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Related Experiment Videos

Subunit interactions in hemoglobin probed by fluorescence and high-pressure techniques.

S Pin1, C A Royer, E Gratton

  • 1Department of Physics, University of Illinois, Urbana 61801.

Biochemistry
|October 2, 1990
PubMed
Summary

Human hemoglobin subunit dissociation was studied using fluorescence and high pressure. Results reveal tetramer to dimer and dimer to monomer transitions, with a dimer dissociation constant of 0.1–1 nM.

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Area of Science:

  • Biochemistry
  • Biophysics
  • Molecular Biology

Background:

  • Human adult oxyhemoglobin (hemoglobin) is a tetrameric protein crucial for oxygen transport.
  • Understanding hemoglobin subunit dissociation is key to comprehending its allosteric regulation and function.
  • Previous studies utilized various techniques, but high-pressure fluorescence offers unique insights.

Purpose of the Study:

  • To investigate human adult oxyhemoglobin subunit dissociation using advanced biophysical methods.
  • To determine the dissociation constants of hemoglobin tetramers and dimers under varying conditions.
  • To assess the impact of pH and organic phosphates on hemoglobin quaternary structure.

Main Methods:

  • Steady-state fluorescence anisotropy and multifrequency phase fluorometry were employed.

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  • Human hemoglobin was labeled with 5-(dimethylamino)naphthalene-1-sulfonyl chloride (DNS-Cl).
  • High hydrostatic pressure was applied to induce and observe subunit dissociation.

    • Main Results:

    • Fluorescence anisotropy changes indicated tetramer to dimer dissociation at pH 7.
    • At pH 9, the dimer form predominated, with inositol hexaphosphate (IHP) slightly destabilizing the tetramer.
    • High pressure induced dimer to monomer dissociation, yielding a dimer dissociation constant of 0.1–1 nM.

    Conclusions:

    • Fluorescence techniques effectively monitor hemoglobin subunit dissociation and conformational changes.
    • Hemoglobin subunit affinities are sensitive to pH and organic phosphate concentrations.
    • Comparisons with other methods suggest conformational heterogeneity in hemoglobin preparations.