Large-Scale Isolation and Fractionation of Tetrahymena Histones. Purification of H 2 B and H 4 Histones

Abstract

The histones of the ciliated protozoan Tetrahymena were isolated and fractionated on a large scale by revised procedures, and the H 2 B and H 4 histones were highly purified for sequence determination. The nuclei of an amicronucleate strain, GL, of Tetrahymena pyriformis were isolated, without cell harvesting, from cultures reaching the stationary state, which were continuously centrifuged through 1.0M sucrose, pH 7.5, containing Nonidet P-40. From the nuclei further purified and washed with 0.15M NaCl, pH 7.5, histones were extracted with 0.25M HCl. Thus 1, 620 liters of cultures yielded 5.20g of histone extract. The histones were first separated into a 0.5M HClO₄ soluble fraction (H 1) and an insoluble fraction (H 2 A, H 2 B, H 3, and H 4). The insoluble fraction was chromatographed on a large column of Bio-Gel P-60 with 20mM HCl containing 250mM NaCI at 15°C, yielding H 2 A+H 3, H 2 B, and H 4 fractions. The H 4 fraction was highly pure; the H 2 B fraction was further purified by the same Bio-Gel P-60 chromatography without NaCl, yielding pure H 2 B.