Evidence for the Formation of Adducts andS-(Carboxymethyl)cysteine on Reaction of α-Dicarbonyl Compounds with Thiol Groups on Amino Acids, Peptides, and Proteins

Nonenzymatic covalent adduction of glucose, or aldehydes derived from glucose or oxidation reactions, to proteins (glycation) has been proposed as a key factor in the vascular complications of diabetes. In conditions of chronic glucose elevation, alpha-dicarbonyl compounds, including glyoxal and methylglyoxal, are also present at elevated levels. These carbonyls react rapidly with nucleophilic groups on Lys and Arg side chains and the N-terminal amino group, to give poorly defined products, often called advanced glycation endproducts. These are present at elevated levels in tissue samples from people with diabetes and have been linked with disease development. As the thiol group of Cys is a powerful nucleophile, we hypothesized that adduction should occur rapidly and efficiently at Cys residues. It is shown here that Cys residues react with dicarbonyl compounds to give thiol-aldehyde adducts, which have been detected by electrospray ionization mass spectrometry. This process is accompanied by loss of the thiol group and formation of stable products. In the case of glyoxal, these reactions give S-(carboxymethyl)cysteine. The percentage conversion of thiol lost to product is substrate-dependent and < or = 32%. S-(Carboxymethyl)cysteine has been quantified by HPLC on thiol-containing, protected amino acids, peptides, and proteins, after exposure to glyoxal. The yield of this product has been shown to increase in a time- and dose-dependent manner with higher glyoxal concentrations and to also be formed on extended incubation of serum albumin with glucose. This novel, stable, advanced glycation endproduct is a potential marker of glycation.