Equilibrium intermediates in the denaturation of human insulin and two monomeric insulin analogs.
The equilibrium denaturation of human insulin in a monomer-inducing solvent and of two monomeric insulin analogs, lysB28proB29 insulin and aspB10desB28-30 insulin, was reexamined [Brems, D. N., Brown, P. L., Heckenlaible, L. A., & Frank, B. H. (1990) Biochemistry 29,9289-9293] by circular dichroism (CD) at additional wavelengths in the near-UV region. Previous denaturation studies were limited by the solubility 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 post-transitional baseline. In the present study, we report the use of an unfolded mimic created by enzymatic digestion of insulin to confirm the slope of the post-transitional baseline. Evidence for equilibrium unfolding intermediates for each of these insulins was indicated by noncoincidence of the denaturation transitions as monitored by tyrosine and helical-dependent CD bands (270 and 224 nm, respectively). Additional evidence 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 two intermediates are significantly populated. An unfolding model is proposed in which the conformation of the least stable intermediate is slightly unfolded only in the C-terminal segment of the B chain. A second more stable intermediate retains minimal secondary structure while containing localized structure proximal to one or more of the disulfide groups. The presence of equilibrium intermediates has important implications for the folding pathway of insulin, in pharmaceutical applications such as formulation stability, and for conformational transitions that accompany receptor binding.
Millican
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Lilly Research Laboratories, Eli Lilly and Company, Lilly Corporate Center, Indianapolis, Indiana 46285.
Brems
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DN
D000818:Animals; D002942:Circular Dichroism; D019791:Guanidine; D006146:Guanidines; D006801:Humans; D007328:Insulin / Q000031:analogs & derivatives / Q000737:chemistry*; D008297:Male; D011489:Protein Denaturation; D017510:Protein Folding; D051381:Rats; D017207:Rats, Sprague-Dawley; D013056:Spectrophotometry, Ultraviolet
D006146:Guanidines; D007328:Insulin; D019791:Guanidine