基于H_(2)O_(2)-丁基玫瑰红B-Fe^(2+)体系的荧光猝灭法测定血清过氧化氢酶的活性

Determination of Activity of Serum Catalase by Fluorescence Quenching Method Based on H_(2)O_(2)-Butyl Rose Red B-Fe^(2+)System

利用H_(2)O_(2)-Fe^(2+)可以快速使丁基玫瑰红B(BRMB)发生荧光猝灭,而过氧化氢酶(CAT)对该猝灭有抑制效应,提出了基于H_(2)O_(2)-BRMB-Fe^(2+)体系的荧光猝灭法测定血清CAT活性的方法。取血清样品10μL,加入1 mmol·L^(-1)H_(2)O_(2)底液0.50 mL,于30℃恒温反应15 min,再依次加入0.02 mmol·L^(-1)BRMB溶液0.70 mL、0.1 mol·L^(-1)冰乙酸-乙酸钠缓冲液(pH 4.2)1.00 mL和0.01 mol·L^(-1)Fe^(2+)溶液0.08 mL,于室温反应20 min后用水定容至10 mL,测定体系在590 nm发射波长处的荧光强度F;以煮死酶液为对照,按照同样的方法测定其在590 nm发射波长处的荧光强度F0。结果表明:CAT活性在0.005~0.05 U·mL^(-1)内与体系荧光强度差值F-F0呈线性关系,检出限(3s/k)为1.2×10^(-3)U·mL^(-1);方法用于6份不同年龄健康人群血清CAT活性分析,测定值为31.50~72.30 U·mL^(-1),与碘量法基本一致,CAT活性的加标回收率为95.2%~102%,测定值的相对标准偏差(n=6)为1.7%~3.3%。

Using the principle that the fluorescence of butyl rose red B(BRMB)can be quickly quenched by H_(2)O_(2)-Fe^(2+),and catalase(CAT)can inhibit this quench process,a method for the determination of activity of serum CAT by fluorescence quenching method based on H_(2)O_(2)-BRMB-Fe^(2+)system was proposed.The serum sample(10μL)was taken,and 0.50 mL of 1 mmol·L^(-1)H_(2)O_(2)base solution was added,and the reaction was performed at 30℃for 15 min.Then 0.70 mL of 0.02 mmol·L^(-1)BRMB solution,1.00 mL of 0.1 mol·L^(-1)glacial acetic acid-sodium acetate buffer solution(pH 4.2)and 0.08 mL of 0.01 mol·L^(-1)Fe^(2+)solution were added successively,and the mixed solution was reacted at room temperature for 20 min.After its volume was made up to 10 mL with water,the fluorescence intensity F of system was measured at the emission wavelength of 590 nm.The boiled dead enzyme solution was used as control,and the fluorescence intensity F_(0) was measured at the emission wavelength of 590 nm using the same method.It was shown that linear relationship between CAT activity and fluorescence intensity difference(F-F_(0))was kept in the range of 0.005-0.05 U·mL^(-1),with detection limit(3s/k)of 1.2×10^(-3)U·mL^(-1).The method was applied to analysis of activities of 6 serum CAT from healthy people of different ages,and the determined values were in the range of 31.50-72.30 U·mL^(-1),which were basically consistent with iodimetry.The spiked recoveries of CAT activity were in the range of 95.2%-102%,and RSDs(n=6)of the determined values ranged from 1.7%to 3.3%.