吡罗红为底物的辣根过氧化物酶催化荧光反应测定葡萄糖

Horseradish-peroxidase Catalytic Fluorescence Reaction with Pyronine B as Substrate for Determination of Glucose

吡罗红在辣根过氧化物酶催化下可被过氧化氢氧化而使其荧光猝灭。在pH 7.2中性介质中,稳态催化速率由酶和底物浓度决定,催化体系服从Michaelis-Menten方程,用Lineweaver-Burk作图法求得米氏常数、最大反应速度、催化常数分别为2.4×10^(-5)mol·L-1,2.5×10^(-6)mol·L-1s-1,75.8 s-1。在最佳反应条件下,荧光F0/F的猝灭程度与过氧化氢浓度在0-3.6×10^(-7)mol·L-1范围内成线性关系,检测限为6.3×10^(-9)mol·L-1;当与葡萄糖氧化酶联用时,可定量检测葡萄糖,线性关系为0-8.0×10^(-7)mol·L-1,检测限为3.4×10^(-8)mol·L-1。方法用于分析人血清中葡萄糖含量,分析结果与苯酚-4-氨基安替比林法基本一致。

Pyronine B was used as a fluorogenic substrate for the determination of hydrogen peroxide and glucose based on the catalytic effect of horseradish peroxidase. The fluorescence of pyronine B was quenched by hydrogen peroxide in the oxidation reaction by the catalysis of HRP at pH 7.2. The steady-state catalytic rate depends upon enzyme and substrate concentrations, and the Michaelis-Menten parameters K-max, V-max and K-cat are 2.4 x 10(-5) mol.L-1, 2.5 x 10(-6) mol.L(-1)s(-1) and 75.8 s(-1), respectively. Under optimum con-ditions, a linear relationship between the fluorescence quenching (F-0/F) and the concentration of hydrogen peroxide was observed in the range of 0 to 3.6 x 10(-7) mol.L-1. The limit of detection (3sigma) was determined to be 6.3 x 10(-9) mol.L-1. By coupling with glucose-oxidasecatalytic reaction, glucose was quantified in the linear range of 0 to 8.0 x 10(-7) mol.L-1 with the limit of detection (3sigma) of 3.4 x 10(-9) mol.L-1. The glucoses in human serum were analyzed with the same results as by the phenol-4-aminoantipyrine method.