IDENTIFICATION OF THE SITE OF GLYCATION OF GAMMA-II-CRYSTALLIN BY (C-14)-FRUCTOSE

Cataract formation in diabetes may be via non-enzymic glycosylation (g lycation) of lens proteins due to increased concentrations of sugars p resent in the lenses of diabetic patients. The objective of this proje ct was to identify the site(s) of glycation of bovine gamma-II-crystal lin by [C-14]fructose. gamma-II-crystallin was isolated from soluble l ens nucleus proteins by gel chromatography, followed by ion-exchange c hromatography and was then glycated by incubation with [C-14]fructose. Radioactively labelled gamma-II-crystallin was cleaved with trypsin. Affinity chromatography of the tryptic peptides gave a single main pea k containing the majority of the radioactivity. This indicated that fr uctose had reacted at a single site on the protein. Amino acid analysi s of this peptide showed it to contain only lysine and a trace amount of glycine. By relating the results of the amino acid analysis to the amino acid sequence of gamma-II-crystallin, it was concluded that the labelled peptide corresponded to the N-terminal dipeptide. The site of glycation of bovine gamma-II-crystallin by fructose was thereby ident ified as the alpha-NH2 group of the N-terminal glycine.