Equilibrium denaturation of human growth hormone and its cysteine-modified forms.

The equilibrium denaturation of human growth hormone (hGH) derived from heterologous gene expression in Escherichia coli was studied. Denaturation was measured by ultraviolet absorbance, intrinsic fluorescence, far ultraviolet circular dichroism, and size exclusion chromatography. The denaturation transitions obtained from each method of detection were coincident, indicating a two-state denaturation mechanism. The denaturation transitions were independent of the concentration of protein. The Gibbs free energy of unfolding is 14.5 +/- 1 kcal/mol. Human growth hormone contains two disulfide bridges between residues 53-165 (large loop) and 182-189 (small loop). The small loop was selectively reduced and cysteines alkylated with iodoacetic acid or iodoacetamide. The tetra-S-carbamidomethylated and tetra-S-carboxymethylated derivatives were also prepared. All S-alkylated hGH forms were indistinguishable from the native conformations in the absence of denaturant by far ultraviolet circular dichroism. The circular dichroism-detected equilibrium denaturation of each derivative was determined and the Gibbs free energy of unfolding of the tetra-S-modified forms was 5.3 +/- 0.5 kcal/mol and of the di-S-alkylated derivatives was 11.2 +/- 0.8 kcal/mol. These results for hGH are different than previously obtained results for bovine, ovine, and rat growth hormones. Stable equilibrium intermediates have been identified for these non-human species of growth hormone. The stable intermediates observed in the denaturation of reduced, alkylated hGH or nonhunam growth hormones are similar and characterized as compact, helical, lacking native-like tertiary structure, and having a tendency to aggregate. The apparent absence of intermediates in the folding of oxidized hGH is due to the relative instability of intermediates compared with their native structures. The hGH conformation is at least 5 kcal/mol more stable than the growth hormones from other species. Reduction and alkylation of the disulfide bridges of hGH diminish the stability differences between the native and intermediate states, such that the denaturation behavior is similar to the nonhuman growth hormones with well-populated intermediates. Most proteins do not demonstrate equilibrium folding intermediates presumably because intermediates are only marginally stable in conditions that disrupt the native state. The folding results with hGH and alkylated hGH substantiate this.