一种用于测定亚皮摩尔量烟酰胺腺嘌呤二核苷酸（NADH）的放射性测量方法。

A radiometric method for the determination of NADH in subpicomole amounts.

作者信息

Weber G, Rosenthal W, Oberdisse E

机构信息

Institute of Pharmacology, Freie Universität Berlin, F.R.G.

出版信息

J Biochem Biophys Methods. 1988 Apr;15(6):295-306. doi: 10.1016/0165-022x(88)90083-8.

DOI:10.1016/0165-022x(88)90083-8

PMID:3379244

Abstract

A radiometric method has been devised for the determination of small quantities of NADH formed in preceding dehydrogenase reactions. In a coupled enzymatic reaction, phosphoglycerate kinase (PGK) catalyzes the transfer of [32P]orthophosphate from [gamma-32P]ATP to 3-phosphoglycerate; the intermediate, 1,3-[1-32P]diphosphoglycerate, is dephosphorylated by glyceraldehyde-3-phosphate dehydrogenase (GAP-DH). [32P]Orthophosphate is released proportionally to NADH and can be measured after adsorption of [gamma-32P]ATP to activated charcoal. With this method, 0.2 pmol of NADH are detectable in the presence of a 10(4)-fold excess of NAD over NADH.

摘要

已设计出一种放射性测量方法，用于测定在前脱氢酶反应中形成的少量NADH。在一个偶联酶反应中，磷酸甘油酸激酶（PGK）催化[γ-32P]ATP中的[32P]正磷酸转移至3-磷酸甘油酸；中间产物1,3-[1-32P]二磷酸甘油酸由甘油醛-3-磷酸脱氢酶（GAP-DH）去磷酸化。[32P]正磷酸与NADH成比例释放，且在[γ-32P]ATP吸附到活性炭上后可进行测量。用这种方法，在NAD比NADH过量10(4)倍的情况下，可检测到0.2皮摩尔的NADH。