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 ß-sheet and helix in its secondary structure and has two domains with similar structures. We assessed the stability of ß-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 ß-alanine and 20.0 mM CaCl 2 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 (∆H cal /∆H vH ) of 1.0 to 0.5 in the pH range of 3.2 to 4.2 suggest protein aggregation. In contrast, ∆H cal /∆H vH 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, ß-trypsin unfolded with Tm = 54 º C and ∆H = 101.8 kcal/mol, and the change in heat capacity between the native and unfolded forms of the protein (∆Cp) was estimated to be 2.50 ± 0.07 kcal mol -1 K -1 . The stability of ß-trypsin calculated at 298 K was ∆G D = 5.7 kcal/mol at pH 3.00 and ∆G D = 15.2 kcal/mol at pH 7.00, values in the range expected for a small globular protein.
The quantitative character of biochemistry imposes some familiarization of the student with analytical practice in laboratory work and data analysis. This article describes the application of an inexpensive home-made precision titrator for titrant standardizations, introducing the students to amino acid and protein pH titrations and data analysis.
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.
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