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Three types of density gradients - neutral metrizamide, alkaline NaOH-metrizamide and alkaline triethanolamine-metrizamide - were used for studying the distribution of histones between the two DNA strands in alkali-denatured chromatin. It was found possible to avoid both protein redistribution and dissociation by using triethanolamine-metrizamide density gradients at pH 10.5. Under these conditions an alkali-denatured mixture of DNA and chromatin was well separated into the original DNA and DNP. When native or sonicated chromatin was denatured at pH 12.2 and centrifuged in a triethanolamine-metrizamide density gradient at pH 10.5 no peak of free DNA appeared. These results show that both DNA strands remain associated with histone molecules upon alkaline denaturation of chromatin.

Distribution of histones in alkali-denatured chromatin studied by isopycnic centrifugation in alkaline metrizamide density gradients.