Titration of RP with 1 mL of 8.4 10-6 mol L-1 NR was
monitored by absorption spectroscopy as shown in Figure 1.
The titration was performed at pH 9 and 25 C. Upon
increasing RP concentration, the absorption peak of NR shifted
to 545 nm. The change of absorption spectra of NR is likely to
be due to the increase of hydrophobic environment upon
binding. A single isosbestic point at 490 nm was observed,
indicating a simple change between bound and unbound states.
The increase in the absorbance intensity at 545 nm was used to
determine the Kd. A directed plot of ΔARP versus [RP]0 was
used to calculate ΔAmax by fitting the hyperbolic graph with
nonlinear regression analysis. The value of ΔAmax was calculated
to be 0.113. The value of ΔARP/ΔAmax or R was used for
calculating the quantity of the bound complex in each titration
and then the concentration of free RP in each titration was
obtained by subtracting the bound fraction from the total
concentration of RP as described in eq 9. The value of Kd was