MONITORING THE PROGRESS OF NONENZYMATIC GLYCATION IN-VITRO

The progress of in vitro non-enzymatic glycation of bovine serum album in was followed by using C-14-glucose and a nitroblue tetrazolium assa y, absorption and fluorescence spectroscopy, SDS gel electrophoresis a nd protease digestion. The number of adducts detectable using both C-1 4-tracers and a fructosamine assay remained low at physiological gluco se concentrations, fewer than five adducts being detectable. When gluc ose concentrations > 1.0 M were used the number of adducts was found t o greatly exceed the number of lysyl residues available in BSA, indica tive of cross-linking between Maillard products. Incubation of BSA wit h glucose concentrations of up to 160 mM for one month produced no obs ervable increase in molecular weight by SDS gel electrophoresis, showi ng that at physiological glucose concentrations, increases in molecula r weight were minimal for short incubation periods, any marked changes (indicated by non-penetration of the 7.5% SDS gel) requiring nine mon ths incubation with greater than or equal to 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 incubatio n. Fluorescence spectroscopy of the modified proteins showed a disappe arance 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 develop ing Maillard products. Protease digestion gave similar patterns with n on-glycated and glycated protein, suggesting that glycation did not bl ock digestion sites, and that partial digestion did not cause signific ant further exposure of susceptible sites. Our results show that while glycation ultimately results in protein conformational changes and th e formation of large molecular weight species, these occur at a relati vely late stage in the maturation of protein Maillard products, after greater than or equal to nine months of incubation with glucose concen trations of greater than or equal to 20 mM. Monitoring of AGE maturati on in vitro is better accomplished by following incorporation of C-14- [UL]-glucose and spectroscopic techniques than by monitoring of molecu lar weight changes. (C) Munksgaard 1994.