Maeda Y, Yamada H, Ueda T, Imoto T
Graduate School of Pharmaceutical Sciences, Kyushu University, Fukuoka, Japan.
Protein Eng. 1996 May;9(5):461-5. doi: 10.1093/protein/9.5.461.
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.
在40℃下，于pH 8.0、4M尿素存在的条件下，通过巯基-二硫键交换反应使还原型溶菌酶复性，反应中添加或不添加添加剂。在不添加添加剂的情况下，还原型溶菌酶的最终折叠产率约为40%。在添加肌氨酸、甘油、硫酸铵、N-乙酰葡糖胺和葡萄糖的情况下，其折叠产率在所有情况下均有所提高。特别是在4M肌氨酸存在的情况下，产率提高到了90%。另一方面，使用差示扫描量热法评估了在0.02M柠檬酸盐缓冲液（pH 6.0）中添加或不添加添加剂时溶菌酶的解链温度。在不添加添加剂的情况下，溶菌酶的解链温度为73.8℃。在添加添加剂的情况下，所有解链温度均高于不添加添加剂时溶菌酶的解链温度。此外，在添加添加剂时，4M尿素存在下还原型溶菌酶折叠产率的增加与无4M尿素时解链温度的增加之间存在良好的相关性。因此，我们得出结论，稳定天然溶菌酶的添加剂在提高4M尿素中还原型溶菌酶的折叠产率方面是有效的。
