Practical Aspects of Radioligand Binding
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Abstract
Radioligand binding has been used for many years to identify new binding sites, characterize receptors, and identify novel ligands. Although various techniques have been developed to improve the efficiency of preparing the biological source of the receptors and for detecting bound radioligand, the principles of the assays remain the same. This unit reviews theory and provides examples of the parameters that can be calculated from radioligand binding data to characterize ligand?receptor interactions. The important aspects of assay development and validation that allow meaningful interpretation are discussed. The selection of a radioligand, buffer and other assay components is critical to developing a useful binding assay. The nature of the binding interaction can also be probed by varying assay conditions.
Keywords: radioligand binding; filtration; membrane preparation; affinity; saturation binding; displacement
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PDF or HTML at Wiley Online LibraryTable of Contents
- Fundamentals of Radioligand Binding Assays
- Guidelines for Establishing a Radioligand Binding Assay
- Analysis of Binding Data
- Troubleshooting
- Literature Cited
- Figures
- Tables
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Figure 1.3.1 Saturation binding to muscarinic receptors on N1E‐115 mouse neuroblastoma cells. Six concentrations of [3 H] N ‐methylscopolamine ([3 H]NMS), with or without 10 µM unlabeled NMS, were incubated with ∼300,000 intact cells/tube for 45 min at 15°C before rapid filtration was performed to separate bound from free. The total binding is the sum of the specific and nonspecific binding. Nonspecific binding is defined as the amount of binding found in the tube containing both the radioligand and unlabeled NMS. View Image -
Figure 1.3.2 The Scatchard (Rosenthal) plot. The specific bound ( B ) is plotted on the x axis, and the ratio of specific bound to free ( B / F ) is plotted on the y axis. The x intercept is the maximal amount of specific binding ( B max ). The slope of the plot is the negative of the inverse of the equilibrium binding dissociation constant ( K d ). View Image -
Figure 1.3.3 Competition between a radiolabeled antagonist and an unlabeled agonist for muscarinic receptors on N1E‐115 cells. [3 H]Quinuclidinyl benzilate ([3 H]QNB; 0.2 nM) was incubated with 200,000 intact cells/tube and various concentrations of carbachol for 75 min at 15°C, and the suspensions were rapidly filtered to terminate the reactions. The level of nonspecific binding was determined using 1 µM atropine. View Image -
Figure 1.3.4 Effects of allosterism or cooperativity on receptor binding. The x axis is the free concentration of the ligand, expressed in relationship to the K d value. The y axis is the fractional specific binding to the receptor, where 1.0 is equivalent to full saturation of the binding sites ( B max ). The n H is the Hill coefficient, a number that can be used to express the degree of cooperativity in the binding reaction. When | n H | > 1, the receptors interact with positive cooperativity; when | n H | < 1, the receptors interact with negative cooperativity. View Image -
Figure 1.3.5 Appearance of binding data using different methods of plotting. Saturation binding (A and B ) and competition binding data (C and D ) are shown. The lefthand panels (A and C ) show specific binding (expressed as fraction bound) plotted versus ligand concentration (expressed as a concentration ratio with respect to the K d ) on the x axis, on arithmetic plots (both axes plot untransformed values). The righthand panels (B and D ) are semilogarithmic plots , which show specific binding plotted on the y axis (untransformed) versus the logarithm of the ligand concentration. View Image -
Figure 1.3.6 Effect of increasing the radioligand concentration in a competition binding assay. This is a semilogarithmic plot of specific binding (expressed on the y axis as fraction of maximal binding) in a hypothetical experiment. The drug D is an unlabeled competitor and the logarithm of its concentration is plotted on the x axis. Curves 1, 2, and 3 represent the appearance of the data when progressively larger concentrations of the radioligand are used. For further explanation, see . View Image -
Figure 1.3.7 Scatchard (Rosenthal) plot of the equilibrium binding of [3 H]NMS to muscarinic receptors on N1E‐115 neuroblastoma cells. The data shown in Figure and Table were used to construct this plot, and a full discussion is given in the text. The linear regression correlation coefficient for this plot ( R ) is 0.979, the K d value (the negative inverse of the slope) is 0.232 nM, and the B max (the x intercept) is 61 fmol/mg. The data in Figure were normalized to mg protein, whereas the data in Figure are expressed in fmol/tube specific binding. View Image -
Figure 1.3.8 Effect of allosterism or cooperativity on the appearance of binding when shown on a Scatchard (Rosenthal) plot. When multiple sites interact in negative cooperativity ( n H < 1), the plot of binding is concave. When multiple sites interact with positive cooperativity ( n H > 1), the Scatchard transform is convex. The normal appearance of the Scatchard plot for simple bimolecular interactions (no cooperativity; n H = 1), is the straight‐line plot. Note that binding of the ligand to multiple independent sites which exhibit different binding affinities will produce a concave Scatchard plot (apparent negative cooperativity). View Image -
Figure 1.3.9 Hill plot for the saturation binding of [3 H]NMS to muscarinic receptors on N1E‐115 cells. The data used to make Figures and , and Table were used for this Hill plot. This is a log‐log plot, in which the logarithm of the ratio of bound to unbound is plotted on the y axis, and the logarithm of the free concentration of the radioligand is plotted on the x axis. The Hill slope is 0.89, which in this case is not significantly different from unity ( P > 0.05). View Image -
Figure 1.3.10 Hill plot for the competition between [3 H]NMS and carbachol for muscarinic receptors on N1E‐115 cells. The binding data shown in Figure were used to construct this plot. The ratio of bound/unbound in this case was expressed using percentages of maximal specific binding (% B ), rather than the B max (as was done in Figure ). The absolute value of the Hill slope in this case was 0.25, which was significantly different from unity ( P > 0.001). The apparent negative cooperativity results from the binding of carbachol to three different sites on N1E‐115 cells with different affinities. View Image -
Figure 1.3.11 Dissociation of [3 H]NMS from muscarinic receptors on N1E‐115 cells. The radioligand (0.56 nM) was incubated with intact N1E‐115 cells (300,000 cells/tube) for 45 min at 37°C, at which time equilibrium is reached. The dissociation of radioligand from the receptors was followed for various periods of time after the addition of 10 µM NMS, and the reactions terminated by rapid filtration. Nonspecific binding was also determined at these various times (not shown) by adding 10 µM NMS to some tubes for the duration of the experiment; the nonspecific binding did not vary with time, and it was subtracted from the total binding to obtain the specific binding, which is plotted. (A ) An arithmetic plot of the time course of dissociation. (B ) Plot of the natural logarithm (ln) of the amount bound at any time ( t ), expressed as a fraction of the amount bound at t = 0 (i.e., B 0 ). Note the inflection point in the lower plot is at about t = 20 min. View Image -
Figure 1.3.12 Association of [3 H]NMS to muscarinic receptors on N1E‐115 cells. The radioligand concentration was 0.56 nM, 300,000 cells/tube were used, and the temperature was 37°C. Panel (A ) is a plot of the untransformed specific binding measured at various times ( t ) after starting the incubations. The plot in panel (B ) is a logarithmic transform of the ratio of the amount bound at equilibrium ( B eq ) to that remaining unbound ( B eq – B t ) at any time ( t ). View Image
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| Key References | |
| Kenakin, 2004. A pharmacology primer: Theory, application and methods. Elsevier, Inc., London, UK. | |
| A complete treatise for the advanced student. | |
| Weiland, G.A. and Molinoff, P.B. 1981. Quantitative analysis of drug‐receptor interactions: I. Determination of kinetic and equilibrium properties. Life Sci. 29:313‐330. | |
| Useful for explaining common plotting methods. | |
| Yamamura, H.I., Enna, S.J., and Kuhar, M.J. 1985. Neurotransmitter Receptor Binding, 2nd ed. Raven Press, New York. | |
| This volume has been a standard in the field for many years and is especially useful for beginners. |
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