Page 263 - The Toxicology of Fishes

Receptor-Mediated Mechanisms of Toxicity 243

A 250
Specific
Bound Ligand 200 Total
Nonspecific
150
100
50
0
0 20 40 60 80 100
Free Ligand Concentration

B 120
100
Bound Ligand 80
60
40
20
0
0.001 0.01 0.1 1 10 100
Free Ligand Concentration

FIGURE 5.5 Idealized ligand binding data. (A) A typical plot of concentration-dependent total and nonspecific binding.
Specific binding is calculated as the difference between total binding and nonspecific binding. (B) Specific binding from
A is shown on a semilog plot to demonstrate that saturating concentrations of ligand are being approached.

Using computer software for performing nonlinear curve fits, it is possible to fit data directly to the
Langmuir isotherm (Equation 5.3) without transformation. This produces more accurate estimates of the
binding constants. The specific binding values are not measured directly, however, so this approach is
still somewhat removed from the ideal of determining binding directly from measured quantities.
Nonspecific binding is by definition unsaturable within the range of ligand concentrations used, and
it typically increases as a linear function of ligand concentration:
=
NSB m L[] (5.5)
where NSB is nonspecific binding and m is the slope of the line fit to the data. Total binding (TB) should
simply be the sum of the specific and nonspecific binding:
[
LR T]
[]
TB = + mL [] (5.6)
L +
[] K D
Therefore, the data collected for the total binding curve can be fit to Equation 5.6 to determine the
binding constants and the slope of the nonspecific binding line. Theoretically, even more accurate
estimates of the binding constants can be achieved by simultaneously fitting the total binding data to
Equation 5.6 and the nonspecific binding data to Equation 5.5. Idealized data showing the typical
relationship among total, specific, and nonspecific binding are shown in Figure 5.5.
The method used to fit the binding data is critical when data are of relatively poor quality, which may
be due to factors beyond the experimenter’s control (e.g., unstable receptor or ligand, difficult tissue
preparations). When the collected data closely fit Equations 5.3, 5.5, and 5.6, the binding constants
estimated by the different methods are nearly identical. This is illustrated in Figure 5.6, where the data in
3
3
panel A were collected from binding of [ H]-2,3,7,8-tetrachlorodibenzo-p-dioxin ([ H]-TCDD) to hepatoma
cells from the fish Poeciliopsis lucida (PLHC-1), which have a relatively unstable aryl hydrocarbon
receptor; the data in panel B were collected using the same technique from dolphin kidney cells (CDKs),
which have a relatively stable receptor. Fitting the data to Equations 5.3, 5.4, or 5.5 and 5.6 (simultaneously)