1. Outline the main reasons for undertaking a competitive inhibitionradioligand binding experiment? These type ofexperiments are used to validate an assay to demonstrate that a drug binds withthe expected potency and to prove that the chosen radio-ligand has identifiedthe correct receptor (Motulsky, 1995-96). Competitive binding curves are alsoemployed to investigate the interaction between low affinity drugs andreceptors. 2. What is the difference between IC50 and Ki? The IC50(half maximal inhibitory concentration) is the concentration of unlabelled drugthat blocks half the specific binding and it is determined by three factors;the Ki (equilibrium dissociation constant) of the receptor for thecompeting drug, the radio-ligand concentration and the radio-ligands affinityfor the receptor (Kd) (Motulsky, 1995-96).
The Ki is the equilibrium dissociation constant for binding of theunlabelled drug, it is proportional to the IC50 i.e. if the Kiis low, the IC50 will also be low (Motulsky, 1995-96). 3. Define the Cheng Prusoff correction. Explain how it is applied incompetitive binding experiments? TheCheng-Prusoff equation allows for the calculation of Ki from the IC50and is as follows:Theequation is based on the assumptions that the Kd value is known frompervious experiments, that the experiment has reached equilibrium and thatbinding is reversible and follows the law of mass action (Motulsky, 1995-96).
Because the IC50 value is not a direct indicator of affinity, theCheng-Prusoff equation is applied to derive the binding affinity of theinhibitor (Ki). 4. What safety measures are taken when working with 3H ligands inthe laboratory?Hydrogen-3 or Tritiumis a beta-emitting radioactive isotope employed in radiolabellingexperiments. Although beta radiationcannot penetrate the outer layer of skin; ingestion, inhalation or absorptionthrough broken skin may result in exposure. Minimum Personal Protective Equipment required when handling radioactivematerial includes disposable gloves, lab coats, and safety glasses. Forcompounds such as tritium which readily penetrate gloves, wear double gloves,changing the outer pair at least every 20 minutes. A designated working areashould have all surfaces covered with spill paper and any aerosol-generatingprocedures should be carried out in a fume hood.
It is recommended that allpersons expected to handle radioactive material are trained, registered, anddocumented on an approved protocol.
