Ellman reagent, thiol assay

Sulfhydryl content can be determined directly by using any thiol assay, typically by using Ellman s reagent, although other more sensitive procedures exist (47), or indirectly by release of some reporter molecule on deprotection of crosslinkers. For example, many reports use the absorbance of 2-thiopyridone at 343 nm after treatment of pyridyldithio-modified proteins with DTT. Extreme caution should be used when interpreting results from these indirect methods since in our experience treatment of antibodies with DTT, even under mild conditions, results in subtle changes in the baseline absorbance at 343 nm and consequent erroneous estimates of thiol content. 

Enzymatic cleavage of the thioester bond (located either in the middle or at the C-terminus of the peptide) produces a free thiol compound that can be easily monitored by very sensitive assays (e.g., Ellman s reagent). 64 ... 

The modified polymer was assayed for reactive amines and thiols using fluo-rescamine and 5,5 -dithiobis(2-nitrobenzoic acid) (Ellman s reagent), respectively. For amine determination, a 10-mg/mL solution of fluorescamine in... 

Non-protein thiol content in the culture media, after precipitation of protein using 0.2M trichloroacetic acid, was assayed using Ellman s reagent (12). with 2-mercaptomethanol used as a standard. 

Ellman s assay was first described " in 1959 and has undergone many minor modifications over the years. " The procedure is based on the reaction of thiols with Ellman s reagent or 5,5 -dithiobis-(2-nitrobenzoic acid) (DTNB) to yield a modified protein thiol (mixed disulfide), and a molecule of 2-nitro-5-thiobenzoic acid (TNB) as shown in below (Figure 2). 

Hatsuo, M., Matsuno, H., Ushida, M., Katayama, K., Saeki, K. and Itoh, N. 2, 4-Dinitrobenzenesulfonyl fluoresceins as fluorescent alternatives to Ellman s reagent in thiol-quantification enzyme assays. Angeiv. Chem. Int. Ed. 44 2922—2925, 2005. 

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