59
Equation 14 of the Appendix gives the elution volumes, binding
ratios and ligand concentrations in terms of dimensionless ratios.
ve vo
vr=?r
. *
1 +
Kt
T
T
1 + Ki
[i] > 0
This equation was used to generate a family of curves shown in Figure
17. The elution volume is corrected for the column void volume and ex
pressed relative to V^_^, the volume at which an enzyme would elute in
the absence of soluble ligand after correction for the column void volume.
The concentration of soluble ligand is expressed relative to Kj. The
two column binding constants are expressed as a ratio of each other.
The point at which all of the lines intersect on the volume axis is that
point at which the concentration of soluble ligand is zero. For ratios
of K]^ijj/K£rp[ greater than one the elution volume will increase with in
creasing ligand concentration; for equal to one there will be
no effect on the elution volume by ligand; finally, when is
less than one the elution volume will decrease with increasing ligand
concentration (Table II). Figures IS through 20 illustrate these three
types of elution patterns expected from this scheme for typical small
zone experiments. We have chosen to refer to them as Case I, Case II
and Case III for column binding ratios greater than, equal to and less
than one, respectively.
Let us examine each case in detail from the standpoint of the en
zyme species. First, Case i (Figure 18) describes the situation in