Modification of histidine residues by 4,5-epoxy-2-alkenals

The reactions of 4,5(E)-epoxy-2(E)-heptenal with 4-methylimidazole and N(alpha)-acetyl-L-histidine methyl ester were studied to characterize the adducts produced in the modification of histidine residues by epoxyalkenals and to develop a methodology for the determination of these adducts in protein hydrolysates. The reaction products, which were isolated and characterized, resulted in the Michael adducts produced in the addition of one of the imidazolic nitrogens to the carbon-carbon double bond of the epoxyalkenal. Only some of the theoretical isomers were produced. Thus, in the reaction with 4-methylimidazole, the main product was 4, 5-epoxy-3-(4-methylimidazol-1-yl)heptanol (88%), although the formation of 4,5-epoxy-3-(5-methylimidazol-1-yl)heptanol (12%) was also observed. On the other hand, the reaction with N(alpha)-acetyl-L-histidine methyl ester produced exclusively N(alpha)-acetyl-1-[1'-(1' ',2' '-epoxybutyl)-3'-hydroxypropyl]-L-histidine methyl ester. This last compound was used to develop a procedure for the determination of 4, 5(E)-epoxy-2(E)-heptenal-histidine adducts in protein hydrolysates. When this procedure was applied to the analysis of bovine serum albumin treated with 0.01-10 mM 4,5(E)-epoxy-2(E)-heptenal, the formation of the adduct was observed and its concentration increased with the concentration of the aldehyde and the incubation time, and was parallel to the histidine losses observed in the protein after acid hydrolysis as well as to the formation of protein carbonyls. In addition, the number of histidine residues lost in the protein was very similar to the number of adduct residues produced, suggesting that the addition reaction is the major mechanism for histidine losses suffered by proteins following their reaction with epoxyalkenals.