Jove
Visualize
Contact Us
JoVE
x logofacebook logolinkedin logoyoutube logo
ABOUT JoVE
OverviewLeadershipBlogJoVE Help Center
AUTHORS
Publishing ProcessEditorial BoardScope & PoliciesPeer ReviewFAQSubmit
LIBRARIANS
TestimonialsSubscriptionsAccessResourcesLibrary Advisory BoardFAQ
RESEARCH
JoVE JournalMethods CollectionsJoVE Encyclopedia of ExperimentsArchive
EDUCATION
JoVE CoreJoVE BusinessJoVE Science EducationJoVE Lab ManualFaculty Resource CenterFaculty Site
Terms & Conditions of Use
Privacy Policy
Policies

Related Experiment Videos

Characterization of erythropoietin dimerization

A M DePaolis1, J V Advani, B G Sharma

  • 1R. W. Johnson Pharmaceutical Research Institute, Bioanalytical Development, Raritan, NJ 08869, USA.

Journal of Pharmaceutical Sciences
|November 1, 1995
PubMed
Summary

Recombinant human erythropoietin (rHuEPO) dimerization, a key aggregation pathway, involves disulfide bond reduction and reoxidation. Understanding this mechanism is crucial for stabilizing rHuEPO formulations.

Related Concept Videos

You might also read

Related Articles

Articles linked to this work by shared authors, journal, and citation graph.

Sort by
Same author

Tracheal diverticulum: a report of 4 cases.

Ear, nose, & throat journal·2009
Same author

Duplication of the clavicle with triplication of the coracoid process.

Skeletal radiology·2003
Same author

A new syndrome of "spondylo-epi-metaphyseal dysplasia: mixed type".

Skeletal radiology·2003
Same author

Spontaneous intraperitoneal expulsion of an unruptured hydatid cyst.

Saudi medical journal·2001
Same author

Possible role of tumor necrosis factor-alpha in erythropoietic suppression by endotoxin and granulocyte/macrophage colony-stimulating factor.

American journal of hematology·1996
Same author

Thermodynamics of the disproportionation of adenosine 5'-diphosphate to adenosine 5'-triphosphate and adenosine 5'-monophosphate, II. Experimental data.

Biophysical chemistry·1991

Area of Science:

  • Biochemistry
  • Protein Chemistry
  • Pharmaceutical Sciences

Background:

  • Recombinant human erythropoietin (rHuEPO) is a therapeutic glycoprotein hormone.
  • Storage under non-ideal conditions can lead to rHuEPO dimerization and aggregation.
  • Understanding the dimerization mechanism is critical for product stability and efficacy.

Purpose of the Study:

  • To elucidate the molecular mechanism of rHuEPO dimerization.
  • To identify the specific sites and bonds involved in dimer formation.
  • To provide insights for developing strategies to prevent rHuEPO aggregation.

Main Methods:

  • Matrix-assisted laser desorption time-of-flight mass spectrometry (MALDI-TOF MS) for molecular mass determination.
  • Endoproteinase LysC peptide mapping to identify structural changes.

Related Experiment Videos

  • N-terminal sequencing to characterize the dimer interface.
  • Fluorescent thiol probe assay to detect free thiols.

    • Main Results:

    • rHuEPO dimer has an average molecular mass of 53.5 kDa, approximately double the monomer (27.8 kDa).
    • LysC mapping revealed new peptide peaks in the dimer, indicating specific cleavage sites.
    • Sequencing identified two types of EPO dimers, suggesting a complex formation pathway.
    • The mechanism involves reduction of the Cys7-Cys161 disulfide bond followed by random reoxidation.

    Conclusions:

    • The primary mechanism for rHuEPO dimerization involves disulfide bond reduction and subsequent intermolecular reoxidation.
    • Aggregation to higher molecular weight species results from random intermolecular reoxidation of Cys7 and Cys161.
    • These findings are essential for improving the stability and shelf-life of rHuEPO therapeutics.