Molar absorption coefficients for the reduced Ellman reagent: reassessment
Molar absorption coefficients for the reduced Ellman reagent: reassessment
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DOI：
10.1016/s0003-2697(02)00506-7
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发表时间：
2003-01-15
影响因子：
2.9
通讯作者：
Reiner, E
中科院分区：
文献类型：
--
作者：
Eyer, P;Worek, F;Reiner, E
用于测定硫醇的Ellman方法基于硫醇与显色DTNB（5，5 '-二硫代双-2-硝基苯甲酸酯）的反应，由此测量5-硫代-2-硝基苯甲酸（TNB）的黄色二价阴离子的形成。Ellman在1959年发表的TNB摩尔吸光系数13.6 × 10（3）M-1 cm（-1）至今几乎被普遍使用。然而，多年来，已经公布了略微不同的值，并且已经进一步表明TNB揭示了热致变色性质。当Ellman方法用于测定酶活性时，如胆碱酯酶测定时，应考虑到这一点。我们的数据表明，TNB的吸收光谱移动到较长的波长时，温度升高，而吸收最大值降低。我们推荐的412 nm处的摩尔吸收系数在25 ℃为14.15 x 10（3）M-1 cm（-1），在37 ℃为13.8 x 10（3）M-1 cm（-1）（0.1 M磷酸盐缓冲液，pH 7.4）。其他温度和波长的摩尔吸收系数也包括在论文中。(C)2003 Elsevier Science（美国）。All rights reserved.
The Ellman method for assaying thiols is based on the reaction of thiols with the chromogenic DTNB (5,5'-dithiobis-2-nitrobenzoate) whereby formation of the yellow dianion of 5-thio-2-nitrobenzoic acid (TNB) is measured. The TNB molar absorption coefficient, 13.6 x 10(3) M-1 cm(-1), as published by Ellman in 1959 has been almost universally used until now. Over the years, however, slightly different values have been published, and it has further been shown that TNB reveals thermochromic properties. This should be taken into account when the Ellman method is used for determination of enzyme activities, such as in cholinesterase assays. Our data show that the absorbance spectra of TNB are shifted to longer wavelengths when temperature increases, while absorbance maxima decrease. Our recommended molar absorption coefficients at 412 nm are 14.15 x 10(3) M-1 cm(-1) at 25degreesC and 13.8 x 10(3) M-1 cm(-1) at 37degreesC (0.1 M phosphate buffer, pH 7.4). Molar absorption coefficients for other temperatures and wavelengths are included in the paper. (C) 2003 Elsevier Science (USA). All rights reserved.