67
determined. For statistical reasons it may be advisable simply to apply
the least squares criterion (i.e., to use the method of simple linear
regression) and not force the line to pass through the origin. Since
the plot of B^/F^ vs. [free binding sites] is used to estimate only one
binding parameter (K^), data sets arising from experimental
preparations differing in total binding site concentration may be merged
prior to the final analysis.
We have extended the analysis presented above to cover a very
limited case of the two ligand-two binding site problem: the method has
been used to "linearize" data resulting from investigation of the
specific mouse brain glucocorticoid receptor in crude cytosol containing
significant amounts of CB6 (transcortin). We have measured the affinity
of "cold" dexamethasone (which has negligible affinity for CBG) for the
specific [ Hjcorticosterone, non-CBG, binding sites (putative receptors)
in CBG-containing brain cytosol in order to compare the resulting
equilibrium constant with that obtained by using [ H]dexamethasone
itself to construct a one-ligand Scatchard plot. The experiment is
performed by first constructing separate sirigle-1 igand binding isotherms
3 3
using [ H]dexamethasone and [ Hjcorticosterone; then an isotherm
3
describing the binding of [ Hjcorticosterone in the presence of a fixed
concentration of dexamethasone is constructed and analyzed. (The same
cytosol preparation is used throughout, of course.) This analysis is
simplified dramatically both by the absence of a significant interaction
of dexamethasone with CBG and by the observation that the one-ligand
[ Hjcorticosterone Scatchard plot is not biphasic, which suggests that
the affinity of corticosterone for CBG is very similar to its affinity
for the putative receptors.