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Separation of rat tissue histone H1 subtypes by reverse-phase h.p.l.c. Identification and assignment to a standard H1 nomenclature.

ABSTRACT: H1 histones from rat liver and rat testis were separated by reverse-phase h.p.l.c. Within 40 min six subfractions (H1(0), H1b, H1a, H1d, H1e + H1c and H1c) and seven subfractions (H1(0), H1b, H1a, H1d, H1e + H1c, H1c and H1t) respectively were isolated by using a linear acetonitrile gradient. Each individual H1 subtype was identified either by comparing the H1 variants (contained in both tissues but in different quantities) or by SDS/PAGE and acetic acid/urea/PAGE. Moreover, all H1 variants were characterized by amino acid analyses. The amino acid compositions of rat histone subfractions H1(0), H1b and H1e were determined for the first time. It was possible to classify unambiguously the H1 subfractions obtained by h.p.l.c. by following the standardized H1 nomenclature for electrophoretic systems recommended by Lennox, Oshima & Cohen [(1982) J. Biol. Chem. 257, 5183-5189]. Incorrect assignments that have been made in various publications are discussed.

SUBMITTER: Lindner H

PROVIDER: S-EPMC1131585 | biostudies-other | 1990 Jul

REPOSITORIES: biostudies-other

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Project description:BackgroundHistone H1 is involved in the formation and maintenance of chromatin higher order structure. H1 has multiple isoforms; the subtypes differ in timing of expression, extent of phosphorylation and turnover rate. In vertebrates, the amino acid substitution rates differ among subtypes by almost one order of magnitude, suggesting that each subtype might have acquired a unique function. We have devised a competitive assay to estimate the relative binding affinities of histone H1 mammalian somatic subtypes H1a-e and H1 degrees for long chromatin fragments (30-35 nucleosomes) in physiological salt (0.14 M NaCl) at constant stoichiometry.ResultsThe H1 complement of native chromatin was perturbed by adding an additional amount of one of the subtypes. A certain amount of SAR (scaffold-associated region) DNA was present in the mixture to avoid precipitation of chromatin by excess H1. SAR DNA also provided a set of reference relative affinities, which were needed to estimate the relative affinities of the subtypes for chromatin from the distribution of the subtypes between the SAR and the chromatin. The amounts of chromatin, SAR and additional H1 were adjusted so as to keep the stoichiometry of perturbed chromatin similar to that of native chromatin. H1 molecules freely exchanged between the chromatin and SAR binding sites. In conditions of free exchange, H1a was the subtype of lowest affinity, H1b and H1c had intermediate affinities and H1d, H1e and H1 degrees the highest affinities. Subtype affinities for chromatin differed by up to 19-fold. The relative affinities of the subtypes for chromatin were equivalent to those estimated for a SAR DNA fragment and a pUC19 fragment of similar length. Avian H5 had an affinity ~12-fold higher than H1e for both DNA and chromatin.ConclusionH1 subtypes freely exchange in vitro between chromatin binding sites in physiological salt (0.14 M NaCl). The large differences in relative affinity of the H1 subtypes for chromatin suggest that differential affinity could be functionally relevant and thus contribute to the functional differentiation of the subtypes. The conservation of the relative affinities for SAR and non-SAR DNA, in spite of a strong preference for SAR sequences, indicates that differential affinity alone cannot be responsible for the heterogeneous distribution of some subtypes in cell nuclei.

Project description:We describe a facile and sensitive reverse-phase h.p.l.c. method for analytical separation of biliary bile pigments and direct quantification of unconjugated bilirubin (UCB) and its monoglucuronide (BMG) and diglucuronide (BDG) conjugates in bile. The method can be 'scaled up' for preparative isolation of pure BDG and BMG from pigment-enriched biles. We employed an Altex ultrasphere ODS column in the preparative steps and a Waters mu-Bondapak C18 column in the separatory and analytical procedures. Bile pigments were eluted with ammonium acetate buffer, pH 4.5, and a 20 min linear gradient of 60-100% (v/v) methanol at a flow rate of 2.0 ml/min for the preparative separations and 1.0 ml/min for the analytical separations. Bile pigments were eluted in order of decreasing polarity (glucuronide greater than glucose greater than xylose conjugates greater than UCB) and were chemically identified by t.l.c. of their respective ethyl anthranilate azo derivatives. Quantification of UCB was carried out by using a standard curve relating a range of h.p.l.c. integrated peak areas to concentrations of pure crystalline UCB. A pure crystalline ethyl anthranilate azo derivative of UCB (AZO . UCB) was employed as a single h.p.l.c. reference standard for quantification of BMG and BDG. We demonstrate that: separation and quantification of biliary bile pigments are rapid (approximately 25 min); bile pigment concentrations ranging from 1-500 microM can be determined 'on line' by using 5 microliters of bile without sample pretreatment; bilirubin conjugates can be obtained preparatively in milligram quantities without degradation or contamination by other components of bile. H.p.l.c. analyses of a series of mammalian biles show that biliary UCB concentrations generally range from 1 to 17 microM. These values are considerably lower than those estimated previously by t.l.c. BMG is the predominant, if not exclusive, bilirubin conjugate in the biles of a number of rodents (guinea pig, hamster, mouse, prairie dog) that are experimental models of both pigment and cholesterol gallstone formation. Conjugated bilirubins in the biles of other animals (human, monkey, pony, cat, rat and dog) are chemically more diverse and include mono-, di- and mixed disconjugates of glucuronic acid, xylose and glucose in proportions that give distinct patterns for each species.

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Separation of rat tissue histone H1 subtypes by reverse-phase h.p.l.c. Identification and assignment to a standard H1 nomenclature.

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