Raman spectroscopic studies of hen egg-white lysozyme at high temperatures and pressures

In situ high-temperature, high-pressure Raman experiments on 3 mM (pH 5) aqueous solutions of hen egg-white (HEW) lysozyme show a decrease in the relative height of the 505 cm-1 band associated with S-S stretching vibrations at 72 degrees C (1 bar). The peak height changes are accompanied by significant band broadening, and the integrated band intensity does not change within experimental error. The effect of increased pressure at 72 degrees C was to hinder broadening of the 505 cm-1 band. HEW lysozyme (2.4 mM, pH 5) was also heated at 76 degrees C, 80 degrees C, and 95 degrees C for different periods of time, and aliquots were quenched to room temperature for Raman and enzymatic activity measurements. After 9 hr at 76 degrees C, the protein exhibits enzyme activity less than 50% of the initial value, and approximately 50% reduction in activity is achieved after 3 hr at 80 degrees C or 1 hr at 95 degrees C. The Raman results suggest that different irreversibly denatured conformations are attained during prolonged exposures at these different temperatures. It is apparent from these studies that the S-S stretch intensity is decreased irreversibly.