A slow fluctuation in the molecular conformation of a soya bean trypsin inhibitor

The kinetics of the hydrogen-deuterium exchange reaction in a trypsin inhibitor (Kunitz) from soya bean have been followed by infrared absorption measurements in aqueous solutions at various temperatures and pH values. It was found that, in every case, 49% of the total peptide hydrogen atoms exchange relatively slowly. This amount corresponds to 83 peptide groups per molecule, and this is considered to be equal to the number of peptide NH groups involved in hydrogen bonds with the carbonyls of other peptide groups in the protein molecule in its native form. Each rate constant (k) determined at pH 2.75 for this category of the NH groups is in good agreement with the value expected from an idea that the breaking of the peptide-peptide hydrogen bonds takes place very slowly, and that this is the rate-determining process in the hydrogen-deuterium exchange reaction. Thus, by ultraviolet absorption measurements at 297 nm, the equilibrium constant of the native and denatured forms has been determined in the temperature range from 42 to 53.5 °C, as well as the reaction rate of reaching equilibrium from an off-equilibrium state. From these data the rate constant (k₁) of the denaturation reaction is determined, and the k₁ value is found to be practically equal to the hydrogen exchange rate constant (k). The Arrhenius plot of this rate constant (k) gives a straight line in the 25 to 55 °C region, and this gives a value of 48.6 kcal/mol for the activation energy of the denaturation reaction. The rate of this reaction is found to be very low at 25 °C; its half-life is about eleven days. Infrared absorption spectra observed in the amide I region suggest that the very slow denaturation of this protein is accompanied by a conformation change from an α-helix to a β-form. The number of the peptide groups involved in this α → β change is estimated to be 9 ± 3.