从哺乳动物红细胞中快速纯化葡萄糖-6-磷酸脱氢酶。

Rapid purification of glucose-6-phosphate dehydrogenase from mammal's erythrocytes.

作者信息

Ninfali P, Orsenigo I, Baronciani L, Rapa S

机构信息

Institute of Biological Chemistry G. Forniani, University of Urbino, Italy.

出版信息

Prep Biochem. 1990;20(3-4):297-309. doi: 10.1080/00327489008050202.

DOI:10.1080/00327489008050202

PMID:2287610

Abstract

A procedure for rapid purification to homogeneity of glucose-6-phosphate dehydrogenase (G6PD) is herein presented. Our method is not new, but represents a simplification of the method of De Flora et al. (Arch. Biochem. Biophys. 169, 362-3, 1975) which consisted of three steps: DEAE-Sephadex, phosphocellulose (P11) and affinity chromatography on 2'5' ADP-Sepharose. These authors eluted the enzyme from the P11 with phosphate and from 2'5' ADP-Sepharose with KC1 and NADP. By our method, the DEAE-Sephadex step is omitted, the G6PD is eluted from P11 with citrate and NADP, and from 2'5' ADP-Sepharose with KC1, NADP and EDTA. The elution of the enzyme from the phosphocellulose was studied in detail and the temperature effect has been described. We report here an application of this method to a rapid microscale purification starting from 3.5-4 ml of rabbit blood, which can be performed in about 8 hours and a macroscale purification starting from 180-200 ml of human blood, which takes a day and a half.

摘要

本文介绍了一种将葡萄糖-6-磷酸脱氢酶（G6PD）快速纯化至同质的方法。我们的方法并非新颖，但它是对De Flora等人（《生物化学与生物物理学文献》169, 362 - 3, 1975）方法的简化，后者包括三个步骤：DEAE - 葡聚糖凝胶、磷酸纤维素（P11）以及在2',5'-ADP - 琼脂糖上的亲和层析。这些作者用磷酸盐从P11上洗脱酶，并用氯化钾和烟酰胺腺嘌呤二核苷酸磷酸（NADP）从2',5'-ADP - 琼脂糖上洗脱酶。通过我们的方法，省略了DEAE - 葡聚糖凝胶步骤，用柠檬酸盐和NADP从P11上洗脱G6PD，并用氯化钾、NADP和乙二胺四乙酸（EDTA）从2',5'-ADP - 琼脂糖上洗脱。详细研究了从磷酸纤维素上洗脱酶的过程，并描述了温度效应。我们在此报告该方法在快速微量纯化中的应用，起始材料为3.5 - 4毫升兔血，约8小时即可完成；以及在大量纯化中的应用，起始材料为180 - 200毫升人血，耗时一天半。