Protein modification by methylglyoxal: chemical nature and synthetic mechanism of a major fluorescent adduct.

The nonenzymatic Maillard reaction of proteins, initiated by the addition of sugars and other aldehydes and ketones, is thought to be an important mechanism in aging and the pathogenesis of diabetic complications. The alpha-dicarbonyl compounds are considered to be key intermediates in this reaction. Methylglyoxal (MG) (pyruvaldehyde), a physiological alpha-dicarbonyl compound, has been shown to modify proteins both in vitro and in vivo. Here we describe a novel fluorescent pyrimidine, N-delta-(5-hydroxy-4,6-dimethylpyrimidine-2-yl)-L-ornithine (argpyrimidine), formed from the Maillard reaction of MG with N-alpha-t-BOC-arginine. We find that the fluorescence spectrum of argpyrimidine is similar to that of methylglyoxal-modified proteins, suggesting that it is a major product in such modified proteins. HPLC-quantification of argpyrimidine in proteins incubated with methylglyoxal revealed a time-dependent formation. We detected significant amounts of argpyrimidine in incubations of N-alpha-t-BOC-arginine with micromolar concentrations of MG, and we find that various sugars and ascorbic acid serve as precursors. Our studies indicate that argpyrimidine is synthesized through an intermediate 3-hydroxypentane-2,4-dione and provide a chemical basis for fluorescence in proteins modified by methylglyoxal. We suggest that enhanced intrinsic fluorescence in diabetic proteins may be due, in part, to methylglyoxal-mediated Maillard reactions.

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 amino groups on proteins by reducing sugars, has been studied for its potential role in aging and the complications of diabetes. One of the major consequences of the advanced Maillard reaction in proteins is the formation of covalently cross-linked aggregates. The chemical nature of the cross-linking structures is largely unknown. Recently, methylglyoxal has been shown to be a potential glycating agent in vivo and suggested to be a common intermediate in the Maillard reaction involving glucose. Methylglyoxal can form enzymatically or nonenzymatically from glycolytic intermediates and by retro-aldol cleavage of sugars. Its elevation in tissues in diabetes and its high potency to glycate and cross-link proteins led us to investigate the chemical nature of its advanced Maillard products. Using an approach in which a synthetic model peptide was reacted with methylglyoxal, we isolated and purified a cross-linked peptide dimer. Characterization of this dimer revealed that the peptides are linked through epsilon amino groups of lysine residues. The actual cross-link was shown to be a methylimidazolium, formed from the reaction of two lysines and two methylglyoxal molecules. We have named this cross-link imidazolysine. Imidazolysine was detected in proteins by high performance liquid chromatography using a postcolumn derivatization method. Proteins incubated with methylglyoxal showed a time-dependent formation of imidazolysine. Quantification of imidazolysine in human serum proteins revealed a significant increase (p < 0.05) in diabetic samples (mean +/- S.D., 313.8 +/- 52.7 pmol/mg protein) when compared with normal samples (261.3 +/- 50.4). These values correlated with glycohemoglobin (p < 0.05). These results provide chemical evidence for protein cross-linking by dicarbonyl compounds in vivo.