In the absence of surfactants, recombinant human growth hormone (rhGH) rapidly forms insoluble aggregates during agitation. The nonionic surfactant Tween 20, when present at Tween:protein molar ratios >4, effectively inhibits this aggregation. Differential scanning calorimetry (DSC) of rhGH solutions showed melting transitions that decreased by ca. 2 degrees C in the presence of Tween. Circular dichroism (CD) studies of the same thermal transition showed that the decrease is specific to the relatively high protein concentrations required for DSC. CD studies showed melting transitions that decreased with lower protein concentrations. Tween has an insignificant effect on the melting transition of rhGH at lower protein concentrations (0.18 mg/mL). Injection titration microcalorimetry showed that the interaction of Tween with rhGH is characterized by a weak enthalpy of binding. For comparison, interferon-g, another protein which has been shown to bind Tween, also shows weak enthalpy of binding. Fluorescent probe binding studies and infrared spectroscopic investigations of rhGH secondary structure support suggestions in the literature (Bam, N. B.; Cleland, J. L., Randolph, T. W. Molten globule intermediate of recombinant human growth hormone: stabilization with surfactants. Biotechnol. Prog. 1996. 12, 801-809) that Tween binding is driven by hydrophobic interactions, with little perturbation of protein secondary structure.

We demonstrate that a surfactant-stabilized molten globule intermediate exists for recombinant human growth hormone (rhGH), is very hydrophobic, and tends to form aggregates. Characterization of this intermediate included equilibrium denaturation measured by electron paramagnetic resonance (EPR) and CD spectroscopy, assessment of aggregation during refolding, and fluorescence studies of its binding to the hydrophobic probe, 1-anilinonapthalene-8-sulfonate (1,8-ANS). We have found that at 4.5 M guanidinium hydrochloride (GuHCl), a molten globule intermediate of rhGH is stabilized and results in significant aggregation upon refolding. This intermediate is populated by the addition of the nonionic surfactant, Tween. This surfactant also reduces the extent of aggregation during refolding of rhGH from 4.5 M GuHCl. Overall, our studies reveal that rhGH forms a molten globule-like intermediate during folding and this intermediate self-associates. This self-association is reduced upon formation of a Tween-rhGH complex. Tween also binds to the native protein. Thus, nonionic surfactants such as Tween may act like molecular chaperones in facilitating protein folding while not altering the native conformation.

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Surfactants are known to stabilize proteins and are often employed as additives in protein formulations. We have developed a method to study the interaction of these formulation additives with proteins by using the partitioning behavior of a spin label. In protein-free formulations, 16-doxyl stearic acid partitions into micelles above the critical micelle concentration (CMC) of the surfactant and gives rise to composite electron paramagnetic resonance (EPR) spectra composed of spectra from "free" label and "rotationally hindered" label. We compute the fraction of micelle-associated label by factor analysis and generate a label partition curve. When protein is added to the formulation, surfactant-protein aggregates form at concentrations below the surfactant's CMC. Partitioning of the label into these aggregates causes the EPR spectrum to reflect hindered rotation of the label at lower surfactant concentrations than in the protein-free solutions. A simple model of label partitioning shows that these partitioning shifts can be correlated to the surfactant:protein binding stoichiometry. We have studied the interactions of various non-ionic surfactants like Brij and Tween with recombinant human growth hormone and recombinant human interferon-gamma and obtained corresponding binding stoichiometries. These binding stoichiometries match those obtained by other techniques. This technique offers a new method for estimating the protein:surfactant binding stoichiometries.

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