Bao Y, Chambers S J, Williamson G
Food Molecular Biochemistry Department, Norwich Laboratory, Norwich Research Park, Colney, United Kingdom.
Anal Biochem. 1995 Jan 1;224(1):395-9. doi: 10.1006/abio.1995.1056.
We have developed a method for assaying the activity of phospholipid hydroperoxide glutathione peroxidase (PHGPx) which is both more sensitive and specific than the spectrophotometric assay. The assay is based on the direct detection of the enzymatic product 1-palmitoyl-2-(13-hydroxy-cis-9, trans-11-octadecadienoyl)-L-3-phosphatidylcholine by HPLC. Under the conditions used, baseline separation is achieved for product and substrate. The utility of the method is demonstrated by the measurement of PHGPx activity in crude extracts from human lenses and from human Hep G2 hepatoma cells. This method is also suitable for measuring the specificity of PHGPx for cofactors apart from glutathione. The assay was used to demonstrate that cysteine alone at pH 7.4 mimics PHGPx activity.
我们已经开发出一种检测磷脂氢过氧化物谷胱甘肽过氧化物酶（PHGPx）活性的方法，该方法比分光光度法更灵敏、更具特异性。该检测基于通过高效液相色谱法直接检测酶促产物1-棕榈酰-2-（13-羟基-顺式-9，反式-11-十八碳二烯酰基）-L-3-磷脂酰胆碱。在所使用的条件下，可实现产物和底物的基线分离。通过测量人晶状体和人肝癌细胞系Hep G2粗提物中的PHGPx活性，证明了该方法的实用性。该方法也适用于测量PHGPx对除谷胱甘肽之外的辅因子的特异性。该检测被用于证明在pH 7.4条件下单独的半胱氨酸可模拟PHGPx活性。
