The use of DNPH‐derivatized protein carbonyls as a marker of oxidative stress in mouse heart and liver

Abstract

Reactive oxygen species (ROS) have been shown to modify proteins, lipids and DNA. These modified biomolecules serve as markers for the quantification of oxidative stress in various physiological and diseased conditions. Identification and quantification of a marker of protein oxidation (i.e., protein carbonyls) in tissues continue to be time consuming and/or costly. To identify and quantify this biomarker, we utilized a mouse model of ALS (G93A) having a mutant human Cu/Zn-superoxide dismutase gene resulting in elevated levels of ROS. Using spectroscopic methods, we previously reported that the G93A mouse has 53% higher protein carbonyls in the skeletal muscle as compared with wild-type littermates. However, these methods require large amounts of sample. In this study, we present a comparison of immunochemical methods, using DNPH-derivatized carbonyls (Oxyblot, Chemicon International vs. ELISA), to assess oxidative stress. A protein with an apparent molecular weight of 28 kDa proved to associate well with established patterns of oxidative stress in these animals. Higher carbonyl content (80%) was found in the liver of G93A versus wild-type mice (5165 ± 741 AU vs. 2869 ± 762 AU, mean ± SEM, P = 0.044). Most importantly, this analysis required, at most, 3 μg of sample and is a more sensitive and biologically relevant measure of oxidative stress as compared with ELISA-based methods. In summary, we have identified and quantified a 28 kDa protein that can serve as a measure of protein oxidative damage.