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series of ligand concentrations to cover 10–90% of maximum binding at a fixed

receptor concentration. The binding data are then analysed using equations 17.4 to

17.11 often in the form of a computer program many of which are available commer-

cially. Most perform linear or non-linear least-squares regression analysis of the

experimental data. Measurements may be made on asteady-state(single measure-

ment) basis or atime-resolved(multiple measurements over a period of time) basis

most commonly by stopped-flow or quench-flow procedures (Section 15.3.3).

Non-specific binding

A general problem in the study of receptor–ligand binding is thenon-specific binding

of the ligand to sites other than the orthosteric or allosteric binding sites. Such non-

specific binding may involve the membrane lipids and other proteins either located in

the membrane or released by the isolation procedure. The characteristic of non-

specific binding is that it is non-saturable but is related approximately linearly to

the total concentration of the ligand. Thus the observed ligand binding is the sum of

the saturable (hyperbolic) specific binding to the receptor and the non-saturable

(linear) binding to miscellaneous sites. Thespecific bindingcomponent is usually

obtained indirectly either by carrying out the binding studies in the presence of an

excess of non-labelled ligand (agonist or antagonist) if a labelled ligand is being used,

or by using a large excess of an agonist or competitive antagonist in other studies.

The presence of the excess unlabelled or competitive ligand will result in the specific

binding sites not being available to the ligand under study and hence its binding

would be confined to non-specific sites (Fig. 17.5). In practice, a concentration of the

competitive ligand of at least 1000 times itsKdorKbmust be used and confirmation that

under the conditions of the experiment, non-specific binding was being studied would

be sought by repeating the study using a range of different and structurally dissimilar

competitive ligands that should give consistent estimates of the non-specific binding.

Total binding

Specific binding

Non-specific binding

[Ligand]

[Ligand bound]

Fig. 17.5Specific and non-specific binding of a ligand to a membrane receptor. Specific binding is normally

hyperbolic and shows saturation. Non-specific binding is linear and is not readily saturated.

682 Cell membrane receptors and cell signalling