Photophysical Characterization of Insulin Denaturation and Aggregation at Hydrophobic Interfaces

Abstract

The peak III/I ratio of pyrene monomer fluorescence was used to characterize surface-induced insulin denaturation and aggregation. An indicator of the hydrophobicity of pyrene's microenvironment, the IIM ratio can be used to study self-association of amphipathic molecules. In sodium lauryl sulfate (NaLS), the III/I ratio was 0.71 below the critical micelle concentration (CMC) and increased to 0.98 when the concentration was above the CMC. When insulin was placed in a polystyrene cuvet for 2 days at 37°C, the IWI ratio decreased from 0.84 to 0.78. The decrease in III/I ratio corresponds to surface-induced denaturation of insulin which exposed pyrene to a more polar environment. Increasing the duration of incubation for up to 12 days increased the III/I ratio to 1.06. Increased hydrophobicity of pyrene's environment correlates with the self-association of insulin. Once the aggregates reached a critical size, they began precipitating as was evident by an increase in the turbidity (absorbance at 600 nm) of solution. The results of this study support previous hypothesis that the aggregation and precipitation of insulin at hydrophobic interfaces is initiated by adsorption and surface-induced denaturation.