GLUCOSE MODIFICATION OF HUMAN SERUM-ALBUMIN - A STRUCTURAL STUDY

Structural changes associated with the exposure of human serum albumin (HSA) to glucose with or without the presence of Cu (II) have been ch aracterized using a bank of methods for structural analysis including circular dichroism (CD), amino acid analysis (AAA), fluorescence measu rements, SDS-PAGE, and boronate binding (which is a measure of Amadori product formation). We show that in the short-term (10 d) incubation mixtures, HSA is resistant to Cu (II)-mediated oxidative damage and th at the early products of glycation of HSA had minimal effects on the f olded structure. Amino acid analysis showed that there was no formatio n of advanced glycation endproducts (AGE), which can be measured by lo ss of lysine. This remained the case in longer term incubation of HSA (56 d) in the hyperglycemic concentration range (5-25 mM glucose) desp ite increased levels of Amadori product (60% boronate binding) and the formation of glycophore (Excitation 350, Emission 425). At high, nonp hysiological concentrations (100 mM and 500 mM) of glucose, glycophore formation increased and 3 and 11 mol Lysine-glucose adduct/mol HSA we re converted to AGE, respectively. This was accompanied by increased d amage to tryptophan and protein-protein crosslinking but only minor te rtiary structural change. In the presence of Cu (II), however, AGE for mation was accompanied by extensive damage to histidine and tryptophan side chains, main chain fragmentation, and loss of both secondary and tertiary structure. Thus, changes in structure appear to be the resul t of oxidation as opposed to glycation, per se. (C) 1997 Elsevier Scie nce Inc.