Norman H A, Pillai P
Weed Science Laboratory, U.S. Department of Agriculture, Beltsville, Maryland, 20705, USA.
Anal Biochem. 1996 May 15;237(1):30-6. doi: 10.1006/abio.1996.0196.
A method is described for the determination of glutathione reductase activity (GR; EC 1.6.4.2) in plant extracts utilizing HPLC quantitation of NADP+ following the reduction of glutathione disulfides. After protein incubation, fluorescence of NADP+ was induced under strongly basic conditions, and the product was directly resolved from the reaction medium by isocratic reversed-phase elution on a silica-coated alumina support which took 2 min. The mobile phase was acetonitrile-water (50:50) delivered at a flow rate of 1.5 ml/min. The adduct (stable for at least 7 days) was detected fluorometrically and quantitated by direct integration of peak area.
描述了一种利用高效液相色谱法（HPLC）对谷胱甘肽二硫化物还原后产生的NADP⁺进行定量，来测定植物提取物中谷胱甘肽还原酶活性（GR；EC 1.6.4.2）的方法。蛋白质孵育后，在强碱性条件下诱导NADP⁺产生荧光，产物通过在硅胶包覆的氧化铝载体上进行等度反相洗脱，直接从反应介质中分离出来，此过程耗时2分钟。流动相为乙腈 - 水（50:50），流速为1.5毫升/分钟。通过荧光法检测加合物（至少稳定7天），并通过峰面积直接积分进行定量。
