Characterization of in vitro protein oxidation using mass spectrometry: a time course study of oxidized alpha-amylase.

ABSTRACT

The study of protein damage by oxidative processes and its influence on protein activity is central to understanding the deleterious effects of oxidative stress on biological systems. This paper will focus on the study of enzyme inactivation by oxidative modifications, utilizing α-amylase from Bacillus species. (BAA) as a model protein. Oxidative stress was induced using metal catalyzed oxidation (MCO). The enzymatic activity of BAA was correlated with the oxidative damage induced to the protein. Off-line nano-HPLC-MALDI-TOF/TOF-MS was used to characterize the oxidative modifications occurring to the protein. Additionally, semi-quantitative analysis was employed in order to evaluate the significance of the various oxidative modifications. BAA oxidation was found to be deleterious to its enzymatic activity. A total of 10 amino acid residues were found to have an oxidation degree above 50%, out of which eight were methionine and tryptophan. Residues in the proximity of key structural elements were found to be particularly susceptible to oxidation. The oxidative process was found to be governed by the nature of the amino acid residues side chain and, to a lesser extent, their location within the three dimensional structure of the protein.