【摘要】 目的观测人胎盘碱性磷酸酶(HPAP)在盐酸胍变性和复性过程中构象与活力的变化,探讨其折叠机制。方法应用荧光光度法检测HPAP构象变化,用分光光度法检测其活力的变化。结果低浓度的盐酸胍(<0.5 mol/L)使酶的荧光强度略有下降,最大发射波长不变,仍为340 nm,酶活力增强;随盐酸胍浓度的增加,其荧光强度升高,最大发射波长明显红移,酶活力迅速下降;当盐酸胍浓度增至3.0 mol/L时,最大发射波长停止于365 nm,酶活力丧失,但其荧光强度在胍浓度增至5.0 mol/L时才停止变化。结论HPAP在变性过程中存在三态;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。更多还原

【Abstract】 Objective To examine the changes of conformation and activity of human placental alkaline phosphatase(HPAP) in denaturation and renaturation of guanidine chloride,and to study its folding mechanism. Methods Spectrofluorimetry and spectrophotometry was used to determine the changes of conformation and activity of HPAP,respectively. Results The fluorescence emission intensity of low concentration guanidine chloride(lower than 0.5 mol/L) decreased a little,without any red shift of emission maximum,and the wave length remained 340 nm,accompanied by a marked increase of HPAP activity.With the increase of guanidine concentration,fluorescence emission intensity increased,emission maximum shifted red and the enzyme was rapidly inactivated.At 3.0 mol/L,emission maximum was red-shifted from 340 nm to 365 nm and the enzyme was completely inactivated.Fluorescence emission intensity stopped to increase at 5.0 mol/L. Conclusion A three-state unfolding mechanism is involved in the denaturation process.The intermediated state HPAP is completely refolding.The denaturated state HPAP is partially refolding and its refolding rate is related to refolding conditions.更多还原