Stability Diagram of Lipoxygenase as Determined From H/D Exchange Kinetics and from Conformational Changes

Abstract

The stability of proteins under different conditions of pressure and temperature has already been the subject of many investigations. An elliptical outline of the stability curve can be suggested. In this study, we obtain the stability diagram of lipoxygenase by following the protein conformation with FTIR. Changes in the amide I ((1700-1600 cm^-1) and amide II (1550 + 1450) region were monitored. Plotting midpoints of pressure and temperature induced cooperative changes gave an elliptical outline. In addition to the inactivation measurements of Ludikhuyze, we were interested to see if the typical behaviour could also be found in the hydrogen deuterium (H/D) exchange kinetics. Herefore, we followed the ratio of intensities at 1550 and 1450 cm^-1, which are proportional to the amount of H, resp. D in peptide bonds of the protein. A similar, but not identical, result was obtained. This shows that FTIR is a well suited technique to follow kinetics and thermodynamic equilibria in the protein under different physical conditions. The results of this work are compare with recent work on the inactivation kinetics at different pressures and temperatures.