Identification of the residues involved in homodimer formation of recombinant human erythropoietin
| Autor: | Kendall Stoney, Thomas W. Strickland, Michael F. Rohde, Patricia L. Derby, Robert S. Rush |
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| Rok vydání: | 2009 |
| Předmět: |
Hot Temperature
Protein Conformation Stereochemistry Dimer Molecular Sequence Data Size-exclusion chromatography Mass spectrometry Biochemistry chemistry.chemical_compound Capillary electrophoresis Protein structure Humans Trypsin Amino Acid Sequence Erythropoietin Polyacrylamide gel electrophoresis Chromatography Chemistry Electrophoresis Capillary Peptide Fragments Recombinant Proteins Kinetics Matrix-assisted laser desorption/ionization Covalent bond Spectrometry Mass Matrix-Assisted Laser Desorption-Ionization Chromatography Gel Electrophoresis Polyacrylamide Gel Dimerization |
| Zdroj: | International Journal of Peptide and Protein Research. 47:201-208 |
| ISSN: | 0367-8377 |
| Popis: | Recombinant human erythropoietin (rHuEPO) is biologically functional when in a monomeric state; upon extensive heating, rHuEPO forms a dimer. The nature of this dimeric linkage was investigated after isolation of the dimer by gel filtration. The dimer fraction was subjected to tryptic digestion, and the peptides were separated by reversed-phase HPLC. SDS-PAGE, N-terminal sequencing, capillary electrophoresis and mass spectrometry (both liquid-chromatographic electrospray and matrix-assisted laser desorption ionization) were employed to compare the tryptic peptides from heat-treated rHuEPO and untreated rHuEPO. Results demonstrated that elevated heat broke the intramolecular disulfide bond between Cys-7 and Cys-161 and an intermolecular disulfide bond then formed from these residues, producing a covalently linked rHuEPO homodimer. Dimer formation was also mathematically modeled and shown to fit a simple equilibrium. |
| Databáze: | OpenAIRE |
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