Riener Christian K, Kada Gerald, Gruber Hermann J
Institute of Biophysics, J. Kepler University, Altenberger Str. 69, A-4040 Linz, Austria.
Anal Bioanal Chem. 2002 Jul;373(4-5):266-76. doi: 10.1007/s00216-002-1347-2. Epub 2002 Jun 6.
Since its introduction in 1959, Ellman's reagent (5,5'-dithio-bis(2-nitrobenzoic acid)) has been the favorite reagent for spectrophotometric measurement of protein sulfhydryls. Meanwhile however, evidence has accumulated that many protein sulfhydryls give an incomplete reaction with Ellman's reagent, even during prolonged assay times. In the present study, the kinetic problem was solved by including cystamine as a "mediator" between the protein sulfhydryl and Ellman's reagent, as previously applied in an enzymatic thiol assay [9]. As an alternative, 4,4'-dithiodipyridine (DTDP) was used in place of Ellman's reagent. Due to its small size, amphiphilic nature, and lack of charge, DTDP quickly reacts with poorly accessible protein sulfhydryls, without any catalysis by cystamine. The DTDP method and the Ellman/cystamine method were both optimized for maximal sensitivity, minimal sample consumption (detection limit 0.2 nmol mL(-1), determination limit 0.6 nmol mL(-1)), and minimal assay time (5 min). In validation experiments, both methods gave identical results and the measured sulfhydryls/protein matched the expected values. Electronic supplementary material to this paper can be obtained by using the Springer Link server located at http://dx.doi.org/10.1007/s00216-002-1347-2.
自1959年引入以来，埃尔曼试剂（5,5'-二硫代双（2-硝基苯甲酸））一直是分光光度法测定蛋白质巯基的首选试剂。然而，与此同时，越来越多的证据表明，即使在延长的检测时间内，许多蛋白质巯基与埃尔曼试剂的反应也不完全。在本研究中，通过加入胱胺作为蛋白质巯基与埃尔曼试剂之间的“媒介物”解决了动力学问题，这一方法先前已应用于酶促硫醇测定[9]。作为替代方案，使用4,4'-二硫代二吡啶（DTDP）代替埃尔曼试剂。由于其体积小、具有两亲性且不带电荷，DTDP能迅速与难以接近的蛋白质巯基反应，无需胱胺催化。DTDP法和埃尔曼/胱胺法均针对最大灵敏度、最小样品消耗（检测限0.2 nmol mL(-1)，测定限0.6 nmol mL(-1)）和最短检测时间（5分钟）进行了优化。在验证实验中，两种方法得到了相同的结果，测得的巯基/蛋白质与预期值相符。本文的电子补充材料可通过位于http://dx.doi.org/10.1007/s00216-002-1347-2的Springer Link服务器获取。
