Effect of ethanol on folding of hen egg-white lysozyme under acidic condition

The equilibrium and kinetics of folding of hen egg‐white lysozyme were studied by means of CD spectroscopy in the presence of varying concentrations of ethanol under acidic condition. The equilibrium transition curves of guanidine hydrochloride‐induced unfolding in 13 and 26% (v/v) ethanol have shown that the unfolding significantly deviates from a two‐state mechanism. The kinetics of denaturant‐induced refolding and unfolding of hen egg‐white lysozyme were investigated by stopped‐flow CD at three ethanol concentrations: 0, 13, and 26% (v/v). Immediately after dilution of the denaturant, the refolding curves showed a biphasic time course in the far‐UV region, with a burst phase with a significant secondary structure and a slower observable phase. However, when monitored by the near‐UV CD, the burst phase was not observed and all refolding kinetics were monophasic. To clarify the effect of nonnative secondary structure induced by the addition of ethanol on the folding/unfolding kinetics, the kinetic m values were estimated from the chevron plots obtained for the three ethanol concentrations. The data indicated that the folding/unfolding kinetics of hen lysozyme in the presence of varying concentrations of ethanol under acidic condition is explained by a model with both on‐pathway and off‐pathway intermediates of protein folding. Proteins 2006. © 2006 Wiley‐Liss, Inc.