Calf Thymus Histone H2A. Purification and Tryptic Peptides1

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 previously 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 fractionated 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.