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.

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.

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

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