Quick measurement of protein sulfhydryls with Ellman's reagent and with 4,4'-dithiodipyridine
Quick measurement of protein sulfhydryls with Ellman's reagent and with 4,4'-dithiodipyridine
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DOI：
10.1007/s00216-002-1347-2
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发表时间：
2002-07-01
影响因子：
4.3
通讯作者：
Gruber, HJ
中科院分区：
文献类型：
--
作者：
Riener, CK;Kada, G;Gruber, HJ
自1959年推出以来，Ellman试剂（5，5 '-二硫代-双（2-硝基苯甲酸））一直是分光光度法测量蛋白质巯基的最受欢迎的试剂。然而，与此同时，越来越多的证据表明，许多蛋白质巯基与Ellman试剂反应不完全，即使在延长的测定时间内也是如此。在本研究中，通过将胱胺作为蛋白质巯基和Ellman试剂之间的“介体”来解决动力学问题，如先前在酶促巯基测定中所应用的那样[9]。作为替代，使用4.4 '-二硫代二吡啶（DTDP）代替Ellman试剂。由于其尺寸小、两亲性和缺乏电荷，DTDP很快与难以接近的蛋白质巯基发生反应，而不受胱胺的任何催化。DTDP法和Ellman/胱胺法均针对最大灵敏度、最小样品消耗量（检测限0.2 nmol mL（-1），测定限0.6 nmol mL（-1））和最短测定时间（5 min）进行了优化。在验证实验中，两种方法得到了相同的结果，并且测得的巯基/蛋白质与预期值匹配。本文的电子补充材料可通过Springer Link服务器获得，网址为http://dx.doi.org/10.1007/ s 00216 -002-1347-2。
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.