A SPECTROPHOTOMETRIC METHOD FOR THE DETERMINATION OF PROTEINS DAMAGEDBY OXIDIZED LIPIDS

The Ehrlich reaction was optimized to determine pyrrolized proteins pr oduced as a consequence of lipid peroxidation and oxidative stress. Th e procedure consisted of the treatment of the modified protein with p- (dimethylamino)benzaldehyde at a controlled acidity and temperature, a nd the determination of adducts produced against the blank obtained in the absence of the reagent. The extinction coefficient of Ehrlich add ucts was calculated by using epsilon-N-pyrrolylnorleucine (Pnl) as sta ndard and was 35,000 M-1 cm(-1). The response was linear and reproduci ble within the range 0.16-20 mu M Pnl. The assay was applied to determ ination of pyrrole content in bovine serum albumin, bovine cu-globulin s, bovine gamma-globulins, and mixtures of them, incubated overnight w ith 1 mM of 4,5(E)-epoxy-2(E)-heptenal, obtaining results similar to t hose from determination of Pnl by capillary electrophoresis after basi c hydrolysis of the protein. The method was also applied to pyrrole de termination in bovine plasma proteins either incubated with epoxyalken als, hydroxyalkenals, lipid hydroperoxides, and secondary products of lipid peroxidation, or oxidized with Fe3+/ascorbate. All these treatme nts produced pyrrolization of plasma proteins and all Ehrlich adducts gave very similar absorbance spectra with the exception of that produc ed in the treatment with hydroxyalkenals. The above results suggest th at protein pyrrolization is a normal consequence of the lipid peroxida tion process and of oxidative stress, and that Ehrlich adducts may be valid to determine this pyrrolization, (C) 1998 Academic Press.