Successive Elution by Ion-Exchange Chromatography of H3–H4 Histone Complexes Differing in Their Degree of Acetylation

On Biorex 70 ion exchanger at neutral pH the histones H3 and H4 are usually eluted by 4Mguanidinium chloride (gdm Cl). In order to protect cysteines and methionines from oxidation we systematically added 2-mercaptoethanol to the elution buffer. This resulted in the two histones being unexpectedly eluted together at around 1Mgdm Cl. The use of a shallower gradient resulted in a division in the peak of histones, with the acetylated species of H3 and H4 being eluted first and the nonacetylated species of H3 and H4 eluted last. When histone H3 or histone H4 was applied alone or when the chromatography was performed at low pH, these histones were eluted in the usual position at about 4Mgdm Cl. These events mean that the simultaneous elution of the histones H3 and H4 at about 1Mgdm Cl involves the formation of H3–H4 complexes. Therefore, the H3–H4 complex may be obtained by ion-exchange chromatography as the H2A–H2B complex was previously; furthermore, the former was fractionated according to postsynthetic modifications. This finding provides a new basis for explaining some of the previous elution profiles of chromatin extracts.