MODIFICATION OF PROTEIN BY THE LIPID PEROXIDATION PRODUCT 4-HYDROXY-2-NONENAL

活性氧很容易与多不饱和脂肪酸相互作用

Reactive oxygen species readily interact with polyunsaturated fatty acids resulting in the formation of cytotoxic aldehydes such as 4-hydroxy-2- nonenal (HNE) and malondialdehyde. In animal tissues, lipid peroxidation increases in a number of degenerative diseases associated with aging and fluorescent material believed to results from interaction of lipid peroxidation products with protein accumulates with age. Modification of protein and other biomolecules by lipid peroxidation products is therefore believed to contribute to the aging process. The mechanisms and relative contributions of several potential reactions are not, however, well understood. Glucose-6-phosphate dehydrogenase (Glu-6-PDH) from Leuconostoc mesenteroides contains no cysteine residues and was chosen as a convenient model for studying the interaction of HNE with nucleophilic amines in protein. Prolonged incubation of Glu-g-PDH with HNE leads to the formation of cross-linked protein. This is accompanied by the appearance of protein-associated fluorescence reminiscent of lipofuscin, with excitation and emission maxima of 340 and 415 nm, respectively. Cross-linked protein is less susceptible than native Glu-6-PDH to proteolysis by the multicatalytic protease, a multienzymic proteolytic complex involved in the intracellular degradation of damaged proteins. In addition, HNE-modified Glu-6-PDH inhibits the multicatalytic protease and can therefore prevent the efficient degradation of oxidized protein. Formation of cross-linked Glu-6-PDH is associated with the nearly exclusively loss of lysine residues. Using chromatographic and mass spectral techniques, we found that an epsilon-amino group of lysine reacts with the double bond (C3) of HNE to form a Michael adduct, which then undergoes reaction with a second lysine residue at the carbonyl moiety (C1) to form a Schiff base cross-link. The structure of the fluorophore is presently under investigation. The results of these and related experiments may provide critical information on the role of lipid peroxidation in disease- and age-related accumulation of damaged protein and impairment of cellular function.