Effect of additives on the renaturation of reduced lysozyme in the presence of 4 M urea.

Abstract

Reduced lysozyme was renatured by sulfhydryl-disulfide interchange reactions at pH 8.0 in the presence of 4 M urea, with or without additives at 40 degrees C. In the absence of additives, the final folding yield of reduced lysozyme was approximately 40%. In the presence of sarcosine, glycerol, ammonium sulfate, N-acetyl glucosamine and glucose, its folding yields increased in all cases. In particular, yields increased up to 90% in the presence of 4 M sarcosine. On the other hand, the melting temperatures of lysozyme with or without additives in 0.02 M citrate buffer (pH 6.0) were evaluated using differential scanning calorimetry. In the absence of additive, the melting temperature of lysozyme was 73.8 degrees C. In the presence of additives, all melting temperatures were higher than that of lysozyme in the absence of additives. Moreover, there was a good correlation on addition of additives between an increase in the folding yield of reduced lysozyme with 4 M urea and an increase in the melting temperature without 4 M urea. Therefore, we conclude that additives, which stabilize native lysozyme, are effective at increasing the folding yield of reduced lysozyme in 4 M urea.