Figure 4

Effects of methylglyoxal on insulin structure and stability. Insulin (3 mg/ml) was incubated with 1 and 5 mM of methylglyoxal at 37°C without stirring for 48 h and compared with non-glycated insulin. Insulin secondary structure was monitored far-UV CD. Circular dichroism spectra were recorded as a function of time at different methylglyoxal concentrations (A - 0 mM; B - 1 mM; C - 5 mM). Spectra were collected at time zero (blue) and after 24 h (red) and 48 h (green) incubation. Deconvolution of the CD spectra are present in Table 2. Protein conformational stability was evaluated for native insulin (D) and glycated insulin (E) by guanidinium hydrochloride equilibrium denaturation curves at pH 7.4 and 37°C monitored by circular dichroism at 222 nm. The curves are non-linear least squares fits to a two-state unfolding model equation [71, 72] representing the entire denaturation curve and using a linear extrapolation method to the experimental circular dichroism data [47]. The insets are the residues plot.