猪脑中硫氧还蛋白还原酶的纯化与表征

Purification and Characterization of Thioredoxin Reductase from Porcine Brain

[目的]用新的方法从猪脑中提取纯化硫氧还蛋白还原酶(TrxR)并对其进行表征。[方法]通过硫酸铵盐析、DEAE-Sepharose Fast Flow弱阴离子交换凝胶色谱柱层析、Blue-Sepharose亲和色谱柱层析、Resource Q强阴离子交换色谱柱层析和Superdex 200 prep grade凝胶过滤色谱柱层析等方法,从猪脑中分离并纯化TrxR。采用聚丙烯酰胺电泳法、等电聚焦法、DTNB还原法和胰岛素还原法对TrxR的性质进行表征。[结果]从猪脑中分离并纯化出TrxR,其纯度大于99%,是酶粗提液的6 000倍,最终产率为13.9%。猪脑TrxR的分子量为56.0 kD,等电点为5.0。以DTNB为底物测得其K m和k cat值分别为62.9μmol/L和467 min-1,以硫氧还蛋白为底物测得其K m和k cat值分别为3.9μmol/L和2 813 min-1。[结论]用新的方法从猪脑中纯化得到较高纯度的TrxR,为进一步研究该蛋白的结构和功能奠定了基础。

[Objective] The aim was to purify and characterize thioredoxin reductase （TrxR） from porcine brain by new methods.[Method]Thioredoxin reductase was purified from porcine brain by ammonium sulfate dialysis,DEAE-Sepharose Fast Flow weak anion exchanger,BlueSepharose affinity column,Resource Q strong anion exchanger and Superdex 200 prep grade gel filtration in sequence.TrxR was collected and characterized by SDS-PAGE,isoelectric focusing analysis,DTNB assay and insulin reduction assay,respectively.[Result] Pure TrxR was obtained with a yield of 13.9％ and a purity of more than 99％,which was 6000-fold purer than that of the homogenate.Molecular size and isoelectronic point of porcine brain TrxR were determined to be 56.0 kD and 5.0,respectively.The kinetic parameters Km and kcat of porcine brain were determined to be 62.9 μmol/L and 467 min 1 using DTNB assay and 3.9 μmol/L and 2 813 min-1 using insulin reduction assay,respectively.[Conclusion] Pure TrxR was purified from porcine brain by new methods.The results will help to further investigate the structure and function of porcine brain TrxR.