Effects of protein concentration on the extent of gamma-ray-mediated oxidative labeling studied by electrospray mass spectrometry.
Exposure of aqueous protein solutions to gamma-rays results in the formation of *OH radicals that readily react with solvent-exposed amino acid side chains. The incorporation of oxygen leads to peak progressions with a spacing of 16 Da in the mass distribution of the polypeptide chain. Unlike some other oxidative labeling strategies, these radiolysis experiments do not require solution additives that could interfere with the analysis or that might cause secondary oxidation processes. Using myoglobin as a model system, we demonstrate that the level of oxidative labeling depends critically on the protein concentration. If ignored, this effect may lead to ambiguities in the interpretation of experiments employing *OH labeling for monitoring solvent-accessible surface areas, protein folding, and protein-protein interactions. We present a simple analysis that allows oxidation to be interpreted as a process with exponential kinetics, characterized by an apparent rate constant of the form kapp=kRAD/([P]tot+B), where kRAD is the primary rate of hydroxyl radical production, B is a constant, and [P]tot is the total protein concentration. While oxidative labeling may trigger some changes in protein conformation, it is found that the magnitude of this effect is surprisingly small, a result that is consistent with observations previously made by others.
Tong
Xin
X
Department of Chemistry, The University of Western Ontario, London, ON, N6A 5B7, Canada.
Wren
J Clara
JC
Konermann
Lars
L
D005609:Free Radicals; D005720:Gamma Rays; D017665:Hydroxyl Radical; D007700:Kinetics; D015336:Molecular Probe Techniques; D011506:Proteins / Q000032:analysis / Q000737:chemistry*; D017382:Reactive Oxygen Species / Q000737:chemistry*; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*
D005609:Free Radicals; D011506:Proteins; D017382:Reactive Oxygen Species; D017665:Hydroxyl Radical