Measurement of binding constants for protein-DNA interactions by DNA-cellulose chromatography.

We describe the application of DNA-cellulose chromatography to the determination of binding constants for the nonspecific interaction between proteins and DNA. The method involves loading a small DNA-cellulose column to a low binding density and subsequently eluting the protein with buffer at a constant salt concentration. The elution is conveniently followed by monitoring the protein fluorescence of the eluate. From the shape of the elution profile, the binding constant for the interaction can be calculated. Employing columns containing double-stranded calf thymus DNA-cellulose, we have measured binding constants in the range of 10(4) to 10(6) M-1. Extension of this range is possible by varying the amount of DNA on the column. For lac repressor, agreement between our measurements and those of Revzin, A., and von Hippel, P.H. [(1977) Biochemistry 16 (second of five papers in a series in this issue)], who employ an absolute boundary sedimentation method, is good. The column method should be useful for the rapid screening of the effect of a large number of variables on protein-DNA binding constants.