Protein Carbonyl Determination Using Biotin Hydrazide

Abstract

Protein oxidation is a recognized component of aging and a consequence of severe or prolonged exposure to reactive oxygen species (ROS). Direct attack of protein by ROS causes formation of protein-bound carbonyl groups (1). These carbonyl functions represent a variety of site-specific modifications, most particularly adipic and glutamic acid semialdehydes (2). Additionally, numerous lipid oxidation products, including αβ-unsaturated γ-hydroxyalkenals can attack proteins yielding protein-bound aldehydes (3). Furthermore, nonemzymatic glycation can yield protein-bound carbonyl functionalities (4). Thus, protein carbonyls represent a possibly convenient indicator of oxidative stress. A variety of techniques have been introduced to measure protein carbonyls in tissue extracts, where they are found to increase exponentially as a function of organism aging (5). All the extant techniques for protein carbonyl determination rely upon reductive amination between the carbonyl group and a probe, typically dinitrophenylhydrazine (DNPH) (5–6). Antibodies specific to the probe can then be used to visualize protein carbonyls.