枸杞果实铜、锌超氧物歧化酶的纯化及其性质的研究

Purification and some Properties of Copper/Zinc Superoxide Dismutase in Lycium Chinense fruit

枸杞果实粗提液中具有Mn-SOD、Fe-SOD和Cu·Zn-SOD三种类型。经氯仿-乙醇处理、丙酮分级沉淀、sepbadex G-100凝胶过滤和DEAE-纤维素柱层析纯化,获得对CN^-敏感的Cu·Zn-SOD。在凝胶电泳染色图谱上,DEAE-纤维素柱层析纯化,获得对CN^-敏感的Cu·Zn-SOD。在凝胶电泳染色图谱上,DEAE-纤堆素柱线性梯度洗脱下来的两个活性组分与酶粗提液同工酶谱上两条Cu·Zn-SOD主区带分别相对应,而且酶活性染色带与蛋白染色带位置对应,说明上述活性组分都已纯化到均一的程度。该酶分子量约为33900道尔顿,亚基分子量约为16700道尔顿。酶的两个活性组分在紫外光区的吸收曲线略有差异,但吸收峰都在255nm。该酶在55℃的温度范围内对热稳定。纯化后的酶平均为4436units/mg protein,纯化了177.6倍,活力回收为12.3%。上述酶没有过氧化氢酶活性。

Detected Mn-SOD,Fe-SOD and Cu·Zn-SOD in crude extract from Lycium Chinense fruit,Purified Cu·Zn-SOD samples was obtained by treatment with chlorofarm-ethanol,precipitation with acetone,gel filtration on Sephadex G-100 and chromatography on DEAE-cellulose[DE52].Two Cu·Zn-SOD constituent were isolated from the soluble fraction in the monogenetic state with the method of linear gradient elution.The molecular weight of the puified emzyme is 33900 daltons as assayed by polyacrylamide gradient gel electrophoresis, that of the enzyme subunit is about 16700 daltons as assayed by sodium dodecyl sulphate polyacrylamide gel electrophoresis. The purified enzyme exhibits one absorption maximum in ultraviolet at 255nm. The enzyme at least up to 55℃ hed stability.DEAE-cellulose Chromatography was used subsequently provided a fraction of Cu·Zn-SOD of 177.6 fold purifition with a specific activity of 4436.4 units/mg protine, on the basis of an assumed 12.3% recovery of total activity.