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Conformation and spin state in methemoglobin.

P Hensley, S J Edelstein, D C Wharton

    The Journal of Biological Chemistry
    |February 10, 1975
    PubMed
    Summary
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    Inositol hexaphosphate (IHP) induces conformational changes in methemoglobin, independent of iron spin state, affecting subunit dissociation and reactivity. These changes differ from the proposed T state, suggesting a distinct allosteric mechanism in hemoglobin.

    Area of Science:

    • Biochemistry
    • Protein Conformation
    • Hemoglobin Research

    Background:

    • Human methemoglobin properties are studied to understand its conformation.
    • The T state conformation, proposed by Perutz, is hypothesized to exist with high spin ligands and inositol hexaphosphate (IHP).
    • Subunit dissociation is a key indicator of the T state, as tetramer-dimer equilibrium differs between oxyhemoglobin (R state) and deoxyhemoglobin (T state).

    Purpose of the Study:

    • To investigate human methemoglobin conformation under various conditions.
    • To determine if methemoglobin exhibits the T state conformation in the presence of high spin ligands and IHP.
    • To analyze the effect of IHP on subunit dissociation and ligand binding kinetics.

    Main Methods:

    • Measurement of tetramer-dimer dissociation constant (K4,2) using ultracentrifugation.

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  • Haptoglobin binding assays to detect hemoglobin dimers.
  • Aggregation studies following dithionite mixing.
  • Kinetic analysis of azide binding to methemoglobin.
  • Investigation of beta93 sulfhydryl group reactivity.
  • EPR spectroscopy for spin state analysis.

    • Main Results:

    • In the absence of IHP, methemoglobin shows extensive subunit dissociation with both high and low spin ligands (K4,2 ≈ 10^-5 M).
    • IHP addition significantly lowers K4,2 for all methemoglobin forms (≈ 10^-5 M), indicating a conformational change independent of spin state.
    • The IHP-induced conformation is not identical to the T state (K4,2 ≈ 10^-12 M) and can occur in ferrous hemoglobin (e.g., CO-hemoglobin).
    • IHP enhances the rate of beta chain reactions and alters beta93 sulfhydryl group reactivity, largely independent of iron spin state.
    • Haptoglobin and aggregation studies qualitatively agree with ultracentrifuge findings on subunit dissociation.

    Conclusions:

    • IHP induces a conformational change in methemoglobin that promotes subunit association but is distinct from the T state.
    • The observed conformational changes are largely independent of the iron's spin state.
    • IHP's effects on subunit dissociation and reactivity are also observed in ferrous hemoglobin, suggesting a general allosteric mechanism.
    • The T state conformation, as defined by Perutz, may not fully represent the conformational states of methemoglobin under these conditions.