Abstract
Spin scavenging combined with chromatographic and mass spectrometric procedures can, in principle, be employed to detect and identify protein-based radicals within complex biological matrices. This approach is based on the well-known ability of stable synthetic nitroxide radicals to scavenge carbon-centered radicals, forming stable diamagnetic addition products. Hence, characterization of these addition products would allow for the identification of specific free radicals. In the present work, we have explored the use of the stable nitroxide radical 4-hydroxy-2,2,6,6-tetramethylpiperidine 1-oxyl (TEMPOL) in scavenging protein-based radicals generated in a horse heart metmyoglobin/hydrogen peroxide (metMb/H(2)O(2)) system. Inclusion of a substoichiometric amount of TEMPOL in the metMb/H(2)O(2) system resulted in a complete loss of peroxyl and tyrosyl radical signals and effectively inhibited the formation of oxidatively damaged heme species, as monitored by electron paramagnetic resonance and reversed-phase liquid chromatography. Scavenging of globin radicals by TEMPOL did not lead to the formation of stable diamagnetic addition adducts; in fact, reversed-phase liquid chromatographic studies and oxygen electrode measurements indicated that TEMPOL acts as a catalyst and is recycled in this system. The oxoammonium cation generated in the course of this reaction initiated secondary reactions resulting in the formation of a free carbonyl on the N-terminal Gly-residue of the protein. This oxidative deamination was confirmed through the combined use of reversed-phase liquid chromatographic purification, tandem MS experiments, and chemical analysis (e.g., by use of 2,4-dinitrophenyl hydrazine). The results reveal the pitfalls inherent in using stable nitroxide radicals such as TEMPOL to identify sites of radical formation on hemoproteins.
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