EQUILIBRIUM INTERMEDIATES IN THE DENATURATION OF HUMAN INSULIN AND 2 MONOMERIC INSULIN ANALOGS

The equilibrium denaturation of human insulin in a monomer-inducing so lvent and of two monomeric insulin analogs, lys(B28)pro(B29) insulin a nd asp(B10)des(B28-30) insulin, was reexamined [Brems, D. N., Brown, P . L., Heckenlaible, L. A., and Frank, B. H. (1990) Biochemistry 29, 92 89-9293] by circular dichroism (CD) at additional wavelengths in the n ear-UV region. Previous denaturation studies were limited by the solub ility of guanidine hydrochloride being only slightly greater than the level of denaturant required to fully unfold human insulin; therefore, only a few data points were available for construction of the posttra nsitional baseline. In the present study, we report the use of an unfo lded mimic created by enzymatic digestion of insulin to confirm the sl ope of the post-transitional baseline. Evidence fbr equilibrium unfold ing intermediates for each of these insulins was indicated by noncoinc idence of the denaturation transitions as monitored by tyrosine and he lical-dependent CD bands (270 and 224 nm, respectively). Additional ev idence for intermediates through multiphasic denaturation transitions was obtained at a wavelength likely related to disulfide conformation, 251 nm. The results suggest that for each of the insulins, at least t wo intermediates are significantly populated. An unfolding model is pr oposed in which the conformation of the least stable intermediate is s lightly unfolded only in the C-terminal segment of the B chain. A seco nd more stable intermediate retains minimal secondary structure while containing localized structure proximal to one or more of the disulfid e groups. The presence of equilibrium intermediates has important impl ications for the folding pathway of insulin, in pharmaceutical applica tions such as formulation stability, and for conformational transition s that accompany receptor binding.