Metal‐catalyzed oxidation of human growth hormone: Modulation by solvent‐induced changes of protein conformation

• Published in: Journal of pharmaceutical sciences Vol. 90; no. 1; pp. 58 - 69
• Main Authors: Hovorka, Susan W., Hong, Jinyang, Cleland, Jeffrey L., Schöneich, Christian
• Format: Journal Article
• Language: English
• Published: New York Elsevier Inc 01.01.2001; John Wiley & Sons, Inc; Wiley; American Pharmaceutical Association
• Subjects: Biological and medical sciences; Catalysis; General pharmacology; human growth homone; Human Growth Hormone - chemistry; Human Growth Hormone - metabolism; Hydroxyl Radical - chemistry; Medical sciences; metal-catalyzed oxidation; Metals - chemistry; Models, Molecular; Oxidation-Reduction; Pharmacology. Drug treatments; Physicochemical properties. Structure-activity relationships; Protein Conformation; protein stability; Protein Structure, Secondary; Protein Structure, Tertiary; Solvents - chemistry; protein conformation; protein stability; human growth homone; metal‐catalyzed oxidation; Stability; Tertiary structure; Somatotropin hormone; Alcohol; Metal; Secondary structure; Radical scavenger; Prevention; Protein hormone; Cosolvent; Adenohypophyseal hormone; Oxidative degradation; Oxidation; Recombinant protein; Physicochemical properties; Conformation
• ISSN: 0022-3549; 1520-6017
• DOI: 10.1002/1520-6017(200101)90:1<58::AID-JPS7>3.0.CO;2-W

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, Cu2+, 3O2) 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, P1/2, 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 P1/2 values. Solvent polarity at P1/2 was determined according to the ET(30) scale. All alcohol/water mixtures at P1/2 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. © 2001 Wiley‐Liss, Inc. and the American Pharmaceutical Association J Pharm Sci 90:58–69, 2001