猪肾γ-谷氨酰转肽酶纯化及其互通性初步研究

目的:从猪肾中纯化γ-谷氨酰转肽酶(GGT),并对其稳定性、互通性进行初步研究,为制备国产GGT参考物建立基础。方法:使用冷丙酮沉淀、硫酸铵处理及层析的方法从猪肾中纯化提取GGT。采用非加速试验方法观察其稳定性,并用国际临床化学协会推荐的参考方法和常规方法,分别对不同基质中的目的蛋白进行测定,根据两者偏离方向和大小与新鲜人血清是否一致判定其互通性。结果:目的蛋白经SDS-PAGE电泳呈单一条带,其GGT特异性比活为150 U/mg。-20℃下在人工基质中保存至少6个月无酶活性损失。高中低不同活性浓度的目的蛋白测定结果未偏离出由多份人血清样本预测的95%置信区间。结论:从猪肾中纯化得到的GGT,具有较高的特异性比活,在人工基质中稳定性和互通性良好。

Primary study on purification of γ-glutamyltransferase from pig kidney and it''s commutability

Objective To purify γ-glutamyltransferase(GGT) from pig kidney and observe it′s commutability and stability.Methods Pig kidney cortex was cut into pieces and homogenized with isotonic saline.Enzyme protein was purified from the treated cortex through DEAE-FF anion exchang and Superdex-75 gel filtration chromatography method.The stability of purified product was observed by means of non-accelerated test.Results We obtained product of GGT protein successfully.The specific activity of GGT protein was 150 U/mg and the recovery rate was 7%.SDS-PAGE showed only one protein band corresponding to the molecular weight of pig kidney GGT.The products dissolved in a matrix containing bovine serum albumin.No loss of activity was observed for 6 months at-20℃.Conclusion Our purification procedure has provided the products of GGT protein with a high specific activity and the protein has satisfied commutability and stability.