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

Abstract

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 β-sheet and helix in its secondary structure and has two domains with similar structures. We assessed the stability of β-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 β-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 (ΔHcal/ΔHvH) of 1.0 to 0.5 in the pH range of 3.2 to 4.2 suggest protein aggregation. In contrast, ΔHcal/ΔHvH 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, β-trypsin unfolded with Tm = 54°C and ΔH = 101.8 kcal/mol, and the change in heat capacity between the native and unfolded forms of the protein (ΔCp) was estimated to be 2.50 ± 0.07 kcal mol-1 K-1. The stability of β-trypsin calculated at 298 K was ΔGD = 5.7 kcal/mol at pH 3.00 and ΔGD = 15.2 kcal/mol at pH 7.00, values in the range expected for a small globular protein.