Determination of thiols and disulfides via HPLC quantification of 5-thio-2-nitrobenzoic acid.
Determination of thiols and disulfides via HPLC quantification of 5-thio-2-nitrobenzoic acid.
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DOI：
10.1016/j.jpba.2008.08.033
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发表时间：
2008-12-15
影响因子：
3.4
通讯作者：
Guan, Xiangming
中科院分区：
文献类型：
--
作者：
Chen, Wei;Zhao, Yong;Seefeldt, Teresa;Guan, Xiangming
关键词：
本工作提出了通过HPLC定量5-硫代-2-硝基苯甲酸（TNB）来测定生物样品中的总硫醇和总二硫化物，所述5-硫代-2-硝基苯甲酸（TNB）来自硫醇与5，5 ′-二硫代双（2-硝基苯甲酸）（DTNB，Ellman试剂）的反应。该方法还通过测量GSH DTNB加合物（GSH-TNB）同时定量谷胱甘肽（GSH）。以326 nm为检测波长，TNB和GSH-TNB加合物的HPLC检测限分别为15 pmol和7.5 pmol。OVCAR-3细胞的回收率研究显示，在测定非蛋白硫醇、蛋白硫醇、非蛋白二硫化物和蛋白二硫化物的程序中，TNB的回收率分别为99.4±1.2%（n = 3）、98.1±5.0%（n = 3）、95.6±0.9%（n = 3）和96.6±2.3%（n = 3）。在测定非蛋白硫醇、蛋白硫醇、非蛋白二硫化物和蛋白二硫化物的方法中，GSH-TNB的回收率分别为99.0±0.3%（n = 3）、95.1±4.9%（n = 3）、96.8±0.6%（n = 3）和95.1±2.9%（n = 3）。TNB的重现性（表示为分析物的相对标准偏差）测定为：非蛋白硫醇为2.8%（n = 6），蛋白硫醇为3.9%（n = 6），非蛋白二硫化物为3.6%（n = 6），蛋白二硫化物为4.6%（n = 6）。GSH-TNB的重现性确定为非蛋白硫醇为1.6%（n= 6），非蛋白二硫化物为2.6%（n = 6）。通过比较NaBH_4还原前后生物样品中GSH的含量，该方法可以提供与巯基谷胱甘肽化相关的信息，为蛋白质谷胱甘肽化研究提供有用的信息。该方法应适用于细胞、亚细胞、蛋白质或其他生物基质样品的巯基和二硫化物定量，并将成为研究巯基氧化还原状态和巯基谷胱甘肽化的有用分析方法。
This work presents an assay for total thiols and total disulfides in biological samples via HPLC quantification of 5-thio-2-nitrobenzoic acid (TNB) derived from the reaction of thiols with 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB, Ellman’s reagent). This method also provides simultaneous quantification of glutathione (GSH) via the measurement of the GSH DTNB adduct (GSH-TNB). By using 326 nm as the detecting wavelength, the HPLC detection limit for TNB and the GSH-TNB adduct was determined to be 15 pmol and 7.5 pmol respectively. A recovery study with OVCAR-3 cells revealed that the recovery yields for TNB in the procedures for determining non-protein thiols, protein thiols, non-protein disulfides, and protein disulfides were 99.4±1.2% (n = 3), 98.1±5.0% (n = 3), 95.6±0.9% (n = 3), and 96.6±2.3% (n = 3) respectively. The recovery yield for GSH-TNB in the procedures for determining non-protein thiols, protein thiols, non-protein disulfides, and protein disulfides were 99.0±0.3% (n = 3), 95.1±4.9% (n = 3), 96.8±0.6% (n = 3), and 95.1±2.9% (n = 3) respectively. The reproducibility, expressed as the relative standard deviation for the analyte, for TNB was determined to be 2.8% (n = 6) for non-protein thiols, 3.9% (n = 6) for protein thiols, 3.6% (n = 6) for non-protein disulfides and 4.6% (n = 6) for protein disulfides. The reproducibility for GSH-TNB was determined to be 1.6% (n= 6) for non-protein thiols and 2.6% (n = 6) for non-protein disulfides. By comparing the amount of GSH determined in a biological sample before NaBH4 reduction with that after the reduction, this method can provide information associated with thiol glutathionylation which would be useful for protein glutathionylation study. This method should be applicable to cellular, subcellular, protein, or other biomatrix samples for thiol and disulfide quantification and will be a useful analytical method in the study of thiol redox state and thiol glutathionylation.
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DOI：
10.1016/0165-022x(86)90102-8
发表时间：
1986-11-01
影响因子：
--
作者：
COTGREAVE, IA;MOLDEUS, P
通讯作者：
MOLDEUS, P
影响因子：
3
作者：
Dursun, E;Timur, M;Ozben, T
通讯作者：
Ozben, T
影响因子：
8.8
作者：
Ferretti, A;D'Ascenzo, S;Podo, F
通讯作者：
Podo, F
影响因子：
7.3
作者：
REGLINSKI, J;SMITH, WE;STURROCK, RD
通讯作者：
STURROCK, RD
影响因子：
2.9
作者：
Eyer, P;Worek, F;Reiner, E
通讯作者：
Reiner, E