文昌鱼酸性磷酸酯酶金属辅基初探

从厦门文昌鱼Branchiostoma belcheri(Gray)分离提纯酸性磷酯酶(EC3.1,3.2),进一步经Sephadex G-75和羧甲基纤维素(CM-52)柱层析纯化,获得聚丙烯酰胺凝胶电泳单一纯酶制剂,比活力为351μm/min mg。酶液吸收峰在280nm,320nm,500nm,表现出含铁离子蛋白的特征吸收峰,用原子吸收法和化学法(邻啡绕啉比色法)分析,证明了文昌鱼酸性磷酸酯酶(ACPase)含有铁离子;还原剂Na_2S_2O_4处理浓度提高,酶活力及荧光强度下降,280nm和500nm峰下降,320nm峰消失,以Na_2S_2O_4测定ACPase失活和变性速度常激,结果表明,Na_2S_2O_4的失活常数大于变性带数。

Metallic Prosthetic Group of Acid Phosphatase from Branchiostoma belcheri (Gray)

Acid phosphatase from Branchiostoma beleheri was isolated and purified by ammonium sulfate fractionation,Sephadex G-75 gel filtration and CM-cellulose chromatography under acid condition. The purified enzyme exhibits absorption peaks at 280 nm and 500 nm, with a shoulder at 320 nm. Iron has been demonstrated in the prosthetic group of the enzyme by atomic absorption spec-troscopy and confirmed with O-phenanthroline colorimetry. Denaturation and inactivation rate constants of ACPase by Na2S2O4 has been determined. Results indicated that the rate constant of inactivation in Na2S2O4 was higher than that of denaturetion. Under Na2S2O4 treatment, change of enzyme conformation in accompaniment with the inactivation has been evidenced in fluorescence and UV absorption spectra.