一种用于丙酮酸的灵敏荧光测定法。
A sensitive fluorimetric assay for pyruvate.
机构信息
Department of Ophthalmology and Visual Sciences, University of Michigan Medical School, Ann Arbor, MI 48105, USA.
出版信息
Anal Biochem. 2010 Jan 1;396(1):146-51. doi: 10.1016/j.ab.2009.09.017. Epub 2009 Sep 11.
Abstract
A sensitive and specific fluorimetric assay for the determination of pyruvate is reported here. This assay is based on the oxidation of pyruvate in the presence of pyruvate oxidase. Hydrogen peroxide generated by pyruvate oxidase reacts with nonfluorescent Amplex Red at a 1:1 stoichiometry to form the fluorescent product, resorufin. The assay is optimized with respect to pH of reaction buffer, enzyme concentration, dye concentration, and the time course. The usefulness of the assay is demonstrated by the accurate measurement of intracellular and extracellular pyruvate concentrations. The limit of detection of the assay is 5 nM.
摘要
本文报道了一种用于测定丙酮酸的灵敏且特异的荧光测定法。该测定法基于在丙酮酸氧化酶存在下丙酮酸的氧化。由丙酮酸氧化酶生成的过氧化氢与非荧光的 Amplex Red 以 1:1 的化学计量比反应，形成荧光产物，即 Resorufin。该测定法在反应缓冲液 pH 值、酶浓度、染料浓度和时间进程方面进行了优化。该测定法通过准确测量细胞内和细胞外丙酮酸浓度得到了验证。该测定法的检测限为 5 nM。
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