The pyridoindole antioxidant stobadine inhibited glycation-induced absorbance and fluorescence changes in albumin.

Abstract

We studied the effect of the pyridoindole antioxidant stobadine on glycation-induced absorbance and fluorescence changes in bovine serum albumin (BSA), used as a model protein. Incubation of BSA (4 mg/ml) with glucose (100-400 mM) in 0.12 M phosphate buffer, pH 7.4, in the presence of 100 microM Cu2+ at 37 degrees C resulted in a time-dependent increase of absorbance (320 nm) and fluorescence (excitation 350 nm, emission 415 nm). The process was found to be dependent on the presence of oxygen and transition metal ions, but equimolar iron could not fully substitute for the activity of copper. The glucose-induced chromo- and fluorophore formation was reduced significantly by stobadine. For 200 mM glucose, in 7- and 14-day incubations, 51%-60% inhibition was obtained at a stobadine concentration of 0.1 mM, and the effect leveled off at higher concentrations of the drug. No inhibition was observed with N-acetyl stobadine, a derivative with restricted antioxidant activity. Since stobadine did not affect the Amadori product formation determined by the thiobarbituric acid (TBA) method as 5-hydroxymethyl furfural (5-HMF) released in boiling oxalic acid, the inhibitory action of stobadine may be explained by its interference with metal-catalyzed oxidation reactions following after the glycation step. The results obtained suggest that antioxidant therapy could be used to limit the damage from adverse glycation-induced processes in diabetes mellitus.