Quantifying Mycotoxin-Albumin Interactions

Fluorescent measurements were performed employing a Hitachi F-4500 fluorescence spectrophotometer (Tokyo, Japan). In order to mimic extracellular physiological conditions, mycotoxin-albumin interactions were studied in PBS (pH 7.4). All measurements were carried out at 25 °C in the presence of air.
Complex formation of 2′R-OTA with HSA was examined applying the Stern-Volmer equation: $\frac{I_0}{I}=1+K_{SV}\times [Q]$ where I and I₀ denote the fluorescence intensities of HSA in the absence and presence of 2′R-OTA, respectively. K_SV (with the unit of L/mol) is the Stern-Volmer quenching constant and [Q] is the molar concentration of the quencher (2′R-OTA). In order to eliminate the inner-filter effect, UV-Vis spectrum of 2′R-OTA was recorded using a Specord Plus 210 spectrophotometer (Analytic Jena AG, Jena, Germany), and fluorescence intensities were corrected applying the following equation [21 (link)]: $I_{\mathit{cor}}=I_{\mathit{obs}}\times e^{(A_{\mathit{ex}}+A_{\mathit{em}})/2}$ where I_cor and I_obs are the corrected and observed fluorescence emission intensities, respectively; while A_ex and A_em are the absorption values of 2′R-OTA at 295 and 340 nm, respectively.
Overall and stepwise binding constants were calculated by non-linear fitting using the fluorescence emission data obtained for all the performed experiments (quenching of the fluorescence of HSA by 2′R-OTA, fluorescence enhancement induced by the energy transfer between HSA and 2′R-OTA, and fluorescence enhancement of 2′R-OTA by HSA) with the Hyperquad2006 program package. To calculate the stability constants associated with the complex formation between HSA and 2′R-OTA, the following equations are implemented in the Hyperquad code [18 (link),22 (link)]: $p\mathrm{HSA}+q\mathrm{OTA}\leftrightarrow {\mathrm{HSA}}_p{\mathrm{OTA}}_q$
${\beta }_{\mathit{pq}}=\frac{\left[{\mathrm{HSA}}_p{\mathrm{OTA}}_q\right]}{{\left[\mathrm{HSA}\right]}^p{\left[\mathrm{OTA}\right]}^q}$
where p and q are the coefficients which indicate the stoichiometry associated with the possible equilibrium in the solution. In the Hyperquad2006 computer fitting program, all equilibrium constants are defined as overall binding constants.
$\mathrm{HSA}+\mathrm{OTA}\leftrightarrow \mathrm{HSA}\mathrm{OTA}{\beta }_1=\frac{\left[\mathrm{HSAOTA}\right]}{\left[\mathrm{HSA}\right]\left[\mathrm{OTA}\right]}$
$\mathrm{HSA}+q\mathrm{OTA}\leftrightarrow {\mathrm{HSA}\mathrm{OTA}}_q{\beta }_q=\frac{\left[{\mathrm{HSA}\mathrm{OTA}}_q\right]}{\left[\mathrm{HSA}\right]{\left[\mathrm{OTA}\right]}^q}$
The relationship between the overall binding constants and the stepwise binding constants calculated by the Hyperquad is the following.
${\beta }_1=K_1;{\beta }_q=K_1\times K_2\dots \times K_q$
The stoichiometry and binding constant of 2′R-OTA-HSA complex were determined by the model associated with the lowest standard deviation.
Fluorescence anisotropy (r) data were determined using the following equation: $r=\frac{(I_{\mathit{VV}}-\mathrm{G}\times I_{\mathit{VH}})}{(I_{\mathit{VV}}+2\times {\mathrm{G}\times I}_{\mathit{VH}})}$ where I_VV and I_VH are fluorescence emission intensities measured in vertical position of polarizer at pre-sample site and at vertical and horizontal position of the post-sample polarizer, respectively, while G is the instrumental factor. Considering the additive behavior of anisotropy, the following equation can be described: $r=f_f\times r_f+f_b\times r_b$ where f_f and f_b are the free and HSA-bound fractions of 2′R-OTA in the solution, respectively, while r_f and r_b are the anisotropies of free and HSA-bound 2′R-OTA, respectively. The free HSA-bound fractions of 2′R-OTA can be described from the rearrangement of Equation (9).
$f_f=\frac{(r-r_b)}{(r_f-r_b)}$
$f_b=1-f_f$
Furthermore, assuming 1:1 stoichiometry of complex formation as well as through the application of Equations (10) and (11), the binding constant (K) can be expressed with the following equation: $K=\frac{f_b/\theta }{f_f\times \left[\mathit{HSA}\right]}$ where [HSA] is the albumin concentration, and θ is the change in quantum yield (I_b and I_f are the fluorescence emission intensities of HSA-bound and free 2′R-OTA, respectively).
$\theta =\frac{I_b}{I_f}$