Assay of protein-bound lipoic acid in tissues by a new enzymatic method
Assay of protein-bound lipoic acid in tissues by a new enzymatic method
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DOI：
10.1006/abio.1998.2615
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发表时间：
1998-05-01
影响因子：
2.9
通讯作者：
Konishi, T
中科院分区：
文献类型：
--
作者：
Akiba, S;Matsugo, S;Konishi, T
建立了测定蛋白质结合硫辛酸的新酶法。通过蛋白酶消化以硫辛酰赖氨酸的形式释放结合的硫辛酰基团，并通过硫辛酰胺脱氢酶（NADH：硫辛酰胺氧化还原酶，EC 1.8.1.4）介导的NADH氧化进行测定。 NADH 氧化与硫辛酰二硫基团的还原相结合。添加谷胱甘肽二硫化物可显着增强 NADH 氧化的荧光动力学，从而循环利用酶介导的硫辛酰/二氢硫辛酰转化。在存在大量过量的谷胱甘肽二硫化物的情况下，NADH 氧化在硫辛酸赖氨酸浓度方面遵循伪一级动力学。在高达5μM的氧化速率和硫辛酸赖氨酸浓度之间获得了良好的线性相关性，并且校准曲线表明检测限可以为100nM硫辛酸赖氨酸。该方法适用于牛、大鼠和兔组织的蛋白酶裂解物来测定硫辛酸赖氨酸水平。发现肾脏和肝脏的硫辛酸赖氨酸含量最高，范围为 3.9 至 4.6 nmol/g 大鼠或兔湿组织或 11.6 至 13.1 nmol/g 牛丙酮粉。 (C) 1998 年学术出版社。
A new enzymatic method for the determination of protein-bound lipoic acid was established. Bound lipoyl groups were liberated in the form of lipoyllysine by protease digestion and assayed by lipoamide dehydrogenase (NADH:lipoamide oxidoreductase, EC 1.8.1.4)-mediated NADH oxidation. NADH oxidation was coupled to reduction of the lipoyl disulfide group. Fluorescence kinetics of NADH oxidation were markedly enhanced by the addition of glutathione disulfide, recycling the enzyme-mediated lipoyl/dihydrolipoyl conversion. In the presence of a large excess of glutathione disulfide, NADH oxidation follows pseudo-first-order kinetics in terms of lipoyllysine concentration. A good linear correlation is obtained between the oxidation rate and lipoyllysine concentration up to 5 mu M and the calibration curve indicates that the detection limit could be 100 nM lipoyllysine. The method was applied to protease lysates of bovine, rat, and rabbit tissues to determine lipoyllysine levels. Kidney and liver were found to have the highest content of lipoyllysine in the range of 3.9 to 4.6 nmol/g rat or rabbit wet tissue or 11.6 to 13.1 nmol/g bovine acetone powder. (C) 1998 Academic Press.