Skip to Main content Skip to Navigation
Journal articles

Fluorimetric screening assay for protein carbonyl evaluation in biological samples

Abstract : Many assays are available for the detection of protein carbonyls (PCs). Currently, the measurement of PC groups after their derivatization with 2,4-dinitrophenol hydrazine (DNPH) is widely used for measuring protein oxidation in biological samples. However, this method includes several washing steps. In this context, we have developed a rapid, sensitive, and accurate fluorimetric method adapted to 96-well microplates for the convenient assessment of protein carbonyl level in biological samples. The method reported here is based on the reaction of carbonyl content in proteins with 7-hydrazino-4-nitroben zo-2,1,3-oxadiazole (NBDH) to form highly fluorescent derivatives via hydrazone formation. PCs were determined using the DNPH and NBDH assays in fully reduced bovine serum albumin (BSA) and plasma and liver homogenates obtained from healthy control rats up the addition of various amounts of HOCl-oxidized BSA (OxBSA). Using the NBDH assay, PC concentrations as low as 0.2 nmol/mg were detected with precision as low as 5%. Matrix-assisted laser desorption/ionization time-of-flight (MALDI–TOF) mass spectroscopy was used to successfully identify the formation of the NBDH adducts after derivatization with standard oxidized peptides. Finally, the two methods were further used for PC determination in plasma and liver samples from diabetic and normal rats, showing that the NBDH assay can be reliably used in biological experiments.
Document type :
Journal articles
Complete list of metadata
Contributor : Sylvia Pietri Connect in order to contact the contributor
Submitted on : Tuesday, December 15, 2015 - 2:29:56 PM
Last modification on : Tuesday, December 14, 2021 - 8:42:05 AM




Pierre Stocker, Ricquebourg Emilie, Nicolas Vidal, Claude Villard, Daniel Lafitte, et al.. Fluorimetric screening assay for protein carbonyl evaluation in biological samples. Analytical Biochemistry, Elsevier Masson, 2015, 482, pp.55-61. ⟨10.1016/j.ab.2015.04.021⟩. ⟨hal-01244060⟩



Record views