Figure 2.

Effect of methylglyoxal concentration on the kinetics of fibril formation of human insulin. (A) Kinetics of fibrillation at different MGO concentrations monitored by ThT fluorescence. The symbols represent the average of ThT fluorescence intensities determined in three experiments, and the lines represent the best fit using the equation 1. Methylglyoxal concentrations used were 0 (•), 0.1 (^■), 0.25 (◇), 0.5 (^□), 1 (×), 2.5 (○) and 5 (+) mM. The decreasing in fluorescence intensities of the curves plateau are correlated with increasing methylglyoxal concentrations. (B) Evaluation of ThT quenching by methylglyoxal and AGEs. Non-glycated insulin fibrils were probed by ThT fluorescence after 8 h incubation (blue). Subsequently insulin fibrils were mixed with methylglyoxal (red) and glycated insulin containing AGEs (green) and probed again by ThT fluorescence. Fluorescence spectra show no quenching of ThT fluorescence induced by either methylglyoxal (red) or AGEs (green). (C) Dependence of the kinetic parameters lag time (C₁) and apparent rate constant (C₂) as a function of methylglyoxal concentration. Lag time is taken as x₀-2τ and the k is given by 1/τ. (D) α- to β- transition of insulin at the indicated methylglyoxal concentrations during the fibrillation process followed by circular dichroism. CD spectra were collected at time 0 h (black), 3 h (blue), 5 h (green) and 7 h (red) incubation. Measurements were all performed at 37°C with agitation of the reaction mixture.