催化分光光度法测定辣根过氧化物酶新方法研究

A Novel Catalytic Spectrophotometric Determination for Horseradish Peroxidase

本文基于微量辣根过氧化物酶 (HRP)对过氧化氢氧化还原型罗丹明B这一显色反应的催化作用 ,建立了一个高灵敏度测定HRP及其酶标记结合物的催化光度新方法 ,探讨了该催化反应体系的反应条件及干扰情况 ,实验表明 β CD能显著增强还原型罗丹明B的稳定性 ,邻苯二胺对该催化反应有催化诱导作用。应用于测定痕量辣根过氧化物酶 ,其检出限为 12pg·mL-1,其线性范围为 15～ 2 5 0pg·10mL-1,RSD为4 2 5 % (n =10 )。

A new catalytic-kinetics spectrophotometric method for the determination of horseradish peroxidase was developed. It was based on. the catalytic effect of horseradish peroxidase (HRP) on the coloration reaction, in which the reduced rhodamine B was oxidized by H2O2 in pH 6.80 phosphate buffer. It was found that reduced rhodamine B stocking solution could be steady in 0.25% beta -CD solution. The kinetic behavior of the reaction and the effects of some experimental conditions were investigated and disussed in detail. The method has been applied to determine HRP with a satisfatory result. The calibration curve is linear over the range 15 - 250 pg.10 mL(-1) of HRP (r = 0.998 9). The limit of detection was 12 pg.10 mL(-1) and the relative standard derivative RSD is 4.25% by determing for 20 pg.10 mL(-1) HRP (n = 10).