8] Nonenzymatic glucosylation of lysine residues in albumin

Abstract

Publisher Summary This chapter describes the preparation and characterization of radiolabeled glycosylamine (GA) and N-substituted-l-amino-l-deoxyketopyranose (KP) derivatives of albumin. It also illustrates procedures for determining kinetic constants involved in their formation and dissociation and for detecting glucose adducts to lysine residues in human serum albumin (HSA) and other proteins. Nonenzymatic glucosylation is a common posttranslational modification of body protein, in which glucose reacts directly with primary amino groups on protein to yield stable covalent adducts. The sugar reacts primarily with the ɛ-amino groups of lysine residues in albumin and other proteins, although reaction at the α-amino terminus also occurs. Since, the rate of hydrolysis of glycosylamines is relatively slow at neutral pH and low temperature, the kinetics of dissociation of GA adducts can be measured by Sephadex G-25 chromatography. The Amadori adduct to the protein (after acid discharge of GA) is stable to extended incubation of the protein at 37° and has an estimated minimum half-life of about 3 weeks.