基于次血红素六肽的过氧化氢和葡萄糖灵敏比色方法

doi:10.11944/j.issn.1000-0518.2019.07.190047

摘要/Abstract

摘要：

采用次血红素六肽(DhHP-6)催化H₂O₂氧化4-氨基安替吡啉-氯代苯酚显色体系,建立了测定H₂O₂和葡萄糖的方法。研究了pH值、底物浓度和DhHP-6浓度对实验的影响,检测了比色方法的反应线性、稳定性、相关性和回收率。在最佳反应条件下,DhHP-6在不同时间及温度条件下,活性要优于过氧化物酶(POD);DhHP-6催化H₂O₂ 的米氏常数(K_m)和最大反应速率(v_max)分别为0.171 mmo/L和4.22×10^-6 mol/s;H₂O₂响应的线性范围为0.39~25.0 mmol/L;高、中、低 3水平测定的变异系数(CV)和加标回收率分别在1.29%~2.16%和94.5%~101.1%之间;与葡萄糖商品试剂盒比较相关系数R²=0.9946;36例血液样品中的葡萄糖浓度在4.26~17.48 mmol/L之间。与葡萄糖检测商品试剂盒之间的两组数据经统计差异不显著(P>0.05)。该方法是一种简单、廉价、方便、灵敏的比色测定方法。

关键词: 过氧化氢, 次血红素六肽, 模拟酶, 比色分析

Abstract:

A simple colorimetric method for determination of H₂O₂ and glucose was established based on the peroxidase-like activity of deuterohemin-Ala His Thr Val Glu Lys(DhHP-6) to catalyze the oxidation of 4-aminoantipyrine(4-APP)-chlorophenol color reaction system in the presence of H₂O₂. The effects of pH, substrate concentration and DhHP-6 concentration were observed, and the reaction linearity, stability, correlation and recovery rate of the colorimetric method were detected. Under optimal reaction conditions, the activity of DhHP-6 is superior to that of peroxidase(POD) under different temperatures and times; the Michaelis constant(K_m) and maximum reaction rate(v_max) of DhHP-6 catalyzing H₂O₂ are 0.171 mmol/L and 4.22×10^-6 mol/s, respectively; the linearity range of H₂O₂ response is 0.39~25.0 mmol/L, the coefficient of variation(CV) and spiked recovery of the high, medium and low levels are between 1.29%~2.16% and 94.5%~101.1%, respectively; and correlation coefficient with the commercial kit is 0.9946; the concentration of glucose in 36 blood samples are 4.26~17.48 mmol/L. There is no significant difference between the two sets of data for glucose assay kit(P>0.05). Therefore, this is a simple, cheap, convenient and sensitive colorimetric determination method.

Key words: hydrogen peroxide, deuterohemin-Ala His Thr Val Glu Lys, mimetic enzyme, colorimetric analysis

石鑫,刘传志,宫平,李伟,侯玥. 基于次血红素六肽的过氧化氢和葡萄糖灵敏比色方法[J]. 应用化学, 2019, 36(7): 847-854.

Xin SHI,Chuanzhi LIU,Ping GONG,wei LI,Yue HOU. Hydrogen Peroxide and Glucose Sensitive Colorimetric Method Based on Deuterohemin-Ala His Thr Val Glu Lys[J]. Chinese Journal of Applied Chemistry, 2019, 36(7): 847-854.

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