酶催化分光光度法测定蛋白质

Spectrophotometric Determination of Protein Based on the Enzymatic Catalytic Reaction

基于血清白蛋白对辣根过氧化物酶(HRP)催化H2O2氧化邻苯二酚紫的反应具有强烈的抑制作用,建立了一种测定血清白蛋白的新方法。在优化的实验条件下:pH4.0NaAc-HAc缓冲介质,(30±0.2)℃恒温反应10min,以430nm为测定波长,本方法测定牛血清白蛋白(BSA)线性范围为(0.4～5.0)×10-7mol/L;检出限为8.6×10-9mol/L。以稳态速率法测定了米氏常数(Km)和米氏速率(Vm),确定其抑制类型为竞争性抑制。方法用于人血清样品中蛋白质的测定,操作简便,灵敏度高,选择性好,结果满意。

An enzymatic method for the determination of protein was proposed based on the inhibition of protein for the catalytic reaction of H2O2 and pyrocatechol violet by horseradish peroxidase （HRP）. Under the optimum conditions （pH 4.0 NaAc-HAc buffer solution, 30 ± 0.2℃ , λmax = 430nm） , the chang of absorbances was linear to the bovine serum albumin （BSA） concentration in the range of （0.4 -5.0） × 10^-7 mol/L. The detection limit was 8.57 × 10^-9mol/L. The Michaelis-menten parameters Km and Vm were measured by steady-state catalytic velocity. The results demonstrate that this inhibitory action of protein is a competitive inhibition. The proposed method has been applied to the determination of total proteins in human serum samples with satisfactory results.