Determination of self association constant between bovine insulin molecules by capillary zone electrophoresis

Abstract: Capillary electrophoresis (CE) is an analytical technique that is very useful for investigating processes that modify the charge and mass of proteins and polypeptide pharmaceuticals. This report explores the ability of CE to determine the aggregation constant between insulin molecules. Bovine insulin is a polypeptide (Mw=5733, pI = 5.3) that has two α-amino groups (Gly and Phe) and one ε–amino group (Lys). Analysis of concentration dependence of electrophoretic mobility of insulin at different conditions yields the association constant for dimerization of insulin. The association constant estimates how tight the peptide molecules are associated. The association constant is a useful factor to evaluate the purity of a peptide or protein sample. The association reaction of bovine insulin molecules was found to be favoured by temperature. The association constants were 7200 M -1, 8000 M -1, and 36000 M -1 at 15 oC, 25 oC and 35 oC, respectively. The interactions between the peptide molecules increase at higher temperature, resulting in stronger association. The association constant was estimated to be 3000 M -1in the presence of dioxane (5%, w/v %) at 25 oC. However, the interaction sites remain to be explored.