Spectral Studies on the Conformational Transitions of Bovine Insulin During Denaturant-induced Unfolding

Transitions among various molecule states and conformational changes of bovine insulin were investigated under different denaturing conditions by means of fluorescence phase diagrams, fluorescence quenching, 1-anilinonaphthalene-8-sulfonate(ANS) binding assay and circular dichroism(CD) spectra. In both guanidine hydrochloride(GuHCl)- and urea-denatured procedures, the spatial structure of insulin molecules changed from ordered states to relative unordered ones with the increasing of denaturant concentration. The GuHCl-denatured process followed a four-state model, for there were two intermediates existed in 2.0 and 6.0 mol/L GuHCl, respectively. Intermediate I-1 is more compact than the normal protein. And intermediate I-2 has lost most of the secondary structures. When GuHCl concentration was above 6.0 mol/L, the fluorophores originally existed in the internal of insulin molecules would expose to the surface. However, the urea-denatured process followed a three-state model, only one intermediate existed in 2.5 mol/L urea. During the urea-denatured procedure, the fluorophores originally existed in the internal of insulin molecules didn't expose to the surface.