Monitoring the progress of non-enzymatic glycation in vitro

The progress of in vitro non-enzymatic glycation of bovine serum albumin was followed by using 14C-glucose and a nitroblue tetrazolium assay, absorption and fluorescence spectroscopy, SDS gel electrophoresis and protease digestion. The number of adducts detectable using both 14C-tracers and a fructosamine assay remained low at physiological glucose concentrations, fewer than five adducts being detectable. When glucose concentrations > 1.0 M were used the number of adducts was found to greatly exceed the number of lysyl residues available in BSA, indicative of cross-linking between Maillard products. Incubation of BSA with glucose concentrations of up to 160 mM for one month produced no observable increase in molecular weight by SDS gel electrophoresis, showing that at physiological glucose concentrations, increases in molecular weight were minimal for short incubation periods, any marked changes (indicated by non-penetration of the 7.5% SDS gel) requiring nine months incubation with > or = 20 mM glucose. Increases in absorption were proportional to both the glucose concentration and the incubation time. Several absorption peaks, at 370, 488 and 554 nm, were consistent in appearance throughout the course of each incubation. Fluorescence spectroscopy of the modified proteins showed a disappearance of the fluorescence associated with peptide bonds and aromatic residues and the appearance of a broad peak at longer wavelengths due to the wide range of absorptive/fluorescent wavelengths of the developing Maillard products. Protease digestion gave similar patterns with non-glycated and glycated protein, suggesting that glycation did not block digestion sites, and that partial digestion did not cause significant further exposure of susceptible sites.(ABSTRACT TRUNCATED AT 250 WORDS)