Application of chromatographic techniques to the fractionation of histones

All the chromatographic fractionations previously reported resolved fractions containing a multiplicity of components. The formic acid gradient eluted poorly separated protein peaks and the sodium chloride - hydrochloric acid procedure could not be repeated by other workers in the same laboratory (Hnilica et al., 1962). The fractions resolved by guanidinium chloride have been shown to be heterogeneous (Rasmussen et al., 1962) and the proportions of the terminally eluted peaks were increased when aggregation was promoted.The phenomenon of histone aggregation is well documented (Cruft et al., 1954; Cruft, Mauritzen & Stedman, 1957; Ui, 1956, 1957; Bijvoet, 1957) and has been extensively studied in the ß- and γ- histones, the arginine-rich and slightly lysine-rich histones, respectively, by Cruft, Mauritzen & Stedman (1958a, b) . Thus ß-histone aggregation is promoted by increases in pH, ionic strength, anion valency, concentration and temperature, while ionic strength increases suppressed the aggregation of γ-histone. Many of the chromatographic procedure therefore, would have promoted aggregation, high pH and ionic strengths, while even at low pH values of the formic acid gradient high concentrations were required to approach pH 1.6.The aggregation of j3 histones is strongly inhibited by the other histones and by the trivalent cations, aluminium and lanthanum (Cruft et al., 1958a,b). If the aggregations of the various components were prevented, the three groups of histones, lysine-rich, relatively lysine-rich and arginine -rich (Cruft et al., 1957; Johns et al., 1960) would be expected to exhibit different degrees of adsorption with ion exchange chromatography.The following investigations were undertaken to apply chromatographic techniques to the fractionation of histones under conditions in which histone aggregations and interactions were suppressed