PROTEIN CROSS-LINKING BY THE MAILLARD REACTION - ISOLATION, CHARACTERIZATION, AND IN-VIVO DETECTION OF A LYSINE-LYSINE CROSS-LINK DERIVED FROM METHYLGLYOXAL

The Maillard reaction, initiated by nonenzymatic glycosylation of amin o groups on proteins by reducing sugars, has been studied for its pote ntial role in aging and the complications of diabetes. One of the majo r consequences of the advanced Maillard reaction in proteins is the fo rmation of covalently cross-linked aggregates, The chemical nature of the cross-linking structures is largely unknown, Recently, methylglyox al has been shown to be a potential glycating agent in vivo and sugges ted to be a common intermediate in the Maillard reaction involving glu cose. Methylglyoxal can form enzymatically or nonenzymatically from gl ycolytic intermediates and by retro-aldol cleavage of sugars, Its elev ation in tissues in diabetes and its high potency to glycate and cross -link proteins led us to investigate the chemical nature of its advanc ed Maillard products, Using all approach in which a synthetic model pe ptide was reacted with methylglyoxal, we isolated and purified a cross -linked peptide dimer, Characterization of this dimer revealed that th e peptides are linked through epsilon amino groups of lysine residues, The actual cross-link was shown to be a methylimidazolium, formed hor n the reaction of tyro lysines and two methylglyoxal molecules, We hav e named this cross-link imidazolysine. Imidazolysine was detected in p roteins by high performance liquid chromatography using a postcolumn d erivatization method, Proteins incubated with methylglyoxal showed a t ime-dependent formation of imidazolysine. Quantification of imidazolys ine in human serum proteins revealed a significant increase (p < 0.05) in diabetic samples (mean +/- S.D., 313.8 +/- 52.7 pmol/mg protein) w hen compared with normal samples (261.3 +/- 50.4), These values correl ated with glycohemoglobin (p < 0.05), These results provide chemical e vidence for protein cross-linking by dicarbonyl compounds in vivo.