猪晶状体醛糖还原酶的纯化及电喷雾质谱分析

Purification and electrospray mass spectrometry of aldose reductase from pig lens.

作者信息

Reymann J M, Rondeau J M, Barth P, Jaquinod M, Van Dorsselaer A, Biellmann J F

机构信息

Laboratoire de Chimie Organique Biologique, Université Louis Pasteur, Strasbourg, France.

出版信息

Biochim Biophys Acta. 1992 Jul 13;1122(1):1-5. doi: 10.1016/0167-4838(92)90119-x.

DOI:10.1016/0167-4838(92)90119-x

PMID:1633191

Abstract

Aldose reductase (alditol: NADP+ 1-oxidoreductase, EC 1.1.1.21) has been purified from pig lens to homogeneity by a rapid and efficient three-step procedure involving poly(ethylene glycol) fractionation, ion-exchange chromatography and chromatofocusing. The homogeneity of the purified enzyme was examined by polyacrylamide gel electrophoresis under native and denaturing conditions, by isoelectric focusing and by high-performance liquid chromatography on a size-exclusion column. The highly purified enzyme is a monomeric protein with a molecular mass of 35,775 +/- 3 Da as determined by electrospray mass spectrometry (ESMS). This purification procedure is particularly suited for the preparation of triclinic single crystals of pig lens aldose reductase, which are currently used in X-ray studies of this enzyme.

摘要

已通过一个快速高效的三步程序从猪晶状体中纯化出醛糖还原酶（醛糖醇：NADP⁺ 1-氧化还原酶，EC 1.1.1.21），使其达到同质状态，该程序包括聚乙二醇分级分离、离子交换色谱和色谱聚焦。通过在天然和变性条件下的聚丙烯酰胺凝胶电泳、等电聚焦以及在尺寸排阻柱上的高效液相色谱法来检测纯化酶的同质性。通过电喷雾质谱（ESMS）测定，高度纯化的酶是一种单体蛋白，分子量为35,775 ± 3 Da。此纯化程序特别适合用于制备猪晶状体醛糖还原酶的三斜单晶，目前该单晶用于对该酶的X射线研究。