GSBS Student Publications

Title

The oxidation of methionine-54 of epoetinum alfa does not affect molecular structure or stability, but does decrease biological activity

UMMS Affiliation

Graduate School of Biomedical Sciences; Department of Biochemistry & Molecular Pharmacology

Date

7-30-2008

Document Type

Article

Medical Subject Headings

Allosteric Regulation; Cell Line; Circular Dichroism; Drug Stability; Epoetin Alfa; Hematinics; Hydrogen Peroxide; Methionine; Oxidation-Reduction; Protein Binding; Protein Folding; Temperature; Thermodynamics; tert-Butylhydroperoxide

Disciplines

Life Sciences | Medicine and Health Sciences

Abstract

Erythropoietin therapy is used to treat severe anemia in renal failure and chemotherapy patients. One of these therapies based on recombinant human erythropoietin is marketed under the trade name of EPREX and utilizes epoetinum alfa as the active pharmaceutical ingredient. The effect of oxidation of methionine-54 on the structure and stability of the erythropoietin molecule has not been directly tested. We have observed partial and full chemical oxidation of methionine-54 to methionine-54 sulfoxide, accomplished using tert-Butylhydroperoxide and hydrogen peroxide, respectively. A blue shift in the fluorescence center of spectral mass wavelength was observed as a linear response to the level of methionine sulfoxide in the epoetinum alfa molecule, presumably arising from a local change in the environment near tryptophan-51, as supported by potassium iodide quenching studies. Circular dichroism studies demonstrated no change in the folded structure of the molecule with methionine oxidation. The thermal unfolding profiles of partial and completely oxidized epoetinum alfa overlap, with a T(m) of 49.5 degrees C across all levels of methionine sulfoxide content. When the protein was tested for activity, a decrease in biological activity was observed, correlating with methionine sulfoxide levels. An allosteric effect between Met54, Trp51, and residues involved in receptor binding is proposed. These results indicate that methionine oxidation has no effect on the folded structure and global thermodynamic stability of the recombinant human erythropoietin molecule. Oxidation can affect potency, but only at levels significantly in excess of those seen in EPREX.

Rights and Permissions

Citation: PDA J Pharm Sci Technol. 2008 May-Jun;62(3):211-23.

Related Resources

Link to Article in PubMed

Journal Title

PDA journal of pharmaceutical science and technology / PDA

PubMed ID

18661870