胍变性人胎盘碱性磷酸酶复性的研究

Study on Renaturation of Quanidine Denatured Human Placental Alkaline Phosphatase

目的:检测胍变性人胎盘碱性磷酸酶复性过程中活力与构象的变化,探讨其折叠机制。材料与方法:应用分光光度法测定复性酶活力,荧光法检测其内源荧光发射光谱。结果:盐酸胍变性平衡态酶用直接稀释法及空气氧气法进行复性,其最适复性条件是:胍稀释浓度0.5mol/L,温度4℃,复性液pH9.0,时间24小时。在此条件下,测得不同浓度盐酸胍变性酶经稀释复性后的酶活力及其内源荧光光谱显示:变性中间态酶复性后的酶活力及内源荧光光谱可恢复至天然态,而变性酶只能部分恢复,且随胍浓度的增加,恢复愈加困难,当胍浓度为6.0mol/L时,变性态酶经复性后的酶活力与内源荧光光谱则完全不能恢复。结论:变性中间态酶可完全复性,而变性态酶仅部分复性,且与复性条件有关。

Objective： To detect change of conformation and activity in the renaturation process of quanidine denatured human placental alkaline phosphatase （HPAP） and investigate the folding mechanism of its peptide. Materials and Methods： To determine activity of human placental alkaline phosphatase in process of renaturation by spectrophotometric method, and emission spectrum of endogeny fluorescence by fluorometric method. Results： Denaturation-equilibrium state HPAP in guanidine hydrochloride solution is renatured by direct dilution and air oxidization, the most suitable renaturation condition is： concentration of guanidine is 0.5 mol/L, 4℃, pH9.0, 24hours. Activity and endogeny fluorescence spectrum of PHAP measured under this condition, which was denatured by different concentration of guanidine hydrochloride and renatured again by dilution, shows that the activity and endogeny fluorescence spectrum of denaturation-intermedial state HPAP are able to renature to nature state, but that of denatured HPAP are only able to renature partly. With increase of quanidine concentration, renaturation becomes more difficult. When quanidine concentration is 6.0 mol/L, the activity and endogeny fluorescence spectrum of denatured HPAP are not completely able to renature. Conclusion： Activity of denaturation-intermedial state HPAP is completely able to renature, activity of denaturation-state HPAP is able to renature partly and related to renaturation condition.