Free radicals generated during the glycation reaction of amino acids by methylglyoxal. A model study of protein-cross-linked free radicals.

The formation of alpha-dicarbonyl compounds seems to be an important step for cross-linking proteins in the glycation or Maillard reaction. To elucidate the mechanism for the cross-linking reaction, we studied the reaction between a three-carbon alpha-dicarbonyl compound, methylglyoxal, and amino acids. Our results showed that this reaction generated yellow fluorescent products as formed in some glycated proteins. In addition, three types of free radical species were also produced, and their structures were determined by EPR spectroscopy. These free radicals are 1) the cross-linked radical cation, 2) the methylglyoxal radical anion as the counterion, and 3) the superoxide radical anion produced only in the presence of oxygen. The generation of the cross-linked radical cations and the methylglyoxal radical anions does not require metal ions or oxygens. These results indicate that dicarbonyl compounds cross-link free amino groups of protein by forming Schiff bases, which donate electrons directly to dicarbonyl compounds to form the cross-linked radical cations and the methylglyoxal radical anions. Oxygen can accept an electron from the radical anion to generate a superoxide radical anion, which can initiate damaging chain reactions. Time course studies suggest that the cross-linked radical cation is a precursor of yellow fluorescent glycation end products.