A new procedure for the isolation and fractionation of histones

An autobiographic conversation with Roger D Kornberg on his work on transcription regulation

“…6 Mild methods failed to resolve the histones entirely from one another, so the paper was ignored. What attracted my attention was the clean separation of the mildly extracted histones into two groups, H2A/H2B and H3/H4.…”

The reconstitution of histone H3H4 tetramer from acid extracted histones in the absence of urea

“…The H3-H4 tetramer containing fractions were pooled and the tetramer precipitated with 0.7 saturation ammonium sulphate. Ht and H5 contamination is removed by this step since these histones are soluble at 0.7 saturation ammonium sulphate [4]. Individual acid denatured histones H3 and I$ were purified as described previously [3] the purification of H3 involved dimerisation with subsequent reduction [7].…”

“…All these methods have in common that the separation conditions are known to seriously interfere with the higher order structures of proteins. The second group of methods is based on the dissociation of natural histone-DNA complexes at high ionic strength and fractionation of the resulting histone-histone complex utilizing the ionic strength and/or pH-dependent dissociation into smaller subunits [4,5]. This latter group of methods yields the Hs-H4 tetramer and the H~A-H~B dimer.…”

Lysosomal origin of chicken erythrocyte nuclear protease

“…Proteolytic degradation is encountered during the purification of histones [ 1 ] and combatted by the inclusion of the inhibitors bisulphite [2] or phenylmethylsulfonyl fluoride (PMSF) [3] in the isolation buffers. It is generally assumed that the proteolytic activity arises from chromatin-bound proteases [4] rather than from contamination of nuclei preparations with proteases originating from other organelles [5].…”

“…Despite the fact that proteolytic activity is absent in avian erythrocytes at pH 7 [10] work on nuclear proteins from this source has been carried out in the presence of the serine protease inhibitor PMSF [3]. Since purification of histone pairs via protamine displacement [2], frequently used for nucleosome reassembly studies, is carried out at pH 5, where proteotysis occurs in avian erythrocytes [10], we have investigated the nature of this proteolytic activity to find suitable inhibitors. Properties of the protease which lend themselves to steps in a purification procedure for this enzyme are also presented.…”