An Assay for lac Repressor
Genetic and physiological experiments investigating properties of the lac operon provided information from which a number of regulatory mechanisms were proposed. These ranged from the logical mechanism of lac repressor binding to DNA and inhibiting transcription to compli-cated translational control mechanisms utilizing tRNA molecules. Clear demonstration of the regulation mechanism required purification of its components and in vitro reconstruction of the lac system.
The most important step in the reconstruction of the lac regulatory system was the ability to detect repressor. Lac repressor, of course, had to be highly purified from lysed cells. If regulation of the lac operon were efficient–and that is the main reason for the existence of regulation–then the cell should contain far fewer molecules of repressor than of the induced gene products. Furthermore, since lac repressor possessed no known enzymatic activity, no easy and sensitive assay for repressor was available. Without the ability to detect repressor, its purification was impossible because any fraction obtained from purification steps that was enriched in repressor could not be identified.
Repressor’s only known property was that it bound inducer, IPTG being one. Therefore Gilbert and Müller-Hill developed an assay of lac repressor based on the protein’s ability to bind to inducer molecules. Equilibrium dialysis can detect a protein that binds a particular small molecule. The protein solution to be assayed is placed in a dialysis sack and dialyzed against a buffer that contains salts to maintain the pH and ionic strength and the small molecule that binds to the protein (Fig. 11.3).
In the case of repressor, radioactive IPTG was used. After equilibrium has been attained, the concentration of free IPTG inside and outside the sack is equal, but in addition, inside the sack are the molecules of IPTG that are bound to repressor. If the concentration of repressor is suffi-ciently high, the increased amount of IPTG inside the sack due to the presence of repressor can be detected. Both the inside and outside concentrations of IPTG can be determined by measuring the amount of radioactivity contained in samples of known volumes taken from out-side and inside the dialysis sack.
Does an equilibrium dialysis assay possess sufficient sensitivity to detect the small amounts of lac repressor that are likely to exist in crude
extracts of cells? The binding reaction between repressor and IPTG is closely approximated by the reaction
where Rf is the concentration of free repressor, IPTG is the concentra-tion of free IPTG, and R⋅IPTG is the concentration of the complex between repressor and IPTG. A dissociation constant KD describes the relations between the concentrations:
Substituting the conservation equation, Rf+R⋅IPTG=Rt, where Rt is the total amount of repressor, and rearranging yields the relation we need. Biochemists have many different names for the equivalent algebraic rearrangements of this equation but usually call the phenomenon Michaelis-Menten binding,
The ratio of radioactivity in the samples obtained from inside and outside the sack is
Normally in liquid scintillation, counting a 5% difference between samples with more than 100 cpm can be readily determined. Thus the quantity
must be greater than 0.05 for detection of lac repressor by this assay.