Paradoxical Acceleration of Dithiothreitol-Induced Aggregation of Insulin in the Presence of a Chaperone

The kinetics of dithiothreitol (DTT)-induced aggregation of human recombinant insulin and the effect of alpha-crystallin, a representative of the family of small heat shock proteins, on the aggregation process have been studied using dynamic light scattering technique. Analysis of the distribution of the particles by size measured in the course of aggregation showed that the initial stage of the aggregation process was the stage of formation of the start aggregates with a hydrodynamic radius (R-h) of about 90 nm. When studying the effect of alpha-crystallin on the rate of DTT-induced aggregation of insulin, it was demonstrated that low concentrations of alpha-crystallin dramatically accelerated the aggregation process, whereas high concentrations of alpha-crystallin suppressed insulin aggregation. In the present study, at the molar stoichiometric ratio (insulin: alpha-crystallin) less than 1:0.5, a pronounced accelerating effect of alpha-crystallin was observed; whereas a ratio exceeding the value of 1:0.6 caused suppression of insulin aggregation. The mechanisms underlying the dual effect of alpha-crystallin have been proposed. It is assumed that heterogeneous nucleation occurring on the surface of the alpha-crystallin particle plays the key role in the paradoxical acceleration of insulin aggregation by alpha-crystallin that may provide an alternative biologically significant pathway of the aggregation process.