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Histone fractionation by high-performance liquid chromatography.

L R Gurley, J G Valdez, D A Prentice

    Analytical Biochemistry
    |February 15, 1983
    PubMed
    Summary

    This study introduces a rapid high-performance liquid chromatography (HPLC) method for histone separation. The technique effectively fractionates histones based on hydrophobicity, enabling quick isolation of key histone types.

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    Area of Science:

    • Biochemistry
    • Chromatography
    • Molecular Biology

    Background:

    • Histones are crucial for DNA packaging and gene regulation.
    • Efficient separation of histone proteins is essential for studying their diverse roles.
    • Traditional histone fractionation methods can be time-consuming and complex.

    Purpose of the Study:

    • To develop a rapid and effective method for histone separation using high-performance liquid chromatography (HPLC).
    • To optimize HPLC conditions for achieving precise histone fractionation based on hydrophobic properties.
    • To facilitate the isolation of specific histone types and variants for further analysis.

    Main Methods:

    • Utilized reverse-phase HPLC with a mu Bondapak C18 column.
    • Employed a linear gradient elution from water to acetonitrile.

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  • Incorporated silylation of silica solid support active groups and trifluoroacetic acid (TFA) in solvents for optimal fractionation.

    • Main Results:

    • Achieved effective histone fractionation based on protein hydrophobicity, with over 90% of applied protein eluted.
    • Determined the elution order of histones: H1, H2B, less/more hydrophobic H2A, H4, less/more hydrophobic H3.
    • Demonstrated facile recovery of salt-free histones via lyophilization due to the volatile eluting solvent.

    Conclusions:

    • The developed HPLC method provides a rapid and efficient system for isolating lysine-rich histones (H1, H2B) and histone H3 variants.
    • The system shows promise for future extension to the fractionation of histone H4 and H2A variants.
    • This HPLC approach offers significant advantages for rapid histone purification and analysis in biochemical research.