IDENTIFICATION OF HYDROGEN-PEROXIDE OXIDATION SITES OF ALPHA-A-CRYSTALLINS AND ALPHA-B-CRYSTALLINS

The alpha-crystallins are the most abundant structural proteins of the lens and, because of their chaperone activity, contribute to the solu bility of the other crystallins. With aging, the lens crystallins unde rgo a variety of modifications which correlate with a loss of solubili ty and the development of cataract. A recent study demonstrating that alpha-crystallins exposed in vitro to FeCl3 and H2O2 exhibit decreased chaperone activity, implicates metal catalyzed oxidations of alpha-cr ystallins in this loss of solubility. The present study has determined that alpha-crystallins incubated with FeCl3 and H2O2 are modified by the nearly complete oxidation of all methionine residues to methionine sulfoxide, with no other detectable reaction products. The modificati ons were identified from the molecular weights of peptides formed by e nzymatic digestion of the alpha-crystallins and located by tandem mass spectrometric analysis of the fragmentation pattern of the modified p eptides. A dominant pattern in the mass spectra of the fragments from peptides with oxidized methionine is loss of 64 Da, which corresponds to loss of CH3SOH from the methionine sulfoxide. These fragments are u seful in identifying peptides that include oxidized methionine residue s.