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. 2014 Aug 1:458:69-71.
doi: 10.1016/j.ab.2014.04.034. Epub 2014 May 6.

Simplified 2,4-dinitrophenylhydrazine spectrophotometric assay for quantification of carbonyls in oxidized proteins

Cristina S Mesquita  1 , Raquel Oliveira  1 , Fátima Bento  1 , Dulce Geraldo  1 , João V Rodrigues  1 , João C Marcos  2
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Simplified 2,4-dinitrophenylhydrazine spectrophotometric assay for quantification of carbonyls in oxidized proteins

Cristina S Mesquita et al. Anal Biochem. .

Abstract

This work proposes a modification of the 2,4-dinitrophenylhydrazine (DNPH) spectrophotometric assay commonly used to evaluate the concentration of carbonyl groups in oxidized proteins. In this approach NaOH is added to the protein solution after the addition of DNPH, shifting the maximum absorbance wavelength of the derivatized protein from 370 to 450nm. This reduces the interference of DNPH and allows the direct quantification in the sample solution without the need for the precipitation, washing, and resuspension steps that are carried out in the traditional DNPH method. The two methods were compared under various conditions and are statistically equivalent.

Keywords: Carbonyl quantification; DNPH assay; Oxidative stress; Protein oxidation.

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