A Fluorescence Analysis of ANS Bound to Bovine Serum Albumin: Binding Properties Revisited by Using Energy Transfer

Abstract

Determination of binding parameters such as the number of ligands and the respective binding constants require a considerable number of experiments to be performed. These involve accurate determination of either free and/or bound ligand concentration irrespective of the measurement technique applied. Then, an appropriate theoretical model is used to fit the experimental data, and to extract the binding parameters. In this work, the interaction between bovine serum albumin (BSA) and 1-anilino-8-naphthalene sulphonate (ANS) is revisited. Using steady state fluorescence spectroscopy, the binding isotherm of BSA/ANS was obtained applying the Halfman–Nishida approach. The binding parameters, site number, and binding site association constants, were determined from the stoichiometric Adair model and Job’s plot. The binding parameters obtained were then correlated to the distance of the respective binding site to the tryptophan residues using the energy transfer technique. This approach, that uses both tryptophans independently from each other, is presented as a tool to help understand the binding mechanism of the albumin fluorescent complex. The results show that ANS molecules bind to BSA in up to five different binding sites. Energy transfer from the tryptophan residues to the BSA/ANS complex shows that the four highest affinity binding sites (>10⁴ M⁻¹) are located at a reasonably close distance (18–27 Å) to at least one of two tryptophan residues, while the lowest affinity binding site (~10⁴ M⁻¹) is located over 34 Å away from the both tryptophans.

Appendix

The critical radius, R ₀, in Förster resonance energy transfer is defined as the distance at which the efficiency of energy transfer is equal to 50%. It can be calculated using the following expression [29]

$$R_{\text{0}} = {\text{9780}} \times \left( {\kappa ^{\text{2}} \phi _{\text{D}}^{\text{0}} n^{ - {\text{4}}} J\left( \lambda \right)} \right)^{{{\text{1}} \mathord{\left/ {\vphantom {{\text{1}} 6}} \right. \kern-\nulldelimiterspace} 6}} $$

(9)

with R ₀ in Å, κ ² is the orientation factor (two-thirds assumed) describing the relative orientation in space of the transition dipoles of the donor (Trp residues) and acceptor (ANS), and $\phi _{\text{D}}^{\text{0}}$ is the donor fluorescence quantum yield in the absence of the acceptor. In the case of tryptophans in BSA this value is 0.15 [35]. A value of 1.33 is assumed for the refractive index, n, of the solution. The spectral overlap integral J(λ) between the donor emission spectrum and the acceptor absorbance spectrum was approximated by:

$$J\left( \lambda \right) = {{\int {F_{\text{D}} \left( \lambda \right)\varepsilon _{\text{A}} \left( \lambda \right)\lambda ^{\text{4}} \operatorname{d} \lambda } } \mathord{\left/ {\vphantom {{\int {F_{\text{D}} \left( \lambda \right)\varepsilon _{\text{A}} \left( \lambda \right)\lambda ^{\text{4}} \operatorname{d} \lambda } } {\int {F_{\text{D}} \left( \lambda \right)\operatorname{d} \lambda } }}} \right. \kern-\nulldelimiterspace} {\int {F_{\text{D}} \left( \lambda \right)\operatorname{d} \lambda } }}$$

(10)

where, F _D(λ) is the normalized fluorescence spectrum of the donor and ε _A(λ) is the absorption molar extinction coefficient.

The distance between the donor and acceptor R _DA can be determined by measuring the change in donor fluorescence. The donor fluorescence intensity was measured in the absence (I _D) and in the presence of acceptor (I _DA). Then, FRET efficiency can be calculated using the following expressions:

$$E{\text{ = 1}} - \frac{{I_{{\text{DA}}} }}{{I_{\text{D}} }}$$

(11)