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[3H]H4, and then treated with Raney nickel. The 3H-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]-estractable form. Treatment of proteins with HNE led to the disappearance of protein sulfhydryl groups. However, less than 10% of the labeled adducts obtained after subsequent reduction with NaB[3H]H4 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.
Uchida
K
K
Laboratory of Biochemistry, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD 20892.
Stadtman
E R
ER
D000111:Acetylcysteine / Q000737:chemistry*; D000447:Aldehydes / Q000737:chemistry* / Q000378:metabolism / Q000494:pharmacology; D001894:Borohydrides; D002851:Chromatography, High Pressure Liquid; D004798:Enzymes / Q000378:metabolism; D005978:Glutathione / Q000737:chemistry*; D008956:Models, Chemical; D011485:Protein Binding; D011506:Proteins / Q000378:metabolism*; D011865:Radioisotope Dilution Technique; D014316:Tritium
D000447:Aldehydes; D001894:Borohydrides; D004798:Enzymes; D011506:Proteins; D014316:Tritium; D005978:Glutathione; C027576:4-hydroxy-2-nonenal; D000111:Acetylcysteine