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 114 are u sually eluted by 4 M guanidinium chloride (gdm Cl). In order to protec t cysteines and methionines from oxidation we systematically added a-m ercaptoethanol to the elution buffer. This resulted in the two histone s being unexpectedly eluted together at around 1 M gdm Cl. The use of a shallower gradient resulted in a division in the peak of histones, w ith the acetylated species of H3 and H4 being eluted first and the non acetylated species of H3 and H4 eluted last, When histone H3 or histon e H4 was applied alone or when the chromatography was performed at low pH, these histones were eluted in the usual position at about 4 M gdm Cl. These events mean that the simultaneous elution of the histones H 3 and H4 at about 1 M gdm Cl involves the formation of H3-H4 complexes . Therefore, the H3-H4 complex may be obtained by ion-exchange chromat ography as the H2A-H2B complex was previously; furthermore, the former was fractionated according to postsynthetic modifications. This findi ng provides a new basis for explaining some of the previous elution pr ofiles of chromatin extracts. (C) 1996 Academic Press, Inc.