Equilibrium denaturation of pituitary- and recombinant-derived bovine growth hormone.
Holladay and co-workers [Holladay, L. A., Hammonds, R. G., & Puett, D. (1974) Biochemistry 13, 1653-1661] reported the presence of an equilibrium intermediate in the guanidine hydrochloride (GdnHCl) induced denaturation of pituitary-derived bovine growth hormone (p-bGH). Since then, numerous reports have appeared demonstrating the inherent heterogeneity in p-bGH. In this report we show that a standard preparation of p-bGH can be separated into two components of almost equal abundance differing in molecular weight by approximately 1000. Each of these two components could give rise to different denaturation transitions which would be interpreted as evidence for equilibrium intermediates. We report here the equilibrium denaturation of bGH produced by Escherichia coli through recombinant DNA technology. The recombinant-derived bGH (r-bGH) is more homogeneous than that derived from pituitary sources and is greater than 95% a single polypeptide entity. Nevertheless, the GdnHCl-induced denaturation profiles of both recombinant bGH and pituitary bGH are very similar. The presence of equilibrium intermediates is verified by the asymmetry of the denaturation transition as measured by size-exclusion high-performance liquid chromatography and by noncoincidence of the denaturation transitions as observed by ultraviolet absorbance, fluorescence intensity, and circular dichroism. These findings conclusively show that the secondary structure of bovine growth hormone is more stable than the tertiary structure and is consistent with a framework model of protein folding.
D000818:Animals; D002417:Cattle; D013006:Growth Hormone / Q000096:biosynthesis / Q000302:isolation & purification*; D019791:Guanidine; D006146:Guanidines; D008970:Molecular Weight; D010902:Pituitary Gland / Q000032:analysis*; D011489:Protein Denaturation; D011994:Recombinant Proteins; D013053:Spectrophotometry
D006146:Guanidines; D011994:Recombinant Proteins; D013006:Growth Hormone; D019791:Guanidine