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 α-dicarbonyl compounds are considered to be key intermediates in this reaction. Methylglyoxal (MG) (pyruvaldehyde), a physiological α-dicarbonyl compound, has been shown to modify proteins bothin vitroandin vivo.Here we describe a novel fluorescent pyrimidine,N-δ-(5-hydroxy-4,6-dimethylpyrimidine-2-yl)-l-ornithine (argpyrimidine), formed from the Maillard reaction of MG withN-α-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 ofN-α-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.