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Analytical biochemistry

A procedure for quantitative determination of tris(2-carboxyethyl)phosphine, an odorless reducing agent more stable and effective than dithiothreitol.


PMID 7978256

Abstract

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.