Construction of hen lysozyme 3-SS, 2-SS and 1-SS derivatives and the analyzes of their structure and function.

我们研究了在变性和还原条件下从包涵体中纯化的鸡溶菌酶三-二硫键衍生物的氧化复性效率与温度的关系。在三-二硫键衍生物中，在真正的溶菌酶中形成二硫键的一对cys残基被ser或ala残基取代：DELTA1（Cys6->Ser，Cys127->Ser），DELTA2（Cys30->Ser，Cys115->Ser）、DELTA3（Cys64->Ser，Cys80->Ser）、DELTA4（Cys76->Ser，Cys94->Ser）、DELTA2Ala（Cys30->Ala，Cys115->Ala）和DELTA4Ala（Cys76->Ala，Cys94->Ala）。DELTA 1的最适复性温度最低（=19 ℃），DELTA 4Ala的最适复性温度最高（=30 ℃），比标准溶菌酶的最适复性温度低16 ~ 5 ℃。在远紫外圆二色性谱中，每一个复性衍生物都发生了可逆的热变性-复性转变，在pH3.9时转变温度最低的是DELTA 1（=39.2 ℃），最高的是DELTA 4Ala（=51.6 ℃）。三个二硫化物衍生物之间的最佳重折叠温度的差异的精确值被发现与那些在重折叠和纯化的衍生物的转变温度。接下来，研究甘油的作用，其已显示出增加3-SS衍生物的重折叠效率（Sawano等人，FEBS Letters 303，11-14（1992））详细检查了DELTA 1。甘油浓度每增加10%，最适复性温度提高3-4 ℃。在甘油存在下，最佳复性温度的增加量几乎等于复性和纯化的DELTA 1的热稳定性的增加量。总的来说，对于三个二硫键衍生物，最佳复性温度与正确复性状态的热稳定性之间存在平行关系。这一发现为进一步优化复性温度提供了依据

We examined the temperature dependence of the efficiency of oxidative refolding of hen lysozyme three-disulfide derivatives which had been purified from inclusion bodies under denaturing and reducing conditions. In the three-disulfide derivatives, a pair of cys residues that form a disulfide bridge in authentic lysozyme had been replaced by ser or ala residues : DELTA1 (Cys6->Ser, Cys127->Ser), DELTA2 (Cys30->Ser, Cys115->Ser), DELTA3 (Cys64->Ser, Cys80->Ser), DELTA4 (Cys76->Ser, Cys94->Ser), DELTA2Ala (Cys30->Ala, Cys115->Ala) and DELTA4Ala (Cys76->Ala, Cys94->Ala). The optimal refolding temperature was lowest for DELTA1 (=19ﾟC) and highest for DELTA4Ala (=30ﾟC), and was about 16 to 5ﾟC lower than that for authentic lysozyme. A reversible thermal denaturation-renaturation transition in the far UV CD spectra was observed for each refolded derivative which was purified to a single peak with reversed-phase HPLC.The transition temperature at pH3.9 was lowest for DELTA1 (=39.2ﾟC) and highest for DELTA4Ala (=51.6ﾟC). Precise values of difference in the optimal refolding temperature among the three-disulfide derivatives were found to correlate with those in the transition temperature of the refolded and purified derivatives. Next, the effect of glycerol, which has been shown to increase the refolding efficiency of 3-SS derivatives (Sawano et al., FEBS Letters 303, 11-14 (1992)), was examined for DELTA1 in detail. The optimal temperature for the refolding increased by 3-4ﾟC with the increase in glycerol concentration by 10%. The amount of increase in the optimal refolding temperature was nearly equal to the amount of the increase in thermal stability, in the presence of glycerol, of refolded and purified DELTA1. Taken together, there exists a parallel relationship for the three-disulfide derivatives between the optimal refolding temperature and the thermal stability of the correctly-refolded state. The finding gives the basis for the optimization of the refolding temperat