Conformational Effects of Cations and pH on Human Growth Hormone in Solution*

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Abstract
Human GH (hGH) binding to membrane receptors is uniquely enhanced by 10 mM Ca++ and Mg++ or 150 tnM Na+ and K+. This study has used polarization of fluorescence (P) and circular dichroism (CD) to identify cation-dependent conformational alterations in hGH. CD indicated that the secondary structure of hGH consisted of 52% a-helix structure, 23% β-structure, and 25% disordered structure at pH 7.5. Increases in pH caused a small increase in a-helix structure to 55% at pH 10.5 with little change in β- structure. Decreases in pH from 7.5 resulted in decreases in ahelix and β-structure to 44% and 8%, respectively, at pH 4.1. Relatively large decreases in ordered structure occurred between pH 6 and 4. This corresponded to the pH optimum of hGH binding to membranes at pH 5.5. Cation-specific changes in structure were observed with increases in cation concentration generally associated with increases in ordered structure, i.e. α-helix and/or β-structure. The cation concentration range in which changes in structure were observed did not correspond with the cation concentration known to alter hGH binding. P was used to estimate the harmonic mean rotational relaxation time (ph) for fluoresceinated hGH (F-hGH) at different cation concentrations. The rotational time for the molecule may be interpreted in terms of molecular size and/or rigidity. Theph calculated for F-hGH at pH 7.5 in 50 mM Tris-HCl was 12.9 nsec. The ph values for F-hGH were 23.1, 17.7, 16.5, and 20.8 nsec in 100 mM KC1, 100 mM NaCl, 10 mM MgCl2, and 1 mM CaCl2, respectively. These values may be compared to the rotational relaxation time for a rigid sphere the size of a hGH molecule (∼20 nsec). Our fluorescence polarization data suggest that while at pH 7.5 in 50 mM Tris-HCl, hGH is a relatively flexible molecule in solution, cations decrease the flexibility of the hGH molecule and induce molecular rigidity in the molecule at physiological concentrations. Alterations observed in the secondary structure of the hGH molecule are apparently not directly associated with binding, since there is a lack of correlation between concentrations required for cation-dependent alterations in CD and cationenhanced hGH binding.