乙醇对锯缘青蟹碱性磷酸酶活力与构象的影响

Effect of ethanol on activity and conformation of alkaline phosphatase from green crab (Scylla serrata)

应用荧光光谱、紫外吸收光谱研究锯缘青蟹碱性磷酸酶经乙醇微扰后的分子构象变化情况。乙醇对酶分子构象有显著的影响 ,酶的内源荧光强度随乙醇浓度增大而增强 ,荧光发射峰逐渐发生红移 ,说明酪氨酸、色氨酸残基的微环境发生明显的变化 ;紫外吸收光谱在 2 78nm吸收峰随乙醇浓度增大而增强。这些结果表明 ,酶蛋白分子中的生色基团残基的微环境发生变化 ;酶的剩余活力随着乙醇浓度增大而迅速下降 ,经乙醇失活的酶在正常的测活体系中监测酶活力 ,发现酶活力可以回升到 92 %以上 ,说明酶的乙醇失活是可逆的 ,可以通过稀释复活。测定乙醇对酶的抑制动力学 ,表明乙醇对青蟹碱性磷酸酶的抑制作用是反竞争性机制 ,其抑制常数Ki为 1 1 .8%。

Conformational changes of green crab alkaline phosphatase in ethanol solutions of different concentrations were measured by fluorescence spectra and ultraviolet absorption spectra. The fluorescence emission peak intensity of the enzyme gradually strengthened with increasing ethanol concentrations, accompanied by the peak being gradually red-shifted. The ultraviolet absorption spectra peak at 278 nm of the enzyme denatured in ethanol solutions of different concentrations increased with increasing ethanol concentrations. Those results were in concordance with the conformational change of the microenvironments of tyrosine and tryptophan. The enzyme activity rapidly declined increasing ethanol concentrations. But when diluted, the inactivated enzyme can be resurrected. The inactivation of the enzyme in ethanol solutions is reversible reaction. The inhibition of ethanol on the enzyme is found to be uncompetitive type and its inhibition constant is found to be 11.8 %.