The lifetime of insulin hexamers

The kinetic stability of insulin hexamers containing two metal ions was inv estigated by means of hybridization experiments. Insulin was covalently lab eled at the N-epsilon-amino group of Lys(B29) by a fluorescence donor and a cceptor group, respectively. The labels neither affect the tertiary structu re nor interfere with self-association. Equimolar solutions of pure donor a nd acceptor insulin hexamers were mixed, and the hybridization was monitore d by fluorescence resonance energy transfer. With the total insulin concent ration remaining constant and the association/dissociation equilibria unper turbed, the subunit interchange between hexamers is an entropy-driven relax ation process that ends at statistical distribution of the labels over 16 t ypes of hexamers differing by their composition. The analytical description of the interchange kinetics on the basis of a plausible model has yielded the first experimental Values for the lifetime of the hexamers. The lifetim e is reciprocal to the product of the concentration of the exchanged specie s and the interchange rate constant: tau = 1/(c.k). Measured for different concentrations, temperatures, metal ions, and ligand-dependent conformation al states, the lifetime was found to cover a range from minutes for T-6 to days for R-6 hexamers. The approach can be used under an unlimited variety of conditions. The information it provides is of obvious relevance for the handling, storage, and pharmacokinetic properties of insulin preparations.