Approximate albumin association constants were determined by conducting a titration procedure that added aliquots of ICB (or ICG) to a solution of BSA (Sigma-Aldrich A964) and monitored quenching of fluorescence from the single tryptophan in the BSA (excitation: 280 nm; slit width: 2 nm). The fluorescent change ΔF was calculated using the equation [F0 -F) where F0 = initial fluorescence intensity and F = fluorescent intensity after each aliquot addition. The approximate dissociation constant (Kd) was determined using GraphPad Prism by fitting the dye concentration dependent ΔF plot to a non-linear one site-specific binding model. Validation experiments showed that light absorption by the added dye at the tryptophan excitation wavelength (280 nm) was negligible before albumin saturation (Figure ), and was assumed to not significantly reduce tryptophan emission enough to alter the titration profile.