Solution, receptor/ligand binding

Affinity capillary electrophoresis (ACE), reviewed by Shimura and Kasai,42 is a method for studying receptor-ligand binding in free solution using CE. The technique depends upon a shift in the electrophoretic mobility of the receptor upon complexation with a charged ligand. Pure receptor preparations or accurate concentration values are not required because only migration times are measured. 

In our discussion of simple receptor/ligand binding, we assumed that all cells have RT receptors and behave identically. In other words, the unique solution to Eqs. (4), (5), or (10) was presumed to describe each and every cell in the system under study. In reality, of course, this solution represents the mean behavior of the system, and individual cells may show some deviations from the mean behavior. In addition to deviations from the mean behavior caused by, for example, slightly different numbers of receptors on individual cells (Mahama and Linderman, 1994a), these deviations may be due to the probabilistic, or stochastic, nature of the system. 

Following the analogy to the case of receptor/ligand binding in solution, kc can be expressed in terms of rate constants for the diffusive step, k+, and the coupling step, kon ... 

Therefore, we can expect that coupling reactions between two membrane-associated species will commonly be diffusion-limited. This result is in contrast to that for receptor/ligand binding in solution, which will not typically be diffusion-limited, and to that for ligand binding to cell surface... 

The properties of the metal in natural systems are finely tuned by the specific and constrained environment. Water activation in hydrolytic enzymes is highly affected by the H-bond network in the receptor pocket, while redox potentials of iron or copper species in catalytic sites are exquisitely controlled by the geometrical constrains existing at the various redox states. In solution, the ligand binding mode can adapt to the redox switch. This results in a specific interlocking of the receptor and metal properties (redox or acid-base). 

Table I describes several of the fluorescent assays that have been used in our lab to study neutrophil activation. Fluorescein-labeled W-formylhexapeptide (FLPEP) has been used to characterize the ki- netics of ligand binding, dissociation, and internalization at 37°C (7,8). FLPEP is added to a suspension of cells, then receptor-bound and free FLPEP in solution are distinguished by adding antibody to fluorescein. This is a high-affinity antibody which binds free FLPEP within 1 s hut does not bind cell-bound FLPEP. When it binds the FLPEP, it quenches the fluorescein fluorescence. Hence the residual fluorescence after antibody addition represents FLPEP that is bound to the cell. Nonspecific binding is determined in cell suspensions that contain an excess of nonfluorescent peptide.

Basis. The rotational mobility of a small ligand is relatively unrestricted in solution (anisotropy approaches 0). The mobility is restricted when the ligand binds to a large immobilized molecule such as the receptor. In a T-format fluorometer, the parallel and perpendicular components of the emission can be examined simultaneously. While precautions must be exercised in working with turbid suspensions, it is nonetheless practical to make continuous measurements of binding and dissociation. 

Fig. 12.1. Thermodynamic cycle for ligand binding. Solutes L and L in solution (below) and bound to the receptor P (above). Vertical legs correspond to the binding reactions. Horizontal legs correspond to the alchemical transformation of L into L . The binding free energy difference can be obtained from either route AAA = AA4 — A A3 = AAi — A An...

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