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A radioligand is a radioactively labeled drug that can associate with a receptor, transporter, enzyme, or any site of interest. Measuring the rate and extent of binding provides information on the number of binding sites, and their affinity and accessibility for various drugs. Radioligand binding can be used to: (1)characterize receptors in their natural environment as well as those transfected into cell lines; (2)study receptor dynamics and localization; (3)identify novel chemical structures that interact with receptors (4)define ligand activity and selectivity in normal and diseased tissues.
tissues. The most common method for detecting the receptors in membrane preparations, tissue slices or in the purified form is to use a radioactive drug which has a high affinity and high degree of selectivity Incubate tissue with radioactive drug under the appropriate experimental conditions, the radioactive drug (D) will bind to the receptor (R) to form a drugreceptor complex (RD).
The amount of drug-receptor complex (RD) can be measured because it is now radioactive.
At Equilibrium
kon koff Kd
Association rate constant or on-rate constant Dissociation rate constant or off-rate constant Equilibrium dissociation constant
c. The radioligand should have a high degree of selectivity for the receptor being studied. d. The radioligand should be chemically stable in the assay media during the binding reaction. e. The radioligand should be pure.
Radioligand binding could be : 1) Specific The site that we want to study is referred to as the SPECIFIC SITE. 2) Non-Specific All other sites are called NONSPECIFIC SITES.
Examples of nonspecific binding sites 1) Other receptors from the same class: [3H] rauwolscine binds to alpha-2A, alpha-2B, and alpha-2C subtypes with similar affinities. If all alpha-2 adrenoceptors are to be studied without differentiating between subtypes then binding of [3H] rauwolscine to all alpha-2 adrenoceptors would be considered specific binding. If only study alpha-2A subtype, then radioligand bound to the alpha-2A subtype would be considered specific binding and binding to the other subtypes would be considered nonspecific binding. 2) Other receptors from a different class: [3H] rauwolscine binding to serotonin receptors as well as to alpha-2 adrenoceptors.
NON-SATURABLE binding sites are sites that are present in essentially infinite amounts. No matter how much ligand you add, not all of the sites will be occupied with ligand. The site is non-saturable.
Examples of saturable sites: Specific binding sites are always saturable All Receptors
Test tubes and glass fiber filters may have saturable, nonspecific binding sites as well as non-saturable binding sites. Examples of non-saturable sites: Low affinity tissue binding sites
Binding is measured in the presence of the highest concentration of radioactive ligand used in a saturation curve. Under these conditions all of the receptors would be occupied by radioactive ligand. Binding is also measured in the presence of the same concentration of radioactive ligand plus increasing concentrations of unlabeled ligand. As the concentration of unlabeled ligand increases the amount of radioactive ligand bound decreases until a plateau is reached. At this point no matter how much unlabeled ligand is added the amount of radioactive ligand bound remains constant. This residual amount of radioligand bound represents binding to nonspecific, non-saturable sites which can't be displaced by unlabeled ligand. The amount of inhibitor to use would be the concentration which completely blocks the saturable binding sites. In this example it would be between 10-9 to 10-8 M.
Stability Radioligand purity must be established periodically. In some cases, the radioligand requires special storage procedures, such as addition of antioxidants or protease inhibitors in the case of a peptide radioligand, to slow or prevent degradation.
Nonspecific binding The physicochemical properties of a ligand determine its level of nonspecific binding due to interactions with lipid membranes and/or filter or scintillation bead material in the assay. Factors such as lipophilicity and aqueous solubility should be taken into consideration when selecting a ligand to radiolabel.
Buffers used In most cases, a homogenization / assay buffer is selected that yields the highest ratio of specific versus nonspecific binding. Common buffers - Tris, Hepes, sodium phosphate and glycylglycine, Krebs, Ringer, or Hanks balanced salt solution (HBSS). Buffers can affect the binding of radioligand to the receptor, using the same buffer as for assays measuring response is best so that the results can be compared. Some binding assays require the presence of special ions. For example, opioid receptor binding is modulated by sodium, GABAA receptor binding by chloride, and the N-methyl-Daspartate (NMDA) glutamate receptor by magnesium.
pH of the assay buffer The affinity of a ligand for a receptor generally varies with the pH. Generally using a physiological pH, such as 7.4, is best so that the results are comparable to what is seen in vivo. Optically active radioligands If a ligand contains a chiral center, use of the stereochemically active form is preferable. With few exceptions, most receptors differentiate between the optical isomers of compounds, with the pharmacologically-active isomer binding with higher affinity than the less-active isomer.
Additional reagents
MgCl2 is used in many receptors binding assays. Initially this was used to help precipitate the radioligand receptor complex during centrifugation. NaCl is often used in adrenergic receptor binding studies to convert the receptor to a form that has a lower affinity for agonists. GTP or its non-hydrolyzeable analog (Gpp(NH)p) are often used when agonist binding is also going to be studied in subsequent competition experiments. Gpp(NH)p converts the receptor into a form that has a low affinity for agonist
Purpose of the Saturation experiment 1)To determine the affinity or Kd of a radioligand for a receptor
Kd is the equilibrium dissociation constant. It is equal to the concentration of radioactive ligand required to occupy 50 % of the receptors.
Results of the saturation experiment can be plotted with BOUND (the amount of radioactive ligand that is bound to the receptor) on the Y axis and FREE (the free concentration of radioactive ligand) on the X axis.
The resulting graph is a hyperbola and is called a saturation curve. Bmax is the density of the receptor in the tissue being studied. Kd is the concentration of ligand required to occupy 50% of the binding sites
Getting an accurate estimate of Kd and Bmax from this graph by eye is difficult. The curve is usually analyzed by nonlinear regression analysis
Rosenthal Plot
The data from the saturation experiment can be plotted with Bound/Free on the Y axis and Bound on the X axis.
Single site binding data can be analyzed by linear regression to give a straight line. The slope of the line is -1/Kd and the X-intercept is Bmax. This is a Rosenthal Plot. Most scientists call it a Scatchard Plot.
Rosenthal Plot
Two-site fit to the data can be visualized more easily with a Rosenthal Plot than with a saturation curve.
2. The concentration of tissue used: It is dependent on the number of binding sites per mg of tissue. The tissue concentrations required in the assay will vary for the same binding site in different tissues and for different receptors in the same tissue.
It is helpful if the tissue concentration is high enough so that at least 1000 cpm are bound when all of the receptors are occupied with radioactive ligand. This allows for reasonable accuracy in counting the radioactivity bound to the tissue at the lowest concentrations of radioligand. It is important that enough tissue be present so that a measurable amount of the radioligand is bound at the lowest radioligand concentration.
However, the tissue concentration not be so high that more than 10% of the radioligand is bound.
The equations used for generation of Kd and Bmax are based on the assumption that the free concentration of radioligand does not change.
It is generally assumed that these conditions are met if less than 10% of the radioligand is bound to the receptor. One way to determine quickly whether 10% of the radioligand is bound to the receptor is to look at the position of Y-intercept in a Rosenthal plot.
If most of the nonspecific binding is to tissue and not to filters used in a filter assay then bound would be total bound. (Note that total bound represents the binding of the radioligand to both specific and nonspecific sites.)
Free = Total Added - (Specific bound + Nonspecific bound) or Free = Total Added - Total bound If the nonspecific binding comes predominately from the binding of the radioligand to the filters used to separate bound from free, then this nonspecific binding is not to the tissue and occurs after the reaction is complete. In this case, only specific binding should be subtracted from total binding to give free. Free = Total added - Specific bound
A time course can be run at the lowest concentration of radioligand under your experimental conditions to demonstrate that equilibrium conditions are present when bound is separated from free.
Less than 10% of the radioligand should dissociate from the receptor in the process of separating bound from free.
Less than 10% of the radioligand should be depleted from the reaction mixture.
The amount of bound radioligand needs to be determined accurately Specific binding must be correctly defined.
When the radioligand has a high affinity for more than one type of receptor.
When the radioactive ligand is an agonist. For example: The G-protein receptors exist in at least two conformations, one with a high affinity for agonist and one with a low affinity for agonist. Unless a reagent, such as Gpp (NH)p, is used to convert all of the receptor molecules to the low affinity form, two binding sites may be detected in the saturation experiments using a radiolabeled agonist
where Bmax1 and Bmax2 are the binding site density for sites 1 and 2
Kd1 and Kd2 are the Kd values for site 1 and site 2
2 Competition Experiments
Many ligands for receptors are not available in a radioactive form. Since they are unlabeled there is no way to directly measure their affinity for the receptor. The affinity of the unlabeled ligand for the receptor can be determined indirectly by measuring its ability to compete with a radioactive ligand for the receptor. In a competition experiment various concentrations of an unlabeled ligand are allowed to compete with a fixed concentration of a radiolabeled ligand for a receptor. As the concentration of unlabeled ligand increases, the amount of radioligand bound to the receptor decreases.
The competitive inhibitor can be either an agonist or an antagonist. It is called a competitive inhibitor because its value is determined by measuring the ability of the unlabeled drug to compete with a radiolabeled drug for the receptor.
The Ki value for an unlabeled drug should be the same as the Kd value obtained for the same drug in radiolabeled form.
Analyses of the results of the competition experiment are simpler and more accurate if the radiolabeled ligand is only binding to one site. Hormone and neurotransmitter receptors exits in conformations which have either high affinity or low affinity for the agonist. A true antagonist has equal affinities for the high and low affinity states of the receptor. The analysis of the data is simpler if the radiolabeled ligand is an antagonist
Competition experiments can be used to determine the affinity of the unlabeled ligand for the receptor The affinity of an agonist for a receptor. The pharmacological characteristics of subtypes of a particular receptor. The classification of receptor subtypes in a tissue
Experimental Conditions for Competition Studies The radiolabeled ligand should have a high affinity for the
receptor being studied. The concentration of radiolabeled ligand used for the competition studies should be between 0.75 and 1.0 times the Kd value for the ligand determined using the same experimental conditions as is planned for use in the competition studies. It is helpful to have at least 10 concentrations of unlabeled ligand for one-site competition studies. There should always be a tube which does not contain any drug but contains the same volume of diluents as used to deliver the unlabeled drug. Increments of 1 and 3 are often used Binding needs to reach steady-state conditions when doing either saturation or competition experiments
If the unlabeled ligand binds to different subtypes of the receptor with different affinities and there is more than one subtype in the tissue you are studying.
If the unlabeled ligand binds to more than one receptor with different affinities. This assumes the radioligand is also binding to more than one receptor.
The characteristics of a competition curve with one-site binding are a sigmoid curve with:
a slope of 1.0. there is a 81 fold difference in the concentration between 90% specific bound and 10% specific bound.
The characteristics of a competition curve with two-site binding is a sigmoid curve with:
Methods used to identify the receptor subtypes in an unknown tissue First, the pharmacological profiles for the known receptor subtypes need to be determined. It is helpful to identify drugs which have a high affinity for each of the subtypes of the receptor. The pharmacological profile for the unknown tissue needs to be determined. In doing this profile, choose drugs which have high affinities for each of the known subtypes. Drugs which have similar affinities for multiple subtypes are not as effective. Compare the pharmacological profile for the unknown tissue with the pharmacological profile of the known subtypes. So for example, suppose a drug has a high affinity for subtype A and low affinity for subtype B and C and it also has a high affinity for the unknown receptor in the tissue you are studying. This would suggest that the subtype in the unknown tissue is subtype A.