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.