Characterization of arginine preventive effect on heat-induced aggregation of insulin.

Aggregation of proteins can affect their efficacy, and is especially important concerning therapeutic proteins such as insulin. Use of additives such as amino acids can counteract this deleterious process. Heat-induced aggregate formation of human insulin was kinetically studied with the use of various concentrations of the protein, at different temperatures, and in the presence of EDTA by UV-visible spectrophotometry. Effect of arginine, lysine, and histidine was then tested on the process at pH 4.8 and 45 °C. Kinetic parameters of the obtained growth curves (parameters t* and t0.5 characterizing the rate of the nucleation stage and the rate of the stage of aggregate growth respectively) were computed in all these conditions, and structure of aggregates was characterized by spectrofluorimetry, and transmission electron microscopy (TEM). Presence of high concentrations of the chelator EDTA increased aggregation. Among used additives, L-arginine (50 mM) most efficiently suppresses the heat-induced amorphous aggregation of insulin, affecting parameters t0.5 and t* presumably by preserving the protein's structure, as observed by the protein intrinsic fluorescence and CD spectra, and smaller formed aggregates in TEM images and dynamic light scattering. Docking experiment and subsequent molecular dynamics simulation indicated possible sites of interaction for arginine with the B-chain of insulin.