| A detailed characterization of the pH titration behavior of the nucleosome, which titrates reversibly from at least pH 5 to pH 11 without denaturation, is studied in either 0.14 or 0.6M KCl by means of potentiometric, spectroscopic, and hydrodynamic methods. We present the first report of the electrometric titration curve of chicken erythrocyte monomer nucleosomes, correlating changes in physical parameters with the titration of specific residues. The lack of a generalized electrostatic perturbation of the titration curve by the anionic charge of the DNA phosphates was interpeted in modeling studies to reflect the counterion condensation ability of the nucleic acid, and the absence of significant selective pK(,a) perturbations suggested that electrostatic interactions are the predominant form of protein-DNA interaction. We also present the first report that, as the pH was increased from neutrality, changes in the circular dichroism spectra indicated that, at about pH 10 in 0.14M KCl, titration of the protein causes a reversible conformational change in the intact particle involving alterations in the DNA secondery structure, while that of the protein is unaltered. In addition, we find that the reversible dissociation of the histones produces a weight average sedimentation rate transition occurring at somewhat more alkaline pH. Isolation of intermediates on preparative sucrose gradients revealed that high pH releases the histones in a fractional manner, resembling the effect of high ionic strength. The lysine-rich histones H2A/2B dissociate from the nucleosome at pH 11, followed by the arginine-rich histones H3/4 at higher pH values. These results indicated a role for lysine, and perhaps arginine and tyrosine residues of the protein, in the maintenance of histone-DNA interactions. A shift of the alkaline pH transitions towards lower pH, as the salt concentration was increased to 0.6M, suggested that electrostatic interactions between protonated lysines and the nucleic acid were being titrated. At the higher salt concentration, where histone dissociation was completed near pH 11, it was confirmed that the reversible effects described above do not require titration of the DNA bases, as detected by 260nm hyperchromicity, or titration of the histone alpha helical secondery structure, as monitored by changes in the 225nm ellipticity, both of which occurred at higher pH values, independent of ionic strength. The disruption of protein alpha helical structure above pH 11 implicated a specific role, heretofore undetected, for arginine, in the maintenance of the protein secondery structure. |
