| Abstract: | Change in aggregation state of insulin upon conjugation with 5-dimethylaminonaphthalene-1-sulfonyl (DNS) group was investigated at neutral pH. DNS group was introduced exclusively into B1 phenylalanine, the N-terminus of the B-chain of insulin. The association state of insulin shifted toward a more highly aggregated one upon conjugation, depending on the mole fraction (d) of DNS group to insulin monomer; at d equal 0.3 the equilibrium between dimer and hexamer was dominant over the range of 1-600 microM, while at d equal 1.0-1.5 DNS-insulin formed a larger aggregate (dodecamer) which is stable over the range of 67-600 microM. The dissociation constant of dimer-hexamer equilibrium at d=0.3 was evaluated to be 2.5 x 10(-10) M2 from the fluorescence anisotropy of the DNS group, which was about one order of magnitude smaller than that of the dimer-hexamer equilibrium in native insulin. Spectroscopic data and fluorescence decay analyses indicated that there exist at least two different environments surrounding the dye bound to B1 phenylalanine and that they are both relatively hydrophilic. It is considered that the major part of DNS group has excitation and emission maxima at longer wavelength with relatively low quantum yield, while the minor part has excitation and emission maxima at shorter wavelengths with relatively high quantum yield. The fluorescence lifetime of the dye was modified by the change in quaternary structure of DNS-lifetime of the dye was modified by the change in quaternary structure of DNS-insulin. Remarkable depolarization of DNS fluorescence was observed at d equal 1.0 and d equal 1.5 due to energy transfer between DNS groups conjugated to B1 phenylalanine in the hexamer or the dodecamer. Critical transfer distance for inter-DNS energy transfer was evaluated to be 15 A. From the molecular model of the insulin crystal, this energy transfer is ascribed to the close proximity, within about 15 A, between DNS groups in dimer units of the hexamer or the dodecamer. |
