74
This equation will yield a straight line when elution volumes are plot
ted as 1/(Ve V') versus the reciprocal of the concentration of soluble
ligand. The values for the intercept and slope will be l/vc^iq(Kj7=iq/K^rTq)-l)
and K-r/V-ยก-^( (K^rqq/K^rrq)-!, respectively. Again the quotient from the slope
divided by the intercept will give Kt, the dissociation constant for the
E*I complex.
Figure 22 shows the data for the elution volumes obtained from small
zone experiments with the carbobenzoxy (CBZ) derivatives of phenylala
nine, tyrosine and tryptophan plotted in this manner.
The data shown represents the triplicate values obtained for each
concentration of I with multiple points shown where the data from the
triplicates varied.
The value for could be determined from the intercept by-
substituting in the value of V;nry[, which was shown to be 1.49 for each
of the CBZ-derivatives shown, From equation 13 in the Appendix, where
[I] = 0 the elution volume is,
(V0 Vm)[E=H]
Vq and VD can be determined from the passage of enzyme through the column
in the absence of soluble ligand. The column void volume, VQ, would be
the elution volume for the autolysis products. Vm is volume of mobile
phase of the column and is determined at the volume at which a very large
molecular weight marker, such as Blue Dextran 2000, will elute. The im
mobilized ligand concentration was obtained from extended solvolysis of
matrix, representing the amount of ligand physically substituted to the
matrix.