Determination of the dissociation constant of valine from acetohydroxy acid synthase by equilibrium partition in an aqueous two-phase system

An aqueous polyethylene glycol/salt two-phase system was used to estimate the dissociation constant, Kdis, of the Escherichia coli isoenzyme AHAS III regulatory subunit, IlvH protein, from the feedback inhibitor valine. The amounts of the bound and free radioactive valine in the system were determined. A Scatchard plot of the data revealed a 1:1 valine–protein binding ratio and Kdis of 133±14 μM. The protein did not bind leucine, and the ilvH protein isolated from a valine resistant mutant showed no valine binding. This method is very simple, rapid and requires only a small amounts of protein compared to the presently used equilibrium dialysis method.