基于H2O2-丁基玫瑰红B-Fe2+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 H2O2-Butyl Rose Red B-Fe2+ System
蒲文静 1白莹 1董娜 1张爱菊 1张小林1
作者信息
摘要
利用H2O2-Fe2+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 H2O2-BRMB-Fe2+体系的荧光猝灭法测定血清CAT活性的方法.取血清样品10 μL,加入1 mmol·L-1 H2O2 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 Fe2+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%.
Abstract
Using the principle that the fluorescence of butyl rose red B(BRMB)can be quickly quenched by H2O2-Fe2+,and catalase(CAT)can inhibit this quench process,a method for the determination of activity of serum CAT by fluorescence quenching method based on H2O2-BRMB-Fe2+ system was proposed.The serum sample(10 μL)was taken,and 0.50 mL of 1 mmol·L-1 H2O2 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 Fe2+ 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 F0 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-F0)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%.
关键词
过氧化氢酶/活性/H2O2/丁基玫瑰红B/荧光Key words
catalase/activity/H2O2/butyl rose red B/fluorescence引用本文复制引用
基金项目
甘肃省教育厅青年博士基金(2021QB-131)
甘肃省高等学校创新基金(2021B-341)
出版年
2024
NETL
NSTL
万方数据