摘要
基于H2O2-辣根过氧化物酶(HRP)-邻苯二胺(OPDA)催化反应体系,建立了测定游离及固定化HRP酶活性的循环连续流动分析法(CCFA)。CCFA实现了反应液、反应过程的循环连续检测。用CCFA法对酶催化反应条件进行研究,得到的最佳反应条件是:pH 5.0,反应温度27℃,66.0μmol/L H2O2,2 mg/LOPDA,缓冲液为0.1 mol/L柠檬酸-柠檬酸钠缓冲液;测定游离HRP的线性范围为0～10 U/L,检出限为0.21U/L,RSD<1.03%。使用CCFA法测定了固定化HRP的活性,并对HRP酶促动力学进行了研究,得到的游离HRP的米氏常数为0.078 mmol/L,最大反应速度为0.26 mmol/L(min),催化常数为5.2×104min-1。CCFA法操作简便、准确度高、节省试剂、易于实现自动化。
Based on H2O2-horseradish peroxidase （ HRP）-O-phenyl-diamine （OPDA） enzymatic reaction system, a new method for determining the activity of soluble and immobilized enzyme-circulating continuous flow analysis（CCFA） was established. CCFA is a method that the reaction mixture solution from a circulately flows a flow-through detector and achieves the reaction process. By using the CCFA system, various conditions of HRP enzymatic reaction were optimizated. The experimental conditions are as follows ： the pH is 5.0, reac- tion temperature is 27℃ , the concentration of H202 and OPDA are respectively 66.0 μmol/L and 2 mg/L, the concentration of citric acid - sodium citrate buffer is 0.1 mol/L. The determination range of HRP was in the range of 0 - 10 U/L, and the detection limit was 0.21 U/L, RSD was 1.0%. The activity of immobilized HRP and the kinetics of soluble HRP were investigated by CCFA. The Michaelis constant of H202 obtained was 0. 078 mmol/L, the maximum reaction rate obtained was 0.26 mmol/（ L min） , and catalytic constant obtained was 5.2 ×10^4/min. The CCFA method has many advantages such as simple operation, high sensitivity and good accuracy.
出处
《分析化学》
SCIE
EI
CAS
CSCD
北大核心
2009年第6期897-901,共5页
Chinese Journal of Analytical Chemistry
基金
四川大学214人才引进振兴计划基金(No.0082204127067)资助项目
关键词
循环连续流动分析
辣根过氧化物酶
活性
Circulating continuous flow analysis, horseradish peroxidase, activity
