Two of the important questions that are always asked of any receptor–ligand interaction are, What is the strength of binding, or affinity, of the interaction, and, What are the rates of association and dissociation? These parameters are generally assessed using purified preparations of proteins. For receptors that are integral membrane proteins in their native state, soluble forms of the proteins are prepared, usually by truncating the proteins to eliminate their membrane-spanning domains. With these purified proteins, binding rates can be measured by following the binding of ligands to receptors immobilized on gold-plated glass slides, using a phenomenon known as surface plasmon resonance (SPR) to detect the binding (Fig. A.31). A full explanation of surface plasmon resonance is beyond the scope of this textbook, as it is based on advanced physical and quantum mechanical principles. In brief, it relies on the total internal reflection of a beam of light from the surface of a gold-coated glass slide. As the light is reflected, some of its energy excites electrons in the gold coating and these excited electrons are affected by the electric field of any molecules binding to the surface of the glass coating. The more molecules that bind to the surface, the greater is the effect on the excited electrons, and this in turn affects the reflected light beam. The reflected light thus becomes a sensitive measure of the number of atoms bound to the gold surface of the slide.
If a purified receptor is immobilized on the surface of the gold-coated glass slide, to make a biosensor ‘chip,’ and a solution containing the ligand is streamed over that surface, the binding of ligand to the receptor can be followed until it reaches equilibrium (see Fig. A.31). If the ligand is then washed out, dissociation of ligand from the receptor can easily be followed and the
The ligand to be tested, in this case peptide:MHC complexes, is immobilized on a special gold-plated surface Soluble T-cell receptors are allowed to ˜ow over the surface and bind to the peptide:MHC complexes Receptor binding reaches equilibrium Unbound receptors are washed away; continued washing removes receptors as they dissociate from the peptide:MHC complexes Ru Time Time Time Ru Ru
Fig. A.31 Measurement of receptor–ligand interactions can be made in real time using a biosensor. Biosensors are able to measure the binding of molecules on the surface of gold-plated glass chips through the indirect effects of the binding on the total internal reflection of a beam of polarized light at the surface of the chip. Changes in the angle and intensity of the reflected beam are measured in ‘resonance units’ (Ru) and plotted against time in what is termed a ‘sensorgram.’ Depending on the exact nature of the receptor–ligand pair to be analyzed, either the receptor or the ligand can be immobilized on the surface of the chip. In the example shown, peptide:MHC complexes are immobilized on such a surface (first panel). T-cell receptors in solution are now allowed to flow over the surface, and to bind to the immobilized peptide:MHC complexes (second panel). As the T-cell receptors bind, the sensorgram (inset panel below the main panel) reflects the increasing amount of protein bound. As the binding reaches either saturation or equilibrium (third panel), the sensorgram shows a plateau, as no more protein binds. At this point, unbound receptors can be washed away. With continued washing, bound receptors now start to dissociate and are removed in the flow of the washing solution (last panel). The sensorgram now shows a declining curve, reflecting the rate at which receptor and ligand dissociation occurs.
Fig. A.32 Polyclonal mitogens, many of plant origin, stimulate lymphocyte proliferation in tissue culture. Many of these mitogens are used to test the ability of lymphocytes in human peripheral blood to proliferate.