Nonenzymatic glycosylation of human serum albumin alters its conformation and function.

Approximately 10% of the albumin in normal human serum is modified by nonenzymatic glycosylation, primarily at the c-amino group of lysine residue 525.Incubation of albumin with glucose under physiological conditions in vitro resulted in glycosylation of the same residue.After separation of glycosylated human serum albumin from the nonglycosylated form by boronate affinity chromatography, the fluorescence emission characteristics of the sole tryptophan residue (Trp 214) were monitored.The quantum yield of tryptophan fluorescence for both in vivo and in vitro glycosylated albumin was reduced 30% relative to nonglycosylated albumin, and the maximal wavelength of the fluorescence emission band was shifted to shorter wavelengths.These observations show that nonenzymatic glycosylation induces a conformational change in human serum albumin.Ligand binding properties of glycosylated and unmodified albumin were compared.Hemin affinity was unaltered by glycosylation of albumin in vivo, whereas the affinity of bilirubin for glycosylated albumin was about 50% its value for the nonglycosylated form.The affinity of the long chain fatty acid cis-parinaric acid for albumin glycosylated in vivo and in vitro was reduced approximately 20-fold relative to nonglycosylated albumin.These differences in affinity suggest that lysine 525 plays a key role in the binding of physiologically important ligands to albumin.Human serum albumin is a single chain polypeptide of 585 residues which comprises about 60% of the plasma protein and is the major contributor to the oncotic pressure of blood.This abundant protein has been thoroughly investigated.Its most striking property is its ability to bind an unusually broad spectrum of ligands (1, 2).These include inorganic cations, organic anions, various drugs, amino acids, and, perhaps most important, physiologically available hydrophobic molecules like bilirubin, hemin, and fatty acids.As a result, albumin is considered a multifunctional plasma transport protein.There is growing evidence that under physiological conditions glucose reacts nonenzymatically with a wide variety of proteins to form stable adducts (3-5).Proteins which are exposed to glucose and have a relatively slow turnover rate are particularly susceptible to nonenzymatic glycosylation.