Fine characterization of glucosylated human IgG by biochemical and biophysical methods.

Nonenzymatic glycosylation of proteins finally generates advanced glycation end products (AGEs). The Schiff's base and Amadori adduct are stages of early glycation. AGE-modified IgG may undergo conformational alterations and the final entity of the process may be involved in the pathogenesis of Rheumatoid Arthritis (RA). In this study, glycation of human IgG was carried out with varying concentrations of glucose. Effect of incubation period on glycation of IgG has also been studied. Amadori adduct was detected by nitroblue tetrazolium (NBT) dye. The glucose mediated structural alterations in IgG were studied by UV, fluorescence, CD, FT-IR, DLS and DSC spectroscopy, and SDS-PAGE. Glycation-induced aggregation in AGE-IgG was reported in the form of binding of thioflavin T and congo red. Furthermore, AGE-modified IgG exhibited hyperchromicity, decrease of tryptophan fluorescence accompanied by increase in AGE specific fluorescence, loss of ß-sheet, appearance of new peak in FT-IR, increase in hydrodynamic size and melting temperature. SDS-PAGE results showed decrease in the band intensity of glycosylated-IgG compared to native IgG. Glycation-induced modifications and aggregation of IgG might be important in the pathogenesis of RA.