Role of protein-bound carbonyl groups in the formation of advanced glycation endproducts

Abstract

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 or fructose. Carbonyls were formed in parallel with AGE-fluorophores, via oxidative Maillard reactions. Neither Amadori nor Heyns products contributed to the DNPH reaction. Fluorophore and carbonyl yields were much enhanced in lipid-associated proteins, but both groups could also be detected in lipid-free proteins. When pre-glycated proteins were incubated in the absence of free sugar, carbonyl groups were rapidly lost in a first-order reaction, while fluorescence continued to develop beyond the 21 days of incubation. Another unexpected finding was that not all carbonyl groups were blocked by aminoguanidine, although there was complete inhibition of reactions leading to AGE-fluorescence. It is suggested that carbonyls acting as fluorophore precursors react readily with aminoguanidine, while others are resistant to this hydrazine, possibly because they are involved in ring closure. Factors influencing the relative rates of acyclisation and hydrazone formation are discussed, together with possible implications for antiglycation therapy.

MALDI based identification of whey protein derived tryptic marker peptides that resist protein glycation

“…Indeed, their reaction with the side chain of proteins, mainly with lysine residues, gives rise to the advanced glycation end products (AGEs). Therefore, the amount of protein bound carbonyls was used as an indicator of the Maillard reaction degree (Liggins & Furth, 1997). As expected, an increase in the amount of protein bound carbonyls due to incubation of whey proteins with glucose was observed (Fig.…”

Effect of α-lipoic acid supplementation on oxidative protein damage in the streptozotocin-diabetic rat

“…The role of increased oxidative stress in the development of oxidative protein damage in diabetes is currently a subject of great interest [9,10]. Among the various oxidative modifications of amino acids in proteins, carbonyl formation may be an early marker for protein oxidation [15].…”

“…Free radicals have previously been shown to be capable of damaging many cellular components such as DNA [6], proteins [9,10,15,17], and lipids [11,16]. The pathogenesis of atherosclerosis may involve both free radicals and peroxidation products [16].…”

“…Indeed, measurement of protein carbonyls has been used as a sensitive assay for oxidative protein damage [15]. Liggins and Furth [9] reported that the carbonyls detected under in vitro conditions were clearly the result of glycoxidation or sugar autoxidation, while some carbonyls may be of purely amino acid origin. Miyata et al [10] hypothesized that both glycoxidation and lipoxidation reactions contribute to the formation of protein carbonyls.…”

The impact of glycated pea proteins on bacterial adhesion

“…A pea protein sample (250 mg) was dissolved in a solution consisting of 39.4 ml 0.2 M phosphate buffer at pH 7.4, 500 mg of glucose and 600 ll 4% of sodium azide and subsequently incubated for 7 days in the temperature of 37°C (Liggins & Furth, 1997). After the glycation period, the samples were dialyzed for 48 h at 4°C against distilled water.…”