A new h.p.l.c. isolation procedure for chicken and goose erythrocyte histones

Abstract

Total chicken erythrocyte histones were separated by reversed-phase h.p.l.c. using a multi-step acetonitrile gradient in a very short time (35 min). The proteins were eluted in the following order: H1, H5, H2B, H2A.2, H4, H2A.1 and H3.2. Applying a special gradient system adapted for the separation of very-lysine-rich histones, chicken erythrocyte H5 was resolved into two subfractions. Their electrophoretic mobilities were identical in both SDS and acetic acid/urea/Triton polyacrylamide-gel electrophoresis, but different in free-flow electrophoresis. Amino-acid-sequence analyses revealed that the two components only differ with respect to position 15, one having glutamine in that position and the other arginine. A separation of histones prepared from goose erythrocytes disclosed no H5 subfractionation. Furthermore, histones obtained from anaemic-chicken blood were analysed by the above-mentioned h.p.l.c. conditions. An alteration in the relation of H1 to H5 was detected, but no further differences in the number and quantity of the histones and histone variants were observed as compared with the corresponding proteins processed from normal-chicken blood.

Enzymes involved in the dynamic equilibrium of core histone acetylation of Physarum polycephalum

“…Chicken crythrocytr whole histones were fractionated as described [37] with tho following modificalions.The separations were performed on a Nucleosil300-5 C, column (0.8 mm internal diameter x 250 mm). The freeze-dried histones were dissolved in 0.2% (v/v) TFA containing 50 mM 2-mcrwptoethanol and 500-800 pg pro tcin samples were injected onto the column.…”

Linker Histone Subtype Composition and Affinity for Chromatin in Situ in Nucleated Mature Erythrocytes

“…The lyophilized proteins were dissolved in water containing 0.2 M 2-mercaptoethanol and injected onto the column. Separation of H1 was performed as described (25)(26)(27). Briefly, linker histones from frog erythrocytes were chromatographed at a constant flow of 1.0 ml/min with a acetonitrile gradient starting at 80% (v/v) solvent A and 20% (v/v) solvent B (solvent A, water containing 0.1% (v/v) trifluoroacetic acid and 15% (v/v) EGME; solvent B, 70% (v/v) acetonitrile, 0.1% (v/v) trifluoroacetic acid, and 15% (v/v) EGME).…”

Reconstitution of native-like nucleosome core particles from reversed-phase-HPLC-fractionated histones

“…It should provide an extremely useful method for the reconstitution of chromatin complexes starting from histone variants or from post-translationally modified histones (acetylated, methylated, phosphorylated or ubiquitinated) [38], which are usually present in small amounts. RP-HPLC can, for instance, resolve the H2A.2 and H2A.1 variants [4] and can be used in conjunction with other HPLC techniques to purify histone fractions with a well-defined extent of acetylation [39]. The histone fractions thus obtained could be combined with sequence-defined DNA templates to further enhance the powerful potential of the reconstituted chromatin complexes that are currently used for the analysis of chromatin structure [17,40] and function [41][42][43].…”

“…In contrast, complete fractionation of histones can be quickly achieved (1-2 h) by RP-HPLC and only microgram amounts of starting sample are required. Furthermore, under optimal conditions, it is possible to separate the histone variants of some of the individual histone fractions, such as H2A.1 and H2A.2 [4]. Several methods of RP-HPLC of histones have been developed [4][5][6][7][8][9][10][11][12][13].…”