[Isolation of histones from the nuclei of the flagellatum Astasia longa]

Abstract

A new procedure for isolation of purified nuclear fraction from the cells of the phytoflagellatum Astasia longa has been developed. The resulting preparations were characterized in terms of their chemical composition. A method of isolation to total histone preparations from the nuclear fractions of A. longa based on the metabolic peculiarities of the phytoflagellatum has been developed. Pure histone fractions were obtained by intensive disruption and thorough washing of the nuclear fraction from the non-histone proteins with weak solutions of salts. The intensive proteolysis of the histones in the course of washing was stopped by an addition of the specific inhibitors of proteinases--phenylmethylsulfanylfluoride and pCMB (pH 7,8--8,0). The use of NaHSO3 or boiling of the nuclear fraction to suppress the histone proteolysis were found to be ineffective. Extraction of the histones by 1 M CaCl2 appeared more productive than the use of mineral acids solutions. The total histone preparation isolated under optimal conditions was found in the chromatin in amounts equivalent to those of DNA; its amino acid composition was typical for histones. During polyacrylamide gel electrophoresis in an acidic system in the presence of urea the histones produced four main bands, whereas in the presence of DS-Na--five bands. In terms of the amino acid composition and electrophoretic properties in different systems the histones of A. longa are similar but not identical to calf thymus histones.