2. Interpreting experimental data. The following questions deal with data collec

Question

2. Interpreting experimental data. The following questions deal with data collected from three different experiments of three different receptor-ligand systems. The data are shown in panels (a)-(c) of Figure 2 (a) Kinetic parameters. To measure the dissociation rate constant, labelled ligand is incubated with cells until complex formation reaches equilibrium (Figure 2(a) below). At this point, the number of complexes is Co. The medium is continually washed away, and the amount of ligand remaining bound to the cell is quantified. Shown in Figure 2(a) are the results for two different ligands. Which ligand (1 or 2) has the higher dissociation rate constant? Briefly explain. (b) Equilibrium binding parameters. To measure the equilibrium dissociation constant, labelled ligand is incubated with cells until complex formation reaches equilibrium. At this point, the amount of is incubated with cells until complex formation reaches equilibium. At this point, the amount of ligand molecules bound to the cell (i.e., complexes, C) and the amount of unbound ligand (i.e., free ligand, L) are quantified. The experiment is repeated at multiple concentrations of ligand. The Scatchard plot in figure (b) shows the data collected for two different ligands. Which ligand (1 or 2) has the higher equilibrium dissociation constant? Briefly explain.

Explanation / Answer

Solution:

(a) Kinetic parameters: Since the concentration of complex, Co is reducing at faster rate in case of ligand-1 than that of the ligand-2, therefore it can be said that the dissociation of the complex in case of ligand-1 is faster than that in the case of ligand-2 and hence the dissociation rate constant. In other words, the dissociation rate constant is higher in case of ligand-1 than that of ligand-2.

(b) Consider the following equilibrium reaction exists as mentioned in the question:

S + L1 ---k1----> SL1

        <---k-1----                          (1)

S + L2 ---k2----> SL2

          <---k-2----                        (2)

Where, S, L1 , L2 and k1, k2 are the receptor, ligands and their corresponding equilibrium constants respectively.

Therefore, k1 = [SL1]/[S][L1] and k2 = [SL2]/[S][L2]

It is clear from the graph (b) that the concentration of receptor-Ligand complex, SL2 is greater than that of receptor-Ligand complex, SL1. Therefore it can be said that the equilibrium constant (k2) for L2 is higher than that of equilibrium constant (k1) of L1. In other words, the equilibrium dissociation constant (k-2) of L2 is lower that of the equilibrium dissociation constant (k-1) of L1.

(c) Figure c, depicts a positive receptor-Ligand system, since it can be seen that the bound increases from initial to the final state (an exponential curve with decreasing bound/free with respect to bound)

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