Metal-catalyzed oxidation of human growth hormone: modulation by solvent-induced changes of protein conformation.
Metal-catalyzed oxidation (MCO) represents a prominent pathway of protein degradation. To evaluate the importance of the integrity of the metal-binding site on MCO, we subjected recombinant human growth hormone (rhGH), to MCO (ascorbate, Cu(2+), (3)O(2)) in the presence of various aliphatic alcohols (ethanol, ethylene glycol, trifluoroethanol, 1-propanol, 2-propanol, 1,2-propylene glycol, 1-butanol, 2-butanol, and tert-butanol). All alcohols inhibited MCO in a concentration-dependent and sigmoidal manner. Half-points, P(1/2), were dependent on the nature of the alcohol. Circular dichroism and fluorescence spectroscopy were used to monitor cosolvent-induced secondary and tertiary structural changes. The presence of alcohols increased the helical content of rhGH and induced a red shift in the tryptophan emission. The midpoints of the tertiary structural change correlated with the P(1/2) values. Solvent polarity at P(1/2) was determined according to the E(T)(30) scale. All alcohol/water mixtures at P(1/2) had rather similar solvent polarities between 54.5 to 56.4 kcal/mol, with the exception of ethylene glycol. On the other hand, no correlation was obtained between the protection against MCO and the hydroxyl radical-scavenging properties of the cosolvent. We conclude that the primary mechanism of MCO inhibition is a cosolvent-induced conformational perturbation of the metal-binding site as opposed to pure radical scavenging.
Hovorka
S W
SW
Department of Pharmaceutical Chemistry, The University of Kansas, 2095 Constant Avenue, Lawrence, Kansas 66047, USA.
Hong
J
J
Cleland
J L
JL
Schöneich
C
C
D002384:Catalysis; D019382:Human Growth Hormone / Q000737:chemistry / Q000378:metabolism*; D017665:Hydroxyl Radical / Q000737:chemistry; D008670:Metals / Q000737:chemistry*; D008958:Models, Molecular; D010084:Oxidation-Reduction; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D012997:Solvents / Q000737:chemistry
D008670:Metals; D012997:Solvents; D019382:Human Growth Hormone; D017665:Hydroxyl Radical