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

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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 In the three-disulfide derivatives, a pair of cys residues that form a disulfide bridge in authentic lysozyme had been replaced by ser or ala residues : DELTA1 (Cys6-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