Abstract
One of the five main histone molecular species, H2A, of calf thymus was fractionated and purified on a large scale for chemical and physical studies. This was achieved by three methods, using different combinations of our CM-cellulose chromatographic technique with other chromatographic systems reported. Method I consists of chromatography first on CM-cellulose and then on Sephadex G-100, Method II first on Amberlite CG-50 and then on CM-cellulose, and Method III on CM-cellulose and then on Bio-Gel P-10. Method I was successful when the starting material obtained by Johns' fractionation methods was contaminated by a small amount of H3 histone. Method II did not suffer from such a limitation but gave a low recovery of H2A on the first chromatography. Method III provided the purest preparations of H2A, together with highly purified H3, H4, and others, and is superior to methods previ-ously reported for the large-scale preparation of H2A and other species from whole histone as regards the simplicity of the procedures and the purity and yield of the products. The preparation obtained by Method I was digested with trypsin [EC 3.4.21.4]. The resulting soluble and insoluble fractions of the digest were frac-tionated by column chromatography to give 20 small peptides and 2 large peptides, respectively, with high recoveries. The sequences of almost all the soluble peptides were determined; these, taking into account the recoveries of these peptides and the compositions of the insoluble peptides (19 and 29 residues), accounted for all the 129 amino acid residues of this histone.
