杂色鲍碱性磷酸酶在DMSO溶液中的活力与构象变化的研究

Effect of Dimethyl Sulfxide(DMSO) on Activity and Conformation of Alkaline Phosphatase from Haliotis diversicolor

以二甲亚砜(DMSO)为效应物,研究其对杂色鲍碱性磷酸酶(ALP)活力的影响,结果表明:该酶的剩余活力随着DMSO浓度增大而迅速下降,当DMSO浓度达40%,酶活力几乎完全丧失.说明DMSO对杂色鲍ALP有明显的失活作用.导致酶活力丧失50%的DMSO浓度为17%.在较低DMSO浓度(<20%)的失活是可逆的反应过程.动力学研究表明,该酶的失活过程属于混合型.进一步测定游离酶(E)和酶底物络合物(ES)与DMSO的结合常数(KI和KIS),结果表明KI<KIS,说明底物存在对酶被DMSO的失活作用有一定的保护作用.应用荧光发射光谱研究杂色鲍ALP经DMSO微扰后的分子构象变化情况,随着DMSO浓度增大,荧光强度逐渐增强,但发射峰未发生明显变化现象,说明酶分子中的生色基团残基的微环境发生了一定的变化.

The effect of dimethyl sulfxide （DMSO） on the activity of Haliotis diversicolor alkaline phosphatase （ALP） was studied. The results showed that the enzyme activity rapidly declined with increasing DMSO concentrations. The inactivation of the enzyme in DMSO solutions was reversible reaction. The inhibition of DMSO on the enzyme was found to be mixed type and the presence of substrate had protection on the enzyme inhibited by DMSO. The conformational changes of the enzyme in different concentrations of DMSO solution were measured by fluorescence absorption spectra. The results showed that the fluorescence emission peak in intensity of the enzyme gradually strengthened with increasing DMSO concentrations. Those results were in concordance with the conformational changes of the microenvironments of tyroslne and tryptophan residues of the enzyme. It can be recognized that conformational integrity is important for maintaining the activity of the enzyme and slight changes at the activity site could lead to inactivation of the enzyme.