Histone fractionation by high-performance liquid chromatography

A method for the rapid chromatography of histones by high-performance liquid chromatography (HPLC) using a reverse-phase μBondapak C₁₈ column containing a packing of octadecylsilane chemically bonded to silica and a linear elution gradient running from water to acetonitrile is described. Two conditions were found to be necessary to achieve histone fractionation: (i) silylation of the active groups of the silica solid support, and (ii) trifluoroacetic acid (TFA) in the eluting solvents. Greater than 90% of the total ³H]lysine-labeled protein applied to the column was eluted from the column. The fractionation of the histones appears to be based on the hydrophobic properties of the proteins. The HPLC histone fractions (identified by their electrophoretic mobilities) were eluted from the column in the following order: H1, H2B, (LHP)H2A, (MHP)H2A + H4, (LHP)H3, and (MHP)H3 (where LHP and MHP refer to the less hydrophobic and more hydrophobic histone variants). Phosphorylated histone species were not resolved from their unmodified parental species. The volatile nature of the water/acetonitrile/TFA eluting solvent facilitated the recovery of salt-free histones from the eluted HPLC fractions by simple lyophilization. This system is very useful for the rapid isolation of the lysine-rich histones, H1 and H2B, and the variants of histone H3. With further development, this system is expected to extend the advantages of HPLC to the fractionation of histone H4 and the variants of histone H2A as well.