Figure 1.
Scatchard plots for ANS interaction with BSA and HSA.
Experimental data (circles) and best fit curve with binding constants (Kbi) and number of binding sites (ni) given on the panels.
Table 1.
Characteristics of ANS – BSA and ANS – HSA interaction.
Figure 2.
Absorption spectra of ANS bound to BSA and HSA.
Curves 1 and 2 represent absorption spectra of ANS in chamber #2 (free ANS in concentration Cf) and in chamber #1 (superposition of absorption spectra of free ANS in concentration Cf, ANS bound to proteins in concentration Cb) after reaching equilibrium. Curve 3 is the absorption spectra of ANS bound to proteins. Curve 4 is the absorption spectra of free dye in a concentration equal to that of bound dye.
Figure 3.
Determination of the molar extinction coefficient of ANS bound to BSA and HSA.
(a) Concentration of ANS bound to proteins (Cb) as a superposition of the concentrations of the dye bound to the sites of mode 1 (Cb1) and mode 2 (Cb2). (b) The dependence on Cb1, Cb2. In the panel experimental data, best-fit curve and the values of molar extinction coefficients εb1 and εb2 obtained by multiple nonlinear regression are presented. (c) Absorption spectra of ANS bound to the sites of mode 1 and mode 2 in the units of the molar extinction coefficient.
Figure 4.
The dependences of fluorescence intensity of quinine sulfate (QS) on its optical density.
are calculated values and
are experimentally recorded and normalized values of fluorescence intensity of QS. Dots are the limiting value of
at
. The dependences of calculated values
and experimentally determined values
on total optical density are given in the Insert. Strait line is the dependence of
on
(
).
Figure 5.
Determination of the fluorescence quantum yield of ANS bound to BSA and HSA.
(a) Optical density of ANS bound to proteins as a superposition of optical densities of the dye bound to the sites of mode 1 (Db1) and mode 2 (Db2). (b) 3D dependence of on Db1 and Db2. Experimental data, best-fit curve and the values of qb1 and qb2, determined by multiple non-linear regression are presented.