ROLE OF PROTEIN-BOUND CARBONYL GROUPS IN THE FORMATION OF ADVANCED GLYCATION ENDPRODUCTS

Several mechanisms have been postulated for the formation of advanced glycation endproducts (AGEs) from glycated proteins; they all feature protein-bound carbonyl intermediates. Using 2,4-dinitrophenylhydrazine (DNPH), we have detected these intermediates on bovine serum albumin, lysozyme and beta-lactoglobulin after in vitro glycation by glucose o r fructose. Carbonyls were formed in parallel with AGE-fluorophores, v ia oxidative Maillard reactions. Neither Amadori nor Heyns products co ntributed to the DNPH reaction. Fluorophore and carbonyl yields were m uch enhanced in lipid-associated proteins, but both groups could also be detected in lipid-free proteins. When pre-glycated proteins were in cubated in the absence of free sugar, carbonyl groups were rapidly los t in a first-order reaction, while fluorescence continued to develop b eyond the 21 days of incubation. Another unexpected finding was that n ot all carbonyl groups were blocked by aminoguanidine, although there was complete inhibition of reactions leading to AGE-fluorescence. It i s suggested that carbonyls acting as fluorophore precursors react read ily with aminoguanidine, while others are resistant to this hydrazine, possibly because they are involved in ring closure. Factors influenci ng the relative rates of acyclisation and hydrazone formation are disc ussed, together with possible implications for antiglycation therapy.