Abstract
The rates of binding and dissociation of the inducer isopropyl-β, d -thiogalactoside to the lactose repressor protein of Escherichia coli were studied by monitoring changes in tryptophan fluorescence in a stopped-flow spectrometer. At pH 8·5 the association rate constant for inducer binding was found to be 1·7×104 m −1 s−1 and that for dissociation to be 0·53 s−1 in 1·0 m -Tris·HCl buffer. These kinetic measurements were made over a range of conditions, and a small but reproducible dependence on pH and ionic strength was observed. The presence of bound calf thymus DNA was found to affect only slightly the rats of inducer binding to the protein. The kinetically determined rate constants were used to calculate dissociation constants for the interaction between repressor and inducer. The calculated values were in agreement with those determined directly by equilibrium dialysis and ultraviolet spectral titration of repressor with inducer. It appears that repressor and inducer interact by a simple equilibrium process, the characteristics of which are similar whether repressor is free in solution or non-specifically bound to DNA.
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