Improved Techniques for the Fractionation of Non-Histone Proteins of Chromatin on Hydroxyapatite

Abstract

The effect of the dissociation medium on the fractionation of chromatin on hydroxyapatite has been studied. Optimal separations of the histones and non-histone protein are only achieved when columns are run in buffers containing high concentrations of sodium ions. We have modified our previously published method such that the chromosomal proteins can be recovered in virtually quantitative yields. Each of the hydroxyapatite fractions has been analysed with respect to nucleic acid content and the proteins have been analysed by two-dimensional gel electrophoresis.Recent evidence suggests that the non-histone proteins of chromatin play an important role in the control of transcription (see review by MacGillivray and Rickwood [l] [6] after the chromatin has been extracted with dilute acid to remove the basic histones. Alternatively, chromatin has been dissociated in sodium dodecyl sulphate [7] or in various salt solutions [8-201 so that after removal of the DNA by centrifugation [7][8][9]12,13,16,17] gel filtration [9-11,18, 191 or precipitation [14,15,20], the chromatin proteins have been obtained as such [I41 or separated from the histones by ion-exchange chromatography [8,[10][11][12][13][15][16][17][18][19][20] or by electrophoresis [7]. Other workers have used variations or combinations of some of these techniques [21-241. However, some of these methods subject the chromatin to extremes of pH and solvent environment which can be destructive to both proteins [25] and DNA [5,19], while others produce selected fractions [2,3,15] or yield low recoveries of non-histone proteins [ l l , 16,18,19]. Our approach has been to use non-rigorous conditions which yet give good yields of non-histone proteins. In our procedure chromatin is dissociated in 2 M NaCl-5 M urea and applied to a column of hydroxyapatite. This ion-exchange medium is not affected by the presence of high concentrations of urea and the phosphate sites which would otherwise bind basic molecules such as histones are effectively blocked by the sodium ions [26,27]. The acidic proteins bind to the Ca2+ sites of the column and are eluted by increasing the phosphate concentration [26]. Thus in the case of chromatin, we found that the histones are not retained and can be recovered in almost quantitative yield, but only 60%-70% of the adsorbed nonhistone proteins could be recovered using phosphate concentrations up to 0.2 M [28,29]. Further increase in the phosphate concentration merely eluted the DNA from the hydroxyapatite. Recently we have found that solutions of high concentrations of guanidinium hydrochloride to be an efficient solvent for non-histone proteins [30,31]. This communication describes the adaption of our previous procedures to include the use of guanidinium hydrochloride in order to obtain a more efficient recovery of nonhistone proteins. MATERIALS AND METHODS ChemicalsAs far as possible these were of AnalaR grade and were largely obtained from British Drug Houses Ltd (Poole, Dorset, U.K.). AnalaR-grade urea was routinely prepared as an 8 M sto...

Mass spectrometry-based strategies for characterization of histones and their post-translational modifications

“…The HAP dissociation method enables chromosomal proteins to be fractionated with high recovery and without nucleic acid contamination. HAP is suitable for large-scale preparation of histones, nonhistone proteins, histone oligmers and/or DNA [79][80][81][82][83].…”

Studies of Epstein-Barr Virus (EBV)-associated Nuclear Antigen: Solubilization from Raji Cell Chromatin with 5 M-Urea-2 M-NaCl and Fractionation on Hydroxyapatite

“…chloride/5 u-urea as described by Rickwood & MacGillivray (1975). The following fractions were obtained: (i) HAP I -eluted from the column with 2 M-NaCI/5 M-urea/t mMphosphate; (2) HAP 2-eluted from the column with z M-NaC1/5 i-urea/5o mM-phosphate; (3) HAP 3-eluted from the column with 2 ~l-NaC1/5 i-urea/2oo mM-phosphate; The preparation and hydroxyapatite fractionation of chromatin were carried out as described by Rickwood & MacGillivray (I975), using 2 x io 9 Raji cells.…”

“…The difference in the absorption patterns of fractions HAP 3 and 4 may be related to the observation that traces of DNA are present in the HAP 4 fraction (Rickwood & MacGillivray, 1975).…”

“…This is based on the separation of histone and non-histone protein fractions and hydroxyapatite (MacGillivray & Rickwood, 1974;Rickwood & MacGillivray, I975). Non-histone proteins have been demonstrated to control, at least in part, the transcriptional activity of isolated chromatin (Gilmour, 1974;MacGillivray & Rickwood, 1975). It is possible that EBNA is involved in the control of transcription of EBV sequences in transformed cells.…”

The Modification of Nuclear Proteins by ADP‐ribosylation

“…The second problem relates to the large number of conflicting reports as to the rate of synthesis of poly(ADP-Rib) at different times of the cell cycle [8] and the difficulty of correlating polymer production and its modification of nuclear proteins with particular events in the cell cycle or with changes in gene activity. For some years these laboratories have been involved in developing procedures for the preparation and analysis of nuclear proteins (see [7,9,10]) and in this paper we describe the application of some of our techniques to study the modification of nuclear proteins by poly-(ADP-Rib) in a number of mouse cells. …”