Cellular Engineering - Principles of Biomedical Engineering - page 263

Native Mutant
L 0 (nM) B/L 0 L 0 (nM) B/L 0
1.00 5.20 0.19 15.35 0.07
0.80 1.38 0.58 4.12 0.19
0.60 0.67 0.90 1.80 0.33
0.40 0.27 1.48 0.85 0.47
0.20 0.10 2.00 0.39 0.51
0.05 0.03 2.00 0.08 0.63
Plot B / L 0 and B for both samples. On the plot, fit linear regression lines and obtain the
slopes and intercepts. From the slopes, the K D value for a native ER- α is 0.415 nM and for
the mutant, it is 1.672 nM. Since K D for the mutant is nearly four times that of the native,
it has four times less affinity for the ligand. Hence, there is a need for a new mutant design.

B (nM)

7.2.2.2 Multiple Site Binding
Many receptor-ligand interactions and enzyme-catalyzed reactions are complex.
For example, some molecules have two or more identical ligand binding sites.
Binding at one site may be independent or dependent on the binding at the second
site. When binding at the second site is not independent, it is called cooperative
binding and it could be positive (i.e., subsequent binding is enhanced) or negative
(i.e., subsequent binding is diminished). One is often an active site, and the others
are control sites that can increase or decrease the affinity or activity of the active
site. Such a protein is referred to as an allosteric protein, indicating that there are
other sites besides the active site. In these scenarios, the B max and K D values deter-
mined by the Scatchard plot are likely to be far from their true values, although

Figure 7.3 Scatchard plot.