The self-association of the separate histone fractions isolated from chicken erythrocytes has been studied in solution at a number of different pH values and ionic strengths. The apparent molecular weights of the histones were determined over a range of macromolecular concentrations using the techniques of osmotic pressure and sedimentation equilibrium. Histone F2c (H5) did not associate under any of the conditions investigated whereas the other histone fractions all appeared to undergo self-association forming dimers, dimers of dimers, etc. The degree of association increased with the pH and ionic strength of the medium. The tendency to aggregate increased in the order; histone F2c (H5) (non-aggregating), histone F2b (H2B), histone F2a2 (H2A), histone F3 (H3), histone F2al (H4) (highly aggregating).In the case of histone F2a2 (H2A) at pH 3.0 and ionic strength 0.1, the apparent weight-average molecular weight was determined at a number of macromolecular concentrations at five different temperatures. The self-association was analysed according to the method of Adams (published by Beckman Instruments Inc. in 1967) and shown to be a monomer-dimer-tetramer equilibrium. The association constants were evaluated at each of the temperatures studied and from their variation with temperature the values of the enthalpy and entropy of association were calculated. The intermolecular association was characterised by only a small change in enthalpy but a large, positive, change in entropy. This suggests that the association of histones at acid pH is due to hydrophobic interactions between the relatively uncharged segments of like polypeptide chains.Several lines of evidence have implicated the histones in the maintenance of the structural and functional integrity of the chromosome (see, e.g. Crick [2]). The amino-acid sequences have shown that all of the histone fractions possess a marked asymmetric distribution of the basic amino acids. It is suggested by Huberman [3] that the positively charged, basic residues of the histone molecule are associated with the negatively charged DNA-phosphates in nucleohistone, and that non-covalent interactions between the non-basic segments of the histone molecules are responsible for maintaining chromatin in its supercoil configuration. Such interactions could be intermolecular or intramolecular. Isolated histones in solution tend to aggregate [4,5] and it is possible that this aggregation is a result of histone-histone interactions of the type normally responsible for maintaining correct chromosomal structure.We present here the results of an investigation of the factors influencing the aggregation of isolated histone fractions in solution. The variation of the apparent molecular weights of the histone fractions with concentration, pH and ionic strength was first studied and has been published in summary already [6]. It was apparent that the analytical procedures of Adams [l] could be applied to such osmotic pressure and sedimentation results to obtain a measure of the stoichiometry and e...

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A new method is described for the large scale preparation of the avian erythrocyte specific histone, F2C. The method is based on the selective extraction of F2C, using perchloric acid, subsequent to the removal of the very lysine-rich histone, Fl, with 0.5 M NaC1.It is now well established that the major histone fractions of mammalian, fish, avian, and some plant somatic cell nuclei are very similar and limited in number, and many methods have been developed for their preparation. For recent reviews see Hnilica [l] and Butler, Johns and Phillips [2].I n chicken erythrocyte nuclei, however, there is a unique histone, not found in other tissues. This histone was first demonstrated by Neelin and Butler This histone, F2C, is of particular interest since it is thought to be implicated in some way with the final condensation and inactivation of the erythrocyte chromatin 16-91, although it has been pointed out [B] that its presence in reticulocytes indicates that it is not directly responsible for the loss of protein synthetic capacity.Murray, Vidali and Neelin [I31 have demonstrated that F2C can be extracted by careful titration of chicken erythrocyte deoxyribonucleoprotein with acid over the range pH 2.0 to pH 1.9. All other methods available for the preparation of this fraction involve the extraction of mixtures of histones and subsequent separations by column chromatography [5,. This paper describes a new method for the direct selective extraction of F2C from chicken erythrocyte deoxyribonucleoprotein, which does not require careful control of pH values and which enables large quantities to be prepared in a pure form in a relatively short time.Mote. The nomenclature F2C, used for the avian erythrocyte specific histone is that used by Hnilica EXPERIMENTAL PROCEDURE AND RESULTSAll preparative procedures were carried out at, between 2 and 4", except for the final washing and drying of precipitates which was at room temperature. The Preparation of Chicken Erythrocyte DeoxyribonucleoproteiyaChicken erythrocyte deoxyribonucleoprotein was prepared essentially as described previously [ 131 using 6 litres of blood, but with the following modifications. The last two washings were made using 0.14 M NaCl only and the volume used was reduced in order to pack the sediment into a single one litre pot. This was then washed three times in absolute ethanol (500 ml), separated into 20 approximately equal parts, and stored under ethanol at -10" until required. The Preparation of F2C from ChickenErythrocyte Deoxyribonucleoprotein One portion of chicken erythrocyte deoxyribonucleoprotein containing approximately 2 g of DNA, prepared as described above was taken for the following preparation; this would be equivalent to approximately 300 ml of chicken blood.The deoxyribonucleoprotein was homogenised at full speed in a M.S.E. Atomix with 500 ml of 0.5 M NaC1, for 15 sec, and centrifuged for 30 min at 200Oxg in an M.S.E. Mistral 6L refrigerated centrifuge. The sediment and the pad which collected on the surface were washed three times ...

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