氢供体组与辣根过氧化酶活性光谱研究  

A Coupling Hydrogen Donor and Spectroscopy Assay of Horseradish Peroxidase Activity

作　　者：王玮[1] 黄琨[1] 杨卫云[1] 赵莹雪[1] 肖保林[1] 洪军[1] 穆萨维姆瓦赫迪 

机构地区：[1]河南大学生命科学学院,河南开封475000 [2]德黑兰大学生物化学与生物物理研究所

出　　处：《广州化工》2011年第11期46-48,70,共4页GuangZhou Chemical Industry

基　　金：河南大学特聘教授科研启动基金;伊朗国家科学基金资助

摘　　要：对邻苯二酚-苯胺和苯酚-氨基安替吡啉构成的两组测定HRP酶活性的体系进行了研究和比较。紫外可见光光谱和高效液相色谱分析表明,在HRP催化过程中,邻苯二酚-苯胺氢供体对会生成为一种粉红色产物,该产物最大吸收波长在510 nm,可用于HRP的活性测量。且与常规使用的苯酚-氨基安替吡啉氢供体对的情况相比,具有更高的灵敏度、更低的检测限值和很好的重复性,在对HRP分别进行20次酶活测量,所得相对标准偏差仅为2.9%。使用邻苯二酚-苯胺氢供体对的方法可计算得到酶动力学参数Km(12.5 mM)和Vmax(12.2 mM min-1mg-1)。In the presence of hydrogen peroxide, the hydrogen donor couples pyrocatechol - aniline was compared with phenol - aminoantipyrine as chromogens for horseradish peroxidase （HRP） assay using UV - visible spectroscopy and high- performance liquid chromatography analysis. During the HRP biocatalytic process pyrocatechol -aniline was converted to a pink - colored reagent with a λmax of 510 nm. A higher sensitivity and lower detection limit was obtained by using pyrocatechol - aniline relative to those of the phenol - aminoantipyrlne couple, which was commonly used for HRP assay. This method had a satisfactory reproducibility, because the relative standard deviation of 2.9% was obtained for 20 HRP activity measurements. Regarding the superiority of pyrocatechol - aniline, it was suggested to be a better hydrogen donor for the HRP spectrophotometric assay. In addition, kinetic parameters of Km （ 12.5 raM） and Vmax （ 12.2 mM min ^-1· mg^-1 ） were calculated for pyrocatechol - aniline

关 键 词：辣根过氧化物酶 氢供体 邻苯二酚 苯胺 酶活测定 

分 类 号：TQ243.12[化学工程—有机化工]