Stocker P, Ricquebourg E, Vidal N, Villard C, Lafitte D, Sellami L, Pietri S
Aix Marseille Université, CNRS, UMR 7273, ICR-SMBSO, 13397 Marseille Cédex 20, France.
Aix Marseille Université, CNRS, UMR 7273, ICR-SMBSO, 13397 Marseille Cédex 20, France.
Anal Biochem. 2015 Aug 1;482:55-61. doi: 10.1016/j.ab.2015.04.021. Epub 2015 Apr 28.
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.
有许多检测蛋白质羰基（PCs）的方法。目前，用2,4-二硝基苯肼（DNPH）对PC基团进行衍生化后进行测量，被广泛用于检测生物样品中的蛋白质氧化。然而，该方法包括几个洗涤步骤。在此背景下，我们开发了一种快速、灵敏且准确的荧光法，适用于96孔微孔板，以便于评估生物样品中的蛋白质羰基水平。本文报道的方法基于蛋白质中的羰基含量与7-肼基-4-硝基苯并-2,1,3-恶二唑（NBDH）反应，通过腙的形成生成高荧光衍生物。在完全还原的牛血清白蛋白（BSA）以及从健康对照大鼠获得的血浆和肝脏匀浆中加入不同量的次氯酸氧化的BSA（OxBSA），使用DNPH和NBDH检测法测定PCs。使用NBDH检测法，能够检测低至0.2 nmol/mg的PC浓度，精密度低至5%。基质辅助激光解吸/电离飞行时间（MALDI-TOF）质谱用于成功鉴定标准氧化肽衍生化后NBDH加合物的形成。最后，这两种方法进一步用于测定糖尿病大鼠和正常大鼠的血浆和肝脏样品中的PCs，结果表明NBDH检测法可可靠地用于生物学实验。
