Preparation of histone variants and high-mobility group proteins by reversed-phase high-performance liquid...

Methods have been developed for the preparation of histone variants and high-mobility group (HMG) proteins by high-performance liquid chromatography (HPLC). The individual HPLC fractions were recovered as a dry powder in 95% yield by direct lyophilization from the column effluent. Perchloric acid-soluble H1 variants and HMG proteins from Chinese hamster cells (line CHO) were separated on a mu Bondapak CN column using a 0-50% linear acetonitrile gradient in water containing 0.2% trifluoroacetic acid (TFA). The proteins were eluted in the following order: HMG-E/G (an HMG-14/17 class proteins from CHO cells), HlO, Hl, HMG-2, and HMG-l. HMG-E/G, Hl, and an unidentified protein were recovered electrophoretically pure. HlO contained contaminants which could be removed by subsequent chromatography on a mu Bondapak C18 Radial-Pak column, but HMG-l and HMG-2 could not be completely resolved. Nucleosomal core histones were fractionated on a mu Bondapak C18 Radial-Pak column using a 30-55% linear acetonitrile gradient containing 0.2-0.3% TFA. They were eluted in the following order: H2B, (LHP)H2A, (MHP)H2A, H4, LHP(H3), and (MHP)H3, (where LHP and MHP refer to less-hydrophobic and more-hydrophobic variants). If the gradient containing 0.3% TFA was interrupted with an isocratic elution at 43% acetonitrile, the H2B, (LHP)H2A, (MHP)H2A, and H4 proteins were completely resolved, thus providing a good preparative method for these proteins. The H2A class of Drosophila histones was also fractionated on a mu Bondapak C18 Radial-Pak column using a 30-35% linear acetonitrile gradient containing 0.2% TFA. Drosophila melanogaster H2A, obtained as a single fraction by chromatography on Biol-Gel P-100, was eluted from the C18 column as three proteins. The order of elution was identified by electrophoresis to be: H2Aox (an oxidized form of H2A), D2 (a Drosophila-specific subtype), and H2A.