Xanthine oxidase-catalyzed crosslinking of cell membrane proteins

Isolated erythrocyte membranes exposed to protease-free xanthine oxidase plus xanthine and ferric iron undergo lipid peroxidation and protein crosslinking (appearance of high molecular weight aggregates on sodium dodecyl sulfate (SDS) gel electrophoresis). Spectrin is more susceptible to crosslinking than the other polypeptides. Thiol-reducible bonds (disulfides) as well as nonreducible bonds are generated, the former type relatively rapidly (detected within 10-20 min) and the latter type more slowly (usually detected after 1 h). Reducible crosslinking is inhibited by catalase, but not by superoxide dismutase, desferrioxamine, butylated hydroxyltoluene, and mannitol; whereas nonreducible crosslinking, like free radical lipid peroxidation, is inhibited by all of these agents except mannitol. Zinc(II) also inhibits lipid peroxidation, but stimulates disulfide bond formation to the virtual exclusion of all other crosslinking. Our results indicate that disulfide formation is dependent on H2O2, but not O2- or iron. However, O2-, H2O2, and iron are all required for lipid peroxidation and nondisulfide crosslinking, suggesting the intermediacy of OH generated via the iron-catalyzed Haber-Weiss reaction. The possible role of malonaldehyde (MDA, a by-product of lipid peroxidation) in the latter type of crosslinking was examined. Solubilized samples of xanthine/xanthine oxidase-treated membranes showed a strong visible fluorescence (emission maximum 450 nm; excitation 390 nm). This resembled the fluorescence of membranes treated with authentic MDA, which forms conjugated imine linkages between amino groups. Fluorescence scanning of SDS gels from MDA-treated membranes showed a strong signal coincident with crosslinked proteins and also one in the low molecular weight, nonprotein region, suggestive of aminolipid conjugates. Similar scanning on xanthine/xanthine oxidase-reacted membranes indicated that all fluorescence is associated with the lipid fraction. Thus, nonreducible protein crosslinks in this system do not appear to be of the MDA-derived, Schiff base type.