PROTEIN MODIFICATION BY METHYLGLYOXAL - CHEMICAL NATURE AND SYNTHETICMECHANISM OF A MAJOR FLUORESCENT ADDUCT

The nonenzymatic Maillard reaction of proteins, initiated by the addit ion of sugars and other aldehydes and ketones, is thought to be an imp ortant mechanism in aging and the pathogenesis of diabetic complicatio ns. The alpha-dicarbonyl compounds are considered to be key intermedia tes in this reaction. Methylglyoxal (MG) (pyruvaldehyde), a physiologi cal alpha-dicarbonyl compound, has been shown to modify proteins both in vitro and in vivo. Here we describe a novel fluorescent pyrimidine, 5-hydroxy-4,6-dimethylpyrimidine-2-yl)-L-ornithine (argpyrimidine), f ormed from the Maillard reaction of MG with N-alpha-t-BOC-arginine. We find that the fluorescence spectrum of argpyrimidine is similar to th at of methylglyoxal-modified proteins, suggesting that it is a major p roduct in such modified proteins. HPLC-quantification of argpyrimidine in proteins incubated with methylglyoxal revealed a time-dependent fo rmation. We detected significant amounts of argpyrimidine in incubatio ns of N-alpha-t-BOC-arginine with micromolar concentrations of MG, and we find that various sugars and ascorbic acid serve as precursors, Ou r studies indicate that argpyrimidine is synthesized through an interm ediate 3-hydroxypentane-2,4-dione and provide a chemical basis for flu orescence in proteins modified by methylglyoxal, We suggest that enhan ced intrinsic fluorescence in diabetic proteins may be due, in part, t o methylglyoxal-mediated Maillard reactions. (C) 1997 Academic Press.