双酶偶联催化分光光度法测定血清次黄嘌呤  

Catalytic Spectrophotometric Quantitation for Hypoxanthine by Conjugating Xanthine Oxidase with Horseradish Peroxidase

作　　者：李忠琴[1] 许小平[1] 王武[1] 

机构地区：[1]江南大学工业生物技术教育部重点实验室

出　　处：《光谱学与光谱分析》2008年第9期2169-2172,共4页Spectroscopy and Spectral Analysis

基　　金：工业生物技术教育部重点实验室基金项目(KLIB-KF200502);福建省自然科学基金项目(2008J0100)资助

摘　　要：依据黄嘌呤氧化酶和辣根过氧化物酶催化反应的特征,研究了以黄嘌呤氧化酶-辣根过氧化物酶-苯酚-4-氨基安替比林为反应显色体系,建立了检测血清和组织中次黄嘌呤浓度的新方法。通过对该测定体系影响因素的考察,确定最佳反应条件为：黄嘌呤氧化酶（XO,EC1.2.3.22）0.32U·mL^-1,辣根过氧化物酶（HRP）7.0U·mL^-1,4-氨基安替比林（AAP）1mmol·L^-1,苯酚（PA）6mmol·L^-1溶于100mmol·L^-1 Tris-HCL缓冲液（pH8.3）;反应温度为37℃,保温时间为8min;检测波长为508nm。测定次黄嘌呤浓度的线性范围为0.2～3.0mmol·L^-1,线性关系良好（r=0.9979）,检测限为0.05mmol·L^-1。方法操作简单易行,测定结果准确可靠。可有效应用于普通实验室和常规临床血液生化检测。A novel technology to determine the concentration of hypoxanthine through the chromogenic reaction of phenic acid （PA）, 4-aminoantipyrine（AAP） and hydrogen peroxide （H2O2）, which was produced via the oxidation of hypoxanthine catalyzed by xanthine oxidase（XO）, under the help of horseradish peroxidase（HRP） was proposed in the present paper, according to the reaction of molybdoenzyme xanthine oxidase （XOD, EC 1.2.3.22） which mainly catalyzes the conversion of hypoxanthine and xanthine to xanthine and uric acid, respectively, and is capable of reducing oxygen to generate the reactive oxygen species （ROS）, superoxide and hydrogen peroxide. The influences of temperature and pH on the system were investigated. The optimal conditions to determine the concentration of hypoxanthine were obtained as follows： XO（0.32 U·mL^-1）, HRP（7.0U·mL^-1）, 4-Aminoantipyrine（1 mmol·L^-1）, and phenic acid（6 mmol·L^-1） were dissolved in 100 mmol·L^-1 Tris-HCl buffer solution （pH 8.3）, and the reaction system was incubated in thermostat of 37 ℃ for 8 min, the absorptive wave length was 508 nm. Under the conditions mentioned above, the linear range of calibration curve was between 0.2 and 3.0 mmol·L^-1, the correlation coefficient was 0.997 9, and the limit of detection was 0.05 mmol·L^-1. All these show that this technology is a potential alternative method to determine the concentration of hypoxanthine in areas like for example in laboratory or clinical serum diagnosis.

关 键 词：黄嘌呤氧化酶 辣根过氧化物酶 次黄嘌呤检测 分光光度 

分 类 号：O657.3[理学—分析化学]