Exposure to endogenous and exogenous agents produces reactive oxidants that induce lipid and DNA peroxidation forming malondialdehyde and base propenal, respectively. These a,� unsaturated aldehydes modify protein and form DNA adducts including M1dG and OPdA. Nuclear formation of base propenal makes it a likely source for nuclear protein modification and a factor for influencing DNA replication and repair in response to oxidative stress. To investigate base propenal (specifically adenine propenal) modification of protein, we chemically characterized its reaction with N-a-acetyllysine and purified human serum albumin. N-a-acetyllysine reaction with base propenal formed two primary products, a lysine-lysine cross-link and an oxopropenyllysine product. HPLC and spectrometric analyses demonstrate as N-a-acetyllysine concentration increases, adenine propenal is depleted and the lysine-lysine cross-link is formed, representing 90% of the starting material. Oxopropenylation was less dependent on N-a-acetyllysine concentration and represented 10% of starting material. The formation of both the oxopropenyllysine product and the lysine-lysine cross-link is hypothesized to form through an amino-enemine intermediate, which we have characterized using mass spectrometry and NMR. Adenine propenal modification of human serum albumin was examined using proteomics-based mass spectrometry, revealing oxopropenylation on 32% of lysine residues. Protein cross-links were also detected. To study nuclear protein modification, colon carcinoma cells were treated with synthesized adenine propenal, nuclear proteins extracted, enriched with biotin hydrazide and analyzed with streptavidin detection. Adenine propenal was found to increase nuclear protein modification. These studies have comprehensively characterized the chemical modification of N-a-acetyllysine as well as human serum albumin by adenine propenal and have demonstrated nuclear protein modification by this reactive electrophile.

Chemical characterization of protein modification by base propenal

246th National Meeting (2013)

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