RENATURATION OF LYSOZYME - TEMPERATURE-DEPENDENCE OF RENATURATION RATE, RENATURATION YIELD, AND AGGREGATION - IDENTIFICATION OF HYDROPHOBIC FOLDING INTERMEDIATES

Renaturation of denatured-reduced hen egg white lysozyme was analyzed at temperatures between 4 and 70°C using the reduced/oxidized glutathione renaturation system. With an increase in temperature to 50°C both renaturation rate constant and renaturation yield increased while formation of aggregates decreased. Denatured-reduced lysozyme and early folding intermediates were less stable against heat than native lysozyme at temperatures above 60°C. Renaturation at 70°C resulted in no reconstitution of lysozyme activity but the highest level of aggregation. Renaturation of denatured-reduced hen egg white lysozyme was further analyzed in the presence of the hydrophobicity-indicating fluorescence dye 1-anilinonaphalene-8-sulfonate at temperatures between 10 and 40°C. The change in fluorescence intensity, the generation of enzyme activity, renaturation yield, and the formation of aggregates were studied. The results showed that early folding intermediates possess a strong hydrophobic nature. With an increase in temperature both the renaturation rate and the decay rate of hydrophobicity-mediated fluorescence increased. Consequently, with increasing temperature, accumulation of hydrophobic folding intermediates and formation of insoluble aggregates decreased, leading to an increase in the renaturation yield. © 1993 Academic Press, Inc.