家兔肝的金属硫蛋白的分离和纯化

Isolation and Purification of Liver Metallothioneins of Rabbit

往皮下注射氯化镉诱导家兔产生金属硫蛋白;取其肝制成匀浆,用无水乙醇和氯仿沉淀杂蛋白,再用三倍体积乙醇沉淀出金属硫蛋白粗品;通过Sephadex G-75柱层析和DEAE-Sephadex A-50离子交换层析,获金属硫蛋白-A和-B纯品。将所得的纯品用原子吸收光谱测定镉离子含量、用高效液相色谱测定纯度、用氨基酸组成分析仪分析氨基酸组成。

In this experiment, rabbit MTs were introduced by ex-posing the rabbit to cadmium chloride throgh hypodermic injection. When the animals were killed, livers and kidneys were got out and made into homogenate. Mixed proteins were precipitated by adding cold anhydrous ethanol and chloroform. To the supernatant obtained by centrifugation, cooling ethanal was added again to make the deposit of MT rough product was then dissolved in the buffer and charged to the Sephadex G-75 column for the primary purification. The MT fraction was collected and again charged to DEAE-Sephadex A-50 ion exchange column with the elution of liner gradien(Tris-HCL buffer). MT-A and MT-B fractions were found. Lyophilizied the product after desalted. About 500mg pur-ifined MT-A and MT-B were finally obtained from the two rabbits.Attomic absorbtion of Cadmium and analysis of Amino Acid composition were also carried out to compare with the present and papers. HPLC was done to determine the purity of the rabbit MTs. Through all the procedures above, it was demonstrated that the MT-A and MT-B were obtioned were quite purity.