The stability of acid-soluble chromosomal proteins from avian erythrocytes

Abstract

The stability of avian erythrocyte histones was examined under the conditions of extraction, chromatography, electrophoresis, and storage, in order to avoid degradation during these operations. Since turbidity in trichloroacetic acid (TCA) was used as a measure of histone integrity, optimal conditions for quantitative assay were established as follows. One volume of histone sample was mixed with five volumes of 1.1 M TCA at room temperature, and the optical density at 400 mμ was measured after 25 min. The relation between turbidity and protein concentration was linear from 0.03 to at least 0.3 mg histone per milliliter and was not related to the kind of histone, except for the lysine-rich fraction which showed slightly less specific turbidity. Under these conditions turbidity was not sensitive to concomitant solutes such as guanidinium chloride, acetic acid, and dilute buffers and acids.With turbidity as the criterion of protein integrity, it was confirmed that brief manipulation in the cold is desirable in most media, including the dilute acids used for extraction. Nevertheless, chromatography at room temperature in concentrated solutions of guanidinium chloride or acetic acid appears to be tolerably safe. The effect of these conditions of manipulation and storage on histone fractions was substantiated by chromatography and starch-gel electrophoresis. Prolonged extraction of avian erythrocyte nuclei at acid pH released additional non-histone basic protein without alteration of authentic histone fractions.