Previous investigations have shown that bovine growth hormone (bGH, somatotropin) unfolds through a reversible multistate process with at least one stable equilibrium intermediate. In extending our knowledge of the folding process for bGH, we demonstrate that a self-associated form of partially denatured bGH is formed during equilibrium unfolding experiments. The self-associated species has been identified by hydrodynamic measurements (size exclusion high-performance liquid chromatography and static and dynamic light scattering) and by measurements of the bGH concentration dependence of aromatic amino acid spectral properties (fluorescence, second-derivative absorption, and circular dichroism). The apparent maximum concentration for self-association occurs when bGH is partially denatured, i.e., at 3.7 M guanidine hydrochloride or 8.5 M urea, and its formation is reversible. Some of the properties of the self-associated species include quenched tryptophan fluorescence, increased tryptophan circular dichroism intensity at 300 nm, polar tryptophan environment, and a weight-average radius of about 5 nm. The self-association of bGH is mediated by specific intermolecular interactions with little increase in molecular size occurring above the saturation level of 4 mg/mL bGH. These phenomena have important implications for the design and interpretation of folding experiments in vitro and may have physiological consequences.
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Havel et al. (1986) studied this question.