45
conducted at different ligand concentrations. These values of B^, when
divided by the incubation time, are taken as estimates of the initial
rate of increase of binding (dBL/dt), which is plotted on the ordinate
as a function of the total ligand S^. To analyze this experimental
strategy we note that since B^ = 0 at t = 0, equation (2-1) reveals that
the initial rate of appearance of bound complex is given simply by
dBL/dt (at t=0) = BQkaSL. (2-21)
Thus, the plot of dB^/dt (initial) as a function of is a line
possessing slope Bq^. In fact, the observed curvature of such a plot
(a decrease in slope at high values of S^) has even been offered as
possible evidence that the glucocorticoid receptor binding process may
involve multiple steps and unobserved transient intermediate states
(Pratt, Kaine and Pratt, 1975). (The most probable explanation of this
anomaly is that the constant incubation time was too long to provide a
reliable estimate of the initial dB^dt at the high ligand
concentrations.) The two aforementioned experimental designs may be
combined, of course, by accumulating the early regions of temporal
binding curves generated at different ligand concentrations. The
individual binding curves will generate independent measurement of k
d
that should not be correlated with the ligand concentration S^.
Information (in the form of the derived values of k ) from the
cl
individual binding curves may also be merged by plotting (l/BgJdB^/dt
(initial) = kfl as the ordinate vs. as the abscissa for each
binding curve. The plot should pass through the origin, and the
resulting slope provides the merged estimate of k .
d