A new procedure for the isolation and fractionation of histones

Introduction The usual method for the isolation of histones, in- volving the extraction of histones from chromatin with dilute sulphuric or hydrochloric acid, may re- sult in the denaturation of various chromosomal com- ponents due to the extreme pH conditions. The ability of histones to regenerate the super-coil configuration of native nucleohistone is sometimes reduced 1,2 by the acid extraction technique. Evidence has been pre- sented 3 that histones to some extend bind to DNA during acid treatment and form part of the so-called non-histones. The present investigation was under- taken to develop a procedure for the isolation and fractionation of histones under mild pH conditions. Nucleoproteins dissociated at high ionic strength can be separated into protein and DNA by ultracentri- fugation 4-91 or gel filtration [8-lo , but we have found these procedures unsuitable for the large scale isolation of histones. The method for histone isola- tion presented here is based on the finding of Mirsky and Ris 1 l that protamine added in vitro to nucleo- protein directly displaces histones. A procedure which avoids extreme pH conditions is described for the large scale isolation and partial fractionation of calf thymus histones. Histones, vir- tually completely displaced from deoxyribonucleo- protein by protamine, and isolated by exclusion chro- matography, appear to be almost identical on disc electrophoresis to histones prepared by acid extrac- tion of deoxyribonucleoprotein. Histones are frac- tionated by gel filtration and ammonium sulphate precipitation into lysine-rich histones (Fl), arginine- rich histones (F3 + F2al), and slightly lysine-rich histones F2a2 and F2b.