Core histone associations in solutions of high salt. An osmotic pressure study.

We have measured the concentration dependence of the osmotic pressure for solutions of core histones in 2.0 M NaC1, pH 8.0, over the concentration range from 1 to 20 mg/ml at 25°C. The ratio of the osmotic pressure to the protein concentration is nearly constant over this range, with a value corresponding to an apparent number average molecular weight of 75,000. Gel filtration of a typical core histone preparation at 5°C at an initial concentration of 4.6 mg/ml resulted in the elution of 10% of the sample by weight as low molecular weight material consisting only of histones H2A + H2B, likely a dimer complex; 90% of the sample eluted at a volume close to that for a cross-linked histone octamer marker. The presence of this amount of low molecular weight material in the sample requires that the remaining material have a number average molecularweight of at least 92,500 to yield a measured value of M,, over the whole sample, of 75,000. The existence of an appreciable amount of histone tetramers is therefore excluded for core histone concentrations greater than about 2 mg/ml. M,, for the purified high molecular weight fraction, however, was only 80,000, indicating that histones H2A + H2B interact with a larger complex in an association equilibrium. At 35”C, the concentration dependence of the osmotic pressure clearly indicated that an association equilibrium occurred. We show that the concentration dependence of the osmotic pressure for a solution of total core histones at 25°C can be described well by a dimer + hexamer octamer equilibrium with K = (1.5 +0.5) X lo5 M-’ and a second osmotic viral coefficient, B = (1.8 k 0.17) X lo-‘ cm3 mol g-‘, where the dimer and hexamer have the compositions (H2A H2B) and (H3 H4)2 (H2A H2B), respectively. We also found s%~+. for the equilibrium mixture was 4.5 and have performed sedimentation equilibrium measurements on the same material as used in the osmometry for comparison with the results of others.