A PROCEDURE FOR QUANTITATIVE-DETERMINATION OF TRIS(2-CARBOXYETHYL)PHOSPHINE, AN ODORLESS REDUCING AGENT MORE STABLE AND EFFECTIVE THAN DITHIOTHREITOL
A PROCEDURE FOR QUANTITATIVE-DETERMINATION OF TRIS(2-CARBOXYETHYL)PHOSPHINE, AN ODORLESS REDUCING AGENT MORE STABLE AND EFFECTIVE THAN DITHIOTHREITOL
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DOI：
10.1006/abio.1994.1290
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发表时间：
1994-07-01
影响因子：
2.9
通讯作者：
HAN, GY
中科院分区：
文献类型：
--
作者：
HAN, JC;HAN, GY
通过测量与5,5'-二硫代双(2 - 硝基苯甲酸)(DTNB)反应后形成的2 - 硝基 - 5 - 硫代苯甲酸酯(NTB)的量，可以方便地测定三(2 - 羧乙基)膦(TCEP)的浓度。该方法利用了TCEP快速且按化学计量比将DTNB还原，生成两当量的NTB这一事实，NTB的阴离子形式在412 nm处的摩尔消光系数为14,150 M⁻¹ cm⁻¹。这种方法足够灵敏，能够检测微摩尔范围内的TCEP浓度，并且已被证明在监测各种条件下TCEP的稳定性或氧化情况时是有用的。TCEP不仅在酸性溶液中非常稳定，而且与在pH高于7.5时容易被氧化的二硫苏糖醇(DTT)不同，它在碱性溶液中也高度稳定。在pH 6 - 9范围内比较了TCEP和DTT还原DTNB的速率。在pH低于8时，TCEP在还原这种二硫键方面比DTT有效得多。在pH 1.5 - 8.5范围内比较了TCEP和DTT还原2,2'-二硫代二吡啶(2,2'-DTDP)的速率。与在pH 1.5时完全无活性的DTT不同，TCEP在该pH下仍然能够有效地还原2,2'-DTDP。因此，如果TCEP和硫醇同时存在，可以通过在极低pH下使用2,2'-DTDP选择性地测定TCEP的浓度。(C) 1994学术出版社
The concentration of tris(2-carboxyethyl)phosphine (TCEP) can be conveniently determined by measuring the amount of 2-nitro-5-thiobenzoate (NTB) formed after reaction with 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB). This method utilizes the fact that TCEP reduces DTNB rapidly and stoichiometrically to generate two equivalents of NTB which, in its anionic form, has a molar extinction coefficient of 14,150 M(-1) cm(-1) at 412 nm. This method is sensitive enough to detect the concentration of TCEP in the micromolar range and has proven useful in monitoring the stability or oxidation of TCEP under various conditions. TCEP is not only very stable in acidic solutions, but unlike dithiothreitol (DTT) which readily oxidizes above pH 7.5, it is also highly stable in basic solutions. The rates of reduction of DTNB by TCEP and DTT are compared in the pH range 6-9. Below pH 8, TCEP is significantly more effective than DTT in reducing this disulfide. The rates of reduction of 2,2'-dithiodipyridine (2,2'-DTDP) by TCEP and DTT are compared in the pH range 1.5-8.5. Unlike DTT which is totally inactive at pH 1.5, TCEP is still capable of reducing 2,2'-DTDP effectively at this pH. Thus, if TCEP and thiols are simultaneously present, the concentration of TCEP can be selectively determined by using 2,2'-DTDP at very low pH. (C) 1994 Academic Press, Inc.