On the determination of availability of ligand binding sites in steady-state systems

Systematic analyses of dose-effect relationships of inhibitors in the presence of substrates in enzyme catalyzed reactions with different reaction mechanisms and different types and mechanisms of inhibition have yielded equation (A), f t = 1 [1+( I 50 I )] , where ft is fractional inhibition and I50 is the concentration of inhibitor, I, that is required to produce a median-effect. Equation (A) has the same form as the Michaelis-Menten equation, v V max = 1 [1+( K m S )] , and thus share the common geometric properties. However, equation (A) is mechanism independent, contains no explicit kinetic constant, and the effect is expressed with respect to the control velocity rather than the maximal velocity. The relationship depicted by equation (A) has the following applications in analyzing experimental data, (i) The median-effect concentration and the apparent kinetic order of interaction can be conveniently determined by graphical methods which transform the dose-effect relationships into linear forms, (ii) The method described by Dixon (1972) for determining the distribution of enzyme species in single-substrate reactions in the presence of a tightly binding inhibitor can be extended to multiplesubstrate reactions with different mechanisms, (iii) The median-effect principle provides an alternative simple way to derive a variety of equations of the mass-action law including the equilibrium binding equation of Scatchard (1949) . It is also shown that by determining the ratios of Kt and I50 of different inhibitors under the same experimental conditions, it is possible to determine whether the inhibitors bind to the same fraction of the total enzyme.