(190bb) Effect of Glycosylation on the Aggregation of Insulin Fragments

Bovine and human insulin have subtle differences in their primary structure. We previously showed that the aggregation kinetics is very different for bovine and human insulin and for their fragments. Experiments showed that the fragments have distinct aggregation kinetics with the bovine one aggregating faster. Pareto analysis conducted at each phase of the experiments showed that temperature and pH are the prominent factors. Favorable conditions for fibrillation were pH 1.6 and a temperature of 60°C. To enhance the peptide resistance to fibrillation, the peptides were further chemically modified using glycosylation. The effect of glycosylation on the propensity to aggregate of fragments of insulin A chains having subtle differences in the primary structure was studied. A two-level full factorial design FF0308 of resolution V consisting of three factors (glycosylation site, anomeric configuration and type of peptide) at two levels was conducted. A monosaccharide, glucose (a and β-D (+)-glucose) was used for glycosylation. The incorporation of saccharides into the peptides was followed by liquid state one-dimensional (1D) ¹H NMR spectroscopy. Studies were carried out using fluorescence and infrared spectroscopy and transmission electron microscopy (TEM). Particle size distribution over time was monitored using a FOQELS particle size analyzer. The anomeric configuration of the sugar has a strong impact on the kinetics of aggregation. An increase in lag times of the glycosylated peptides in comparison with the non-glycosylated ones was found.