Conformational changes in the reversed phase liquid chromatography of recombinant human growth hormone as a function of organic solvent: the molten globule state.

As a continuation of a previous paper on the retention behavior of recombinant human growth hormone (rhGH) in reversed phase chromatography at pH 6.5 (Oroszlan, P., et al. Anal. Chem. 1992, 64, 1623-1631) the effect of 1-propanol (1-PrOH) and acetonitrile on the conformation of rhGH at this pH has been investigated by circular dichroism (CD), second-derivative UV spectroscopy, fluorescence anisotropy, fluorescence quenching, and fluorescence lifetime measurements. Addition of 1-PrOH up to a concentration of 10% (v/v) does not cause any significant changes in protein structure. However, above this concentration, a transition from the native to a new state is observed; the transition is completed above 30% (v/v) of 1-PrOH, the composition for completion being dependent on temperature. This change in structure correlates with retention changes observed in reversed phase chromatography. The new rhGH conformation retains much of the alpha-helicity and possesses a slightly expanded hydrodynamic radius relative to native rhGH. Second-derivative UV spectroscopy suggests that the hydrogen bond between Trp 86 and Asp 169, spanning two alpha-helices, remains intact. On the other hand, the near-UV CD intensity changes from positive to negative in the Trp region of the spectrum, signaling an alteration in the Trp environment. In addition, fluorescence quenching measurements with trichloroethanol reveal greater accessibility to solvent of the Trp residue after the conformational transition has occurred. From the results, it is concluded that a molten globule state (compact state retaining much of the secondary structure of the native state but with a disrupted tertiary structure) is produced with the addition of > 30% (v/v) 1-PrOH.(ABSTRACT TRUNCATED AT 250 WORDS)

Conformational effects in the reversed-phase chromatographic behavior of recombinant human growth hormone (rhGH) and N-methionyl recombinant human growth hormone (Met-hGH).

This paper examines the retention behavior of recombinant DNA-derived human growth hormone (rhGH) in reversed-phase chromatography and its separation from the closely related N-methionyl variant (Met-hGH). It is first shown that retention for rhGH decreases with increasing column temperature when 1-propanol (1-PrOH) is used as organic modifier. On the other hand, retention increases with temperature when acetonitrile (CH3CN) is employed. The differences in behavior for the two organic modifiers could be related to conformational changes in the protein as determined by solution and adsorption intrinsic fluorescence spectroscopy. Specifically, desorption and elution of rhGH using 1-PrOH could be correlated with a solvent-induced conformational change, with retention decreasing with increasing temperature due to the increasing ease of structural alteration. On the other hand for CH3CN the increase in retention correlated with temperature rise was related to a partial structural change yielding a more hydrophobic species. In this case, a surface-driven process is suggested. The work then turned to the separation of rhGH and Met-hGH where it was found for both organic modifiers optimum separation occurred at 45 degrees C and pH 6.5. Separate studies revealed that during the conformational change Met-hGH appeared more hydrophobic than rhGH since protein-protein aggregation was observed at a lower 1-PrOH concentration. It is suggested that this hydrophobic difference, which was optimized under the conditions cited above, resulted in the separation. The study demonstrates the importance of conformational changes in retention behavior and separation of protein samples.