Detection of oxidized lipid-modified erythrocyte membrane proteins by radiolabeling with tritiated borohydride.

Human erythrocyte ghosts treated with tert-butyl hydroperoxide or ADP-Fe3+ incorporated radioactivity on reduction with tritiated borohydride. The tritium incorporation closely correlated with membrane lipid oxidation as assessed by the formation of thiobarbituric acid-reactive substances and fluorescent substances. Treatment of ghosts with the inducers in the presence of butylated hydroxytoluene, thiourea, or desferrioxamine suppressed the tritium incorporation in the subsequent reduction. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the tritiated ghost proteins showed that the label was incorporated into the intermolecularly cross-linked and the uncross-linked proteins of bands 1, 2, 3, 4.1, 4.2, 5 and 6, and into the noncross-linked glycophorin A (PAS-1). Glycophorin A was hardly cross-linkable but modified during membrane lipid oxidation. Possible candidates for producing borohydride-reducible functions in the proteins are various mono- and bifunctional aldehydes, as well as those for producing fluorescence and cross-links. A part of thiobarbituric acid-reactive or fluorescent substances may be involved in borohydride reduction and tritium labeling.