SELECTIVE CLEAVAGE OF THIOETHER LINKAGE IN PROTEINS MODIFIED WITH 4-HYDROXYNONENAL

The peroxidation of polyunsaturated fatty acids leads to numerous products including 4-hydroxynonenal (HNE). That 4-hydroxy-2-alkenal compounds react with sulfhydryl groups of proteins to form thioether adducts possessing a carbonyl function has been established [Schauenstein, E. & Esterbauer, H. (1979) Ciba Found. Symp. 67, 225-244]. Taking advantage of the fact that Raney nickel catalyzes cleavage of thioether bonds, we have developed a procedure to quantitate the amount of HNE moiety bound to protein by means of a thioether linkage. Adducts of HNE with N-acetylcysteine and glutathione were prepared, labeled with NaB[H-3]H-4, and then treated with Raney nickel. The H-3-labeled product was recovered in 85-90% yield from both HNE-N-acetylcysteine and HNE-glutathione adducts in a solvent [10% (vol/vol) methanol/ chloroform]-extractable form. Treatment of proteins with HNE led to the disappearance of protein sulfhydryl groups. However, <10% of the labeled adducts obtained after subsequent reduction with NaB[H-3]H-4 could be released in a solvent-extractable form upon treatment with Raney nickel. This and the observation that HNE reacts with proteins lacking a sulfhydryl group attests to the fact that HNE can react with amino acid residues other than cysteinyl residues.