Hydrogen peroxide induced oxidation of peroxisomal malate synthase and catalase

Peroxisomes contain oxidases that produce H2O2, which can result in protein oxidation. To test the vulnerability of peroxisomal proteins to oxidation in vivo the organelles were isolated from castor bean endosperm incubated with H2O2. When peroxisomes were exposed to H2O2 in vivo, the peroxisomal proteins exhibited an increase in carbonylation as detected in avidin blots of biotin hydrazide derivatized samples. Biotin-tagged peptides from trypsin digests of the proteins were analyzed by mass spectroscopy and compared to the masses of peptides from the same protein that had not been biotin-tagged and from proteins not exposed to excess H2O2. H2O2 exposure was found to increase the activity of catalase (CAT), and to increase the number of oxidized peptides found in CAT and malate synthase (MS). CAT had 10 peptides that were affected by in vivo exposure to H2O2 and MS had 8. These sites of oxidation have definable locations within the proteins' structures. (C) 2009 Elsevier Inc. All rights reserved.