偶联酶催化分光光度法测定黄嘌呤.pdfVIP

《化学通报》在线预览版 偶联酶催化分光光度法测定黄嘌呤 李忠琴 1，2 邬敏辰 1 许小平 1，2# 王武 1 # ( 江南大学工业生物技术教育部重点实验室 无锡 214036 ； 福州大学化学化工学院 福州 350002) 摘 要 研究了以黄嘌呤氧化酶-辣根过氧化物酶-苯酚-4-氨基安替比林反应显色体系，测定不同样液中黄嘌呤浓度的的新方法。确定该测定方法 的最佳反应条件为：黄嘌呤氧化酶(XO) 0.32 U/mL，辣根过氧化物酶(HRP) 7.0U/mL，4-氨基安替比林(AAP) 1mmol/L，苯酚(PA) 6mmol/L 溶于 100mmol/L Tris-HCL 缓冲液(pH8.4) ；反应温度为 37 ℃，保温时间为 20min ；检测波长为 508nm 。本方法测定黄嘌呤浓度的线性范围为 0.2~10.0mmol/L，线性关系良好(R=0.9978)，检测限为 0.05 mmol/L 。该方法操作简单易行，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。 关键词 黄嘌呤氧化酶 辣根过氧化物酶 酶活测定 分光光度 Catalytic Spectrophotometric quantitation for Xanthine By Conjugating Xanthine oxidase with Horseradish Peroxidase 1,2 1 1,2# 1 Li Zhong-qin Wu Mingchen Xu Xiao-ping Wang Wu (#The Key Laboratory of Industry Biotechnology, Ministry of Education, Southern Yangtze University, Wuxi 214036; College of Chemistry and Chemical Engineering, Fuzhou University, Fuzhou 350002) Abstract A novel technology to determine the concentration of Xanthine through the chromogenic reaction of Phenic Acid(PA), 4-Aminoantipyrine(AAP) and hydrogen peroxide (H O ), which was produced via the oxidation of Xanthine catalyzed by Xanthine oxidase(XO), under the help of Horseradish 2 2 Peroxidase(HRP) was proposed in this paper. The influences of temperature and pH on the system were investigated. The optimal conditions to determine the concentration of xanthine were obtained as follows: XO(0.32 U/mL), HRP(7.0U/mL), 4-Aminoantipyrine(1mmol/L), and Phenic Acid(6mmol/L) were dissolved in 100mmol/L Tris-HCl buffer solution (pH8.4), the reaction system was incubated in thermostat of 37 ℃ for 20min, the absorptive wave