Titration of RP with 1 mL of 8.4 Â 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 beduetotheincreaseofhydrophobic 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.AdirectedplotofΔARP versus [RP]0 was used to calculate ΔAmax by fitting the hyperbolic graph with nonlinear regression analysis. The value of ΔAmax was calcu- lated 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.