Concepts of Pharmaceutical Science

Problem 1, Part 2. At time zero, 1 ml samples of the same tissue membranes used in problem 1 are mixed with 10 nM of radioligand L*, and then the counts bound to the membranes are determined at 2 minute intervals. After subtracting nonspecific binding (determined in other samples), the following data are obtained:

Time (min)
Specific CPM bound
0
0
2
155836
4
272312
6
359369
8
424437
10
473071
12
509421
14
536590
16
556897
18
572075
20
583419

e. Use 10 nM [LR]eq = 3.70563828 pMole (derived from a Scatchard plot) and the K-1 value from problem 1a to calculate K+1 (unit = L/mole-1 min-1), and then from both values, calculate Kd. Show your graph and your work. The values for the radioligand, the volume, and the counting efficiency are the same as above and throughout this entire problem set questions 1-3. Hint: remember the difference between moles and molar.

f. Calculate the T½ of the ligand binding to the receptor at 10 nM ligand. T½ of association = ln(2)/Kon

g. What would the graph look like for [L*] nM? Plot the data and calculate the T½ of binding under these conditions.

h. If you were setting up an equilibrium binding assay at 1 nM radioligand would 1 hour of binding be sufficient? Explain your answer. Also give an estimate of how long you would expect to wait to have 87.5% of binding at equilibrium (remember T1/2 = time to reach 50% of binding at equilibrium, so 87.5% = 3 x T1/2, or Percent binding (1 - 1/(2x)), where x is the number of elapsed T1/2s as long as x>0). How long would it take to reach approximately 99% of the equilibrium binding?