A boundary sedimentation velocity method for determining nonspecific nucleic acid-protein interaction binding parameters

Abstract

An absolute method for measuring affinity constants and binding site sizes for the non-sequence-specific binding of protein to single- or double-stranded polynucleotide chains is described, based on the difference in the rate of sedimentation of free protein and the protein-nucleic acid complexes. In the form outlined, the technique can measure binding constants ranging from 10 3 to 10 6 m −1; methods for extending the range of measurable binding constants are also described. Data obtained for the noncooperative binding of ribonuclease to DNA are presented as fully worked-out examples of the method, and the extension of this approach to other noncooperative and cooperative protein-nucleic acid binding systems is discussed.