Dissociation Constant (Kd)

The dissociation constant is defined as the off-rate constant divided by the on-rate constant, which gives it units of concentration. At a free ligand concentration equal to the dissociation constant, exactly half the binding sites are filled at equilibrium; at ten times that concentration, roughly ninety per cent are. A ligand with a constant of one nanomolar therefore half-saturates its target at one nanomolar free, and the number carries no information about what happens after binding. The equivalent constant for a competing inhibitor is the inhibition constant, obtained from a displacement experiment by correcting for the concentration and affinity of the tracer used.

Measurement methods differ in what they return. Saturation binding gives the constant and the site density together. Surface plasmon resonance reports the on- and off-rates separately, so two ligands with identical constants can be distinguished by residence time. Isothermal titration calorimetry adds the thermodynamic breakdown. All of them are conditional on temperature, buffer ionic strength, pH and whether the target sits in a membrane, a detergent micelle or a whole cell.

Paired with a free plasma concentration, the constant converts exposure into occupancy, which is the step that makes a pharmacokinetic curve mean something pharmacologically. Compared between two targets it becomes a selectivity ratio, and compared with achievable concentrations it says whether an off-target interaction is theoretical or real.

The frequent misuse is treating it as potency. Where receptor reserve exists, a full response can arise from a small fraction of receptors occupied, putting the functional half-maximal concentration well below the binding constant, sometimes by more than an order of magnitude. Values quoted without the assay conditions that produced them should not be compared.