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. 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 0.2 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.
Aix Marseille Université, CNRS, UMR 7273, ICR-SMBSO, 13397 Marseille Cédex 20, France. Electronic address: p.stocker@univ-amu.fr.
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Stocker
Aix Marseille Université, CNRS, UMR 7273, ICR-SMBSO, 13397 Marseille Cédex 20, France.
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Ricquebourg
Aix Marseille Université, CNRS, UMR 7273, ICR-SMBSO, 13397 Marseille Cédex 20, France; SARL YELEN, 13820 Ensuès la Redonne, France.
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Vidal
Centre de Recherches en Encologie Biologique et Onco-Pharmacologie (CRO2), Aix-Marseille Université, Institut National de la Santé et de la Recherche Médicale (INSERM), 13385 Marseille, France.
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Villard
Centre de Recherches en Encologie Biologique et Onco-Pharmacologie (CRO2), Aix-Marseille Université, Institut National de la Santé et de la Recherche Médicale (INSERM), 13385 Marseille, France.
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Lafitte
Centre de Recherches en Encologie Biologique et Onco-Pharmacologie (CRO2), Aix-Marseille Université, Institut National de la Santé et de la Recherche Médicale (INSERM), 13385 Marseille, France.
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Sellami
Aix Marseille Université, CNRS, UMR 7273, ICR-SMBSO, 13397 Marseille Cédex 20, France.
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Pietri
D000595:Amino Acid Sequence; D000818:Animals; D001798:Blood Proteins / Q000737:chemistry / Q000378:metabolism*; D002417:Cattle; D003921:Diabetes Mellitus, Experimental / Q000378:metabolism; D004140:Dinitrophenols / Q000737:chemistry; D005470:Fluorometry / Q000379:methods*; D006834:Hydrazines / Q000737:chemistry; D008099:Liver / Q000737:chemistry / Q000378:metabolism*; D010069:Oxadiazoles / Q000737:chemistry; D010084:Oxidation-Reduction; D050050:Protein Carbonylation; D051381:Rats; D012710:Serum Albumin, Bovine / Q000737:chemistry; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods
C542965:7-nitrobenzoxadiazole; D001798:Blood Proteins; D004140:Dinitrophenols; D006834:Hydrazines; D010069:Oxadiazoles; D012710:Serum Albumin, Bovine
Fluorescence; MALDI–TOF; NBDH; Oxidized BSA; Protein carbonyls