Fluorimetric screening assay for protein carbonyl evaluation in biological samples

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...

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Bibliographic Details
Published in:Analytical biochemistry 2015-08, Vol.482, p.55-61
Main Authors: Stocker, P., Ricquebourg, E., Vidal, N., Villard, C., Lafitte, D., Sellami, L., Pietri, S.
Format: Article
Language:English
Subjects:
Amino Acid Sequence
Animals
Blood Proteins - chemistry
Blood Proteins - metabolism
Cattle
Chemical Sciences
Diabetes Mellitus, Experimental - metabolism
Dinitrophenols - chemistry
Fluorescence
Fluorometry - methods
Hydrazines - chemistry
Liver - chemistry
Liver - metabolism
MALDI–TOF
NBDH
Oxadiazoles - chemistry
Oxidation-Reduction
Oxidized BSA
Protein Carbonylation
Protein carbonyls
Rats
Serum Albumin, Bovine - chemistry
Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization - methods
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Summary: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-nitrobenzo-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 0.2nmol/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.
ISSN:0003-2697
1096-0309
DOI:10.1016/j.ab.2015.04.021