Modification of proteins by lysine-directed bifunctional aldehydes derived from lipid peroxidation

Lipid peroxidation derived aldehydes have been implicated in the modification of proteins during oxidative stress, which occurs in a variety of age-related degenerative diseases, including atherosclerosis, diabetes and Alzheimer disease. Most focus has been on reactive bifunctional aldehydes such as 4-hydroxy-2-alkenal, 4-oxo-2-alkenal, 2-hydroxyaldehydes, and 2-oxoaldehydes, and on modification and cross-linking of protein lysine residues by these agents.; Short chain 2-hydroxyaldehydes can modify proteins and give rise to both protein cross-linking and fluorophore generation. Using primary amines to model the lysine side-chain residues, the major ex/em 327/390 nm fluorophore formed from 2-hydroxybutanal or 2-hydroxyheptanal was shown unambiguously to be a 4,5-dialkyl-3-hydroxypyndinium derivative, representing condensation of two molecules of 2-hydroxyalkenal with a single amine. A mechanism involving intramolecular aldol cyclization and 2e oxidation following azadiketone formation was proposed for formation of the 3-hydroxypyridinium core structure. In addition the major cross-linking product was shown to be a 4-alkylimidazolium derivative. To determine the occurrence of the fluorophore in certain degenerative disorders, an immunogen for the 2-hydroxyheptanal-derived 3-hydroxypyridinium was prepared and used in the production of the corresponding polyclonal rabbit antibody, which was used to detect the epitope in 2-hydroxyheptanal-adducted LDL and in oxidized LDL.; Glyoxal can modify proteins and give rise to protein cross-linking in the form of the imidazolium, GOLD. Here a novel amide cross-link, GLAD, was identified in the reaction of glyoxal with simple amines and protein. Favorable formation of GLAD over GOLD using a high ratio of amine to glyoxal and in proteins suggests that GLAD might be more physiologically important than GOLD in the pathogenesis of aging and diabetes. Mechanistic studies indicated that formation of GLAD and CML proceeds via both deprotonation/tautomerization and an intramolecular Cannizzaro reaction.; A 1:1 mixture of Nα-(2,4-dinitrophenyl (DNP- d₀))lysine and Nα-(2,4-dinitro-3,5,6-trideuteriophenyl) (DNP-d₃))lysine, used as a protein-based lysine surrogate, was used to explore the chemical nature of 4-hydroxy-2-E-nonenal (HNE) and 4-oxo-2-E-nonenal (ONE) modification by HPLC-MS. This method enabled us not only to detect the previously characterized cross-link and non-cross-link modifications, but also to find novel cross-link and non-cross-link modifications such as maleimide and 3-aminosuccinimide derived from the oxidative cleavage of ONE-amine adducts. Also this method provides some insight to the relative frequency of different modifications and some metastable intermediates that do not survive isolation. (Abstract shortened by UMI.)...