CHEMICAL ASPECTS OF NONENZYMIC GLYCOSYLATION OF PROTEINS

Nonenzymic glycosylation, a post-translational protein modification reaction, is simply a reflection of the potential of the 'aldehydic' function of aldoses to form a reversible Schiff-base adduct with the amino functions and the subsequence intramolecular rearrangement, Amadori rearrangement, to form a more stable ketoamine adducts. The in vivo nonenzymic glycosylation can be considered as a continuous, cumulative chemical insult to body proteins and possibly nucleic acids and phospholipids. The glucose dependent cross-linking of tissue proteins, and the accelerated aging in diabetes, has been described, as the consequence of enhanced nonenzymic glucosylation of body proteins. The major objectives of the present proposal are a) to understand the stereochemical features of the protein in the vicinity of the susceptible Epsilon-amino group(s) that facilitate the Amadori rearrangement and b) to determine the reactivity of the carbonyl function of the ketoamine linkages towards the free amino groups of proteins to understand whether the glucose-dependent cross-linking reaction seen with nonenzymically glucosylated protein is a chemical consequence of such a reactivity. Model tetrapeptides containing an internal lysine residue will be synthesised, and the influence of the neighboring amino acid residues on the rate of nonenzymic glucosylation of their Epsilon-amino group will be investigated. A relatively simple in vitro model protein system of nonenzymic glucosylation will be developed using Ribonuclease-A. Reactivity of the carbonyl function of the ketoamine linkages towards dansylhydrazine and also for reductive amination with dansyl ethylenediamine will be investigated. Attempts will be made to develop these reactions for a quantitative new fluorescent assay to estimate nonenzymic glucosylation. The reactivity of the carbonyl functions of ketoamines with the amino groups of proteins to form stable fluorescent products will be investigated. Fluorescent/cross-lined products will be isolated by RPHPLC of a complete enzymatic digest of cross-linked RNase-A and characterized. Fluorescent adduct from crytallin and collagen will be isolated by similar procedures to establish their identity with the synthetic material. The results of these studies will increase our basic knowledge about the chemical aspects of nonenzymic glucosylation of proteins and the chemical consequences, and thus its contribution in the pathophysiology of diabetes mellitus and aging process.