Oxidative stress results in the damage of several biological components, particularly membrane lipids in vivo. 4-Hydroxyalkenals are known as the major cytotoxic aldehydes generated in oxidative lipid damage and show many biological effects such as high toxicity to Ehrlich ascites tumor cells, the lysis of erythrocytes and inhibition of the synthesis of DNA and protein. These biological effects of 4-hydroxyalkenals have been attributed to reactions with specific amino acid side-chains in protein. Hence, to obtain insight into the molecular basis of protein modification during lipid peroxidation, we have tried to develop procedures which can quantitate the 4-hydroxyalkenals moiety attached to protein molecule. Reaction of protein with 4-hydroxyalkenals (4-hydroxynonenal) resulted in the loss of enzyme activity, the disappearance of protein sulfhydryl group, and the appearance of protein-linked carbonyl groups. After reduction of carbonyl groups to labeled hydroxy derivatives by treatment with sodium borotritide, the labeled lipid adduct was cleaved from the protein by means of Raney nickel catalysis. The amount of labeled lipid material released by this treatment is therefore a measure of the amount of lipid aldehyde that had been conjugated to the protein in thioether linkage. 4-Hydroxynonenal also showed facile reactivity with lysine and histidine residues in proteins. We have succeeded in detecting the modified lysine and histidine residues after acid hydrolysis of proteins. Because these modified amino acids can be labeled with [3H]NaBH4, their microanalysis has become feasible.
