Effect of the pH of isolation on the quantities and properties of histones extractable from calf thymus and rat liver nuclei

Abstract

The loss of histone from rat liver and calf thymus nuclei has been followed by analyzing nuclei for ammonia-precipitable and ammonia-soluble histone as the pH of isolation is lowered from 5.8 to 1.2. The total histone of rat liver nuclei isolated at pH 5.8 is approximately the same as that of nuclei isolated at pH 3.8, but there is a rapid loss of ammonia-precipitable histone between pH 3.8 and pH 3.4. Thereafter histone is lost at a slower rate down to pH 1.2, below which no more histone is extracted. Nuclei isolated from rat liver at pH 3.8 and then subjected to various lower pH values down to pH 1.2 behave similarly to nuclei in homogenates, in regard to histone loss. Histones remaining in rat liver nuclei after subjection of homogenates or pH 5.8 nuclei to pH values down to pH 3.0 have approximately the same amino acid composition as the total histone of pH 3.8 or pH 5.8 rat liver nuclei. Undamaged histone cannot be extracted from pH 3.8 or 5.8 rat liver nuclei by lowering the pH to 3.4, apparently because the histone is heavily damaged by proteolytic enzymes as it is extracted. The curve showing the total histone content of rat liver nuclei isolated at different pH values, taken together with the curve showing the quantity of histone remaining in pH 3.8 rat liver nuclei after lowering the pH to various extents, suggests the possibility that histone may be extracted from the nuclei in at least two different classes. The ratio of ammonia-soluble to ammonia-precipitable histone increases as the pH of isolation of rat liver nuclei decreases. The curve showing the quantity of histone left in calf thymus nuclei as the pH of isoation is lowered indicates that down to pH 2.5, no histone is lost. Below this pH value there is a steady loss of histone down to pH 1.0, where the histone remaining in the nuclei becomes essentially zero. The above findings are discussed in relationship to a previously proposed idea concerning the quantity of total histone in different types of nuclei and the possibility that histones can be divided into two classes, depending upon the ease of their extraction.