MECHANISM OF AUTOXIDATIVE GLYCOSYLATION - IDENTIFICATION OF GLYOXAL AND ARABINOSE AS INTERMEDIATES IN THE AUTOXIDATIVE MODIFICATION OF PROTEINS BY GLUCOSE

Glycation and oxidation reactions contribute to protein modification i n aging and diabetes. Formation of dicarbonyl sugars during autoxidati on of glucose is the hypothetical first step in the autoxidative glyco sylation and subsequent browning of proteins by glucose [Wolff, S. P., and Dean, R. T. (1987) Biochem. J. 245, 243-250]. In order to identif y the dicarbonyl sugar(s) formed during autoxidation of glucose under physiological conditions, glucose was incubated in phosphate buffer (p H 7.4) at 37 degrees C under air (oxidative conditions) or nitrogen wi th transition metal chelators (antioxidative conditions). Dicarbonyl c ompounds were analyzed spectrophotometrically and by HPLC after reacti on with Girard-T reagent. Carbohydrates were analyzed by gas chromatog raphy-mass spectrometry. Both dicarbonyl sugar and arabinose concentra tions increased with time and glucose concentration in incubations con ducted under oxidative conditions; only trace amounts of these product s were detected in glucose incubated under antioxidative conditions. H PLC analysis of adducts formed with Girard-T reagent indicated that gl yoxal was the only a-dicarbonyl sugar formed on autoxidation of glucos e. Glyoxal and arabinose accounted for greater than or equal to 50% of the glucose lost during a 21 day incubation. Neither glucosone nor it s degradation product, ribulose, was detectable. Reaction of glyoxal w ith RNase yielded the glycoxidation product, N epsilon-(carboxymethyl) lysine, while arabinose is a source of pentosidine. Our results implic ate glyoxal and arabinose as intermediates in the browning and crossli nking of proteins by glucose under oxidative conditions. They also pro vide a mechanism by which antioxidants and dicarbonyl trapping reagent s, such as aminoguanidine, limit glycoxidation reactions and support f urther evaluation of these types of compounds for inhibition of chemic al modification and crosslinking of proteins during aging and diabetes .