis of Cm-p-benzamidino-azo-beta-trypsin provided peptides containing modified tyrosine residues. The major product, Ser-146 to Lys-156, which corresponded to labeling at Tyr-151, was recovered in 35% yield, and its structure was demonstrated by amino acid analysis, Edman degradation, and mass spectrometry. Yields of labeled Tyr-151, Tyr-39, and Tyr-172, identified by peptide analysis, were in the proportion of 100:7:3. Tyr-151-(p-benzamidino)-azo-beta-trypsin is permanently activated, but can be further activated by substrates. Values of kcat, Ks', and kcat' vary from two to three times the equivalent values for trypsin. Berenil (4,4'-diazoamino-bis-benzamidine), a parabolic competitive inhibitor of beta-trypsin, was a hyperbolic competitive inhibitor of azo-beta-trypsin. Thus, Tyr-151, part of subsite S'2, affects the catalytic process and, when modified covalently, permanently activates trypsin. Equilibrium binding with berenil supported the kinetic data obtained with substrates. This permits the integration of protein modification, kinetics, equilibrium binding, and crystallographic data to demonstrate a fine interaction between subsites S1-S3 and S'2 in trypsin and azo-beta-trypsin, resulting in subtle structural changes when the native enzyme is covalently modified at Tyr-151.

Pharmacological demonstration of a barrier for protease, peptides and acetylcholine in the endometrium of the rat.

Contractions of the rat uterus in response to trypsin, kallikrein, bradykinin, angiotensin II, oxytocin and acetylcholine, were abolished when an inside-out preparation was used. Sensitivity to Ba++, however, was preserved. In preparations in which the endometrium was mechanically removed, all above cited agonists elicited contractions. By treating the uterus with both collagenase and hyaluronidase, acetylcholine was able to induce a contraction when applied to the endometrium side of the uterus. The results show that a barrier for protease, peptides and acetylcholine is present in the mucosa of the rat uterus.

Kallikrein-kinin system in the angiogenesis.

Using the bioassay to investigate the presence of mediators in the angiogenesis exudate to explain its property to cause vascular permeability and fall in the blood pressure we found the presence of histamine, bradykinin, prostaglandin E2 and angiotensin in the exudate. However, the role of these mediators in the angiogenesis process needs to be investigated.

The substituent effect on complex formation between alpha-trypsin and para-substituted benzamidinium ions: a thermodynamic study.

1. Dissociation constants, Ki, were determined spectrophotometrically by measuring the absorbance at 410 nm, using N alpha-benzoyl-D,L-arginine-para-nitroanilide (Bz-D,L-Arg-Nan) as substrate. The Ki values for the complexes of alpha-trypsin with each of the para-derivatives of the benzamidinium ion -NH2, -CH3, -H, -F, -Cl, -Br, -COOEt, and -NO2 were measured at six temperatures (8, 15, 20, 25, 29 and 33 degrees C), in order to determine the thermodynamic parameters delta G0, delta H0, delta S0, and delta C0P for complex formation. 2. The standard enthalpy change (delta H0) was constant (-12.45 kcal/mol) and all other parameters were also negative. The large negative values obtained for the standard heat capacity change (delta C0P) suggest that the process occurs with a conformational adaptation in the enzyme structure. 3. The apparent partial specific volumes of free alpha-trypsin and alpha-trypsin bound to benzamidinium ion indicated that there is a decrease of approximately 0.10 cm3/g in the enzyme volume when the inhibitor binds. This contraction is consistent with the release of about 130 water molecules per enzyme molecule.

Tonin and kallikrein-kinin system.

The kininogenase activity of tonin has been demonstrated by Ikeda and Arakawa, 1984. Tonin of the rat submandibular gland contracts the rat uterus independent of addition of the substrate. On repetition, the same dose of enzyme elicited desensitization. When a double dose was used the contraction again occurred. After desensitization to tonin the contraction to kallikrein was reduced about 80% of the control. The desensitization to kallikrein lightly reduced the contraction to tonin. When the muscle was desensitized to trypsin tonin did not evoke contraction. These experiments suggest the presence of two different substrates in the uterus, one more specific to kallikrein and the other for tonin. The experiments with the parallel uterus preparation strongly suggest release of kinin in the process of contraction of the uterus by tonin.

The substituent effect on complex formation between alfa-trypsin and para-substituted benzamidinium ions: A thermodynamic study

Dissociation constants, Kj, were determined spectrophotometrically by measuring the absorbance at 410 nm, using N alfa-benzoyl-D,L-argomome-para-nitroanilide (Bz-D,L-Arg-Nan) as substrate. The Ki values for the complexes of alfa-trypsin with each of the para-derivatives of the benzamidinium ion -NH2, -CH3, -H, -F, -Cl, -Br, -COOEt, and -NO2 were measured at six temperatures (8,15,20,25, 29 and 33§C), in order to determine the thermodynamic parameters for complex formation. The standard enthalpy change was constant and all other parameters were also negative. The large negative values obtained for the standard heat capacity change suggest that the process occurs with a conformational adaptation in the enzyme structure. The apparent partial specife volumes of free alfa-trypsin and alfa-trypsin bound to benzamidinium ion indicated that there is a decrease of approximatelly 0.10 cm**3/g in the enzyme volume when the inhibitor binds. This contraction is consistent with the release of about 130 water molecules per enzyme molecule

Crossed desensitization between plasma kallikrein and trypsin.

The addition of rat plasma kallikrein or trypsin to the bath containing rat uterus caused contraction. On repetition, the same amount of the enzyme, after 4-5 additions, elicited desensitization. When a double dose of the enzyme was used the contraction again occurred. However, after desensitization to kallikrein the response for trypsin remained in altered, but after the desensitization for trypsin the uterus did not respond to kallikrein. Chymotrypsin, in spite of did not cause contraction, became the uterus insensitive to kallikrein and trypsin. It seems that bradykinin is not involved in the mechanism of contraction. The desensitization may be due to the release of inhibitors specific for kallikrein or trypsin; the effect of chymotrypsin may also be due to release of similar inhibitors.

Autolysis of beta-trypsin at pH 3.0.

Bovine beta-trypsin was prepared from commercial crystalline trypsin by SP-Sephadex equilibrium chromatography (D.D. Schroeder and E. Shaw, Journal of Biological Chemistry, 243: 2943-2949, 1968) and by a modification which included 0.1 M NaCl in the eluting buffer to reduce the buffer volume (time) required. SDS-PAGE analysis indicated that beta-trypsin prepared by both methods contained appreciable amounts of alpha-trypsin. The following relative amounts of alpha-trypsin were detected at each step of the process: 4.4% eluted from the column into pH 3.0 buffer; 10.7% after concentration on SP-Sephadex; 14.9% after desalting at pH 3.0; 16.1% after lyophilization. These data show that even at pH 3.0, a condition under which trypsin is believed to be inactive, and which is employed for preparation and storage of trypsin solutions, appreciable partial proteolysis can occur to generate the two-chain alpha form from beta-trypsin.

Kinetic evidence for a two-state, hybrid model for the trypsin activation by modifiers.

Kinetic investigation of the activation of alpha- and beta-trypsins by tosyl-L-arginine methyl ester, in the presence of benzamidine at concentrations higher than 1 mM, indicates that each could exist as a two-state hybrid allosteric system, based on at least two enzyme forms, E and E* (or E and F). Form E* shows much higher affinity for ligands, thus giving rise to ternary complexes of the types SE*S, ME*S, ME*M, SE*M, where the right position identifies binding at the active site, whereas the left position represents binding to the allosteric site. Using a large number of experimental points, measured at different combinations of substrate and modifier concentrations, it was possible to evaluate quantitatively the parameters needed for the description of the model with considerable precision. At high concentrations, benzamidine competes with tosyl-L-arginine methyl ester for an allosteric site, blocking substrate activation of beta-trypsin, but allowing activation of alpha-trypsin. These results imply the participation of an induced fit step during ternary complex formation that gives rise to complexes of the form E*S2 or E*SI, where E* stands for the conformationally changed E* in the complex. The overall picture is that of a two-state model combined with induced-fit. Negative cooperativity (nH = 0.80) was found for the binding of benzamidine (over 1 mM) to trypsin in the presence of substrate. The proposed model is allowing the design of experiments that should lead to an understanding of the mechanism of trypsin activation by modifiers.