Page 261 - The Toxicology of Fishes

Receptor-Mediated Mechanisms of Toxicity 241

The relationship expressed by Equation 5.3 involves several assumptions about the nature of the
association. The most important assumptions for the experimental measurement of binding affinity are:

• The system must have reached equilibrium; that is, association and dissociation are occurring
at equal rates when binding is measured, and the fraction of ligand bound has reached a steady
state. Generally, this must be determined empirically by measuring binding at a single ligand
concentration over several time points.
• Binding of one ligand molecule does not effect binding of another, which includes two major
components: (1) Removal of a ligand molecule from the available pool by binding does not
alter the concentration of free ligand; that is, the fraction of total ligand that is bound to the
receptor is relatively small. (2) Binding is not cooperative, an assumption that is likely to be
violated in instances where multiple ligand molecules bind to a single receptor or where receptor
proteins form complexes and will often result in nonlinear Scatchard plots (see below).

Deviation from these assumptions can be accommodated by modifications of Equation 5.3 which fall
outside the scope of this chapter but are described elsewhere (Kenakin, 1999; Kenakin et al., 1992).

Measurement of Affinity
Although binding assays utilizing fluorescence techniques are seeing increased use in pharmacological
research (Hovius et al., 2000), binding affinity is most commonly measured in prepared cells or tissues
of interest using radiolabeled ligands. The goal of such ligand binding assays is twofold. First, free
ligand must be separated from bound ligand. Second, ligand bound to the receptor must be separated
from that bound to nonspecific binding sites within the cell or tissue. Several procedures to achieve the
first goal are discussed in the next section. To achieve the second goal, binding assays assume that
nonspecific binding is of lower affinity and cannot be saturated in the range of ligand concentrations
used. In contrast, the specific receptor binds ligand with much higher affinity but is present in vastly
smaller quantities, which can be saturated at relatively low ligand concentrations. Labeled ligand can
be displaced from high-affinity, low-capacity receptors but not from low-affinity, high-capacity nonspe-
cific binding sites by an excess of unlabeled ligand; therefore, the definition of high-affinity (specific)
binding sites (i.e., receptors) is binding sites that can be saturated within the concentrations of ligand
used. High-affinity binding is determined by subtracting the amount of ligand bound in the presence of
unlabeled competitor from the amount of ligand bound in the absence of competitor.
This means that the actual data collected from an experiment consist of two binding plots, a total
binding curve measuring binding at several ligand concentrations in the absence of competitor and a
nonspecific binding curve determined in the presence of competitor. The specific binding curve is simply
the difference between these two. It is important to note that specific binding in radioligand experiments
is not a directly determined value, but a derived one.
Possessing both a method to determine specific binding and a theoretical relationship to describe it
(Equation 5.3), it should be possible to fit data to the relationship. By measuring the amount of receptor
occupied at several different free ligand concentrations, the values of R and K can be determined.
T
D
First, it is necessary to determine whether sufficient data have been collected to perform a proper binding
analysis. The data should include several points at concentrations of free ligand above the K . This can
D
be assessed visually by plotting specific binding vs. free ligand concentration on a semilog plot but not
with a linear scale of free ligand, which can be misleading (Munson and Rodbard, 1983). Figure 5.3
shows two plots of the same idealized specific binding curve. The data on the left are plotted with a
linear x-axis and seem to indicate that the last few points are beyond the inflection point (i.e., K ) of
D
the curve and that a saturating ligand concentration is being approached; however, the semilog plot on
the right reveals that the inflection point has not been reached, and R and K (hereafter collectively
T
D
called the binding constants) cannot be estimated properly. The experiment must be repeated with
additional treatments using higher concentrations of ligand.
When sufficient data have been collected, the binding constants can be determined. The classic method
for estimating these values, and one still in use, is the Scatchard plot (Scatchard, 1949). Because Equation