MOLTEN GLOBULE INTERMEDIATE OF RECOMBINANT HUMAN GROWTH-HORMONE - STABILIZATION WITH SURFACTANTS

We demonstrate that a surfactant-stabilized molten globule intermediat e exists for recombinant human growth hormone (rhGH), is very hydropho bic, and tends to form aggregates. Characterization of this intermedia te 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 intermedi ate of rhGH is stabilized and results in significant aggregation upon refolding. This intermediate is populated by the addition of the nonio nic surfactant, Tween. This surfactant also reduces the extent of aggr egation during refolding of rhGH from 4.5 M GuHCl. Overall, our studie s reveal that rhGH forms a molten globule-like intermediate during fol ding and this intermediate self-associates. This self-association is r educed 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 alteri ng the native conformation.