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.