5.3 The use of fluorescence spectroscopy associated with molecular docking

Data from fluorescence spectroscopy offers a description of the complex in terms of binding constant, number of binding sites, cooperativity, and the thermodynamics of the complex formation. On the other hand, molecular docking offers details about the binding site environment, the ligand conformation, and the interactions that stabilize the complex. Some authors associated the fluorescence quenching data with the results from molecular docking in order to give a complete description of the biological system.

He and co-workers [55] verified the tryptophan fluorescence quenching of HSA caused by alpinetin. The authors used the van't Hoff model to calculate the thermodynamic parameters and concluded that the hydrophobic interactions were predominant to stabilize the complex. With molecular docking technique, the authors selected the best binding site of alpinetin in the HSA and showed that the binding site was close to tryptophan, which explains the static quenching. The authors also showed that the binding site was a hydrophobic cavity, which explains the data obtained by van't Hoff equation.

With a similar methodology, Kim and co-workers [56] showed the relationship between the number of OH groups in flavonoids and the affinity for mushroom tyrosinase by fluorescence quenching. Molecular docking studies showed that the dicopper catalytic site of tyrosinase is a preferential binding site for flavonoids, which explains the inhibitory activity of the polyphenolic compounds.

Figure 8. (a) Binding site of isovitexin in subdomain IIA of HSA (b) Binding site microenvironment [57].

Figure 9.

(a) Location of the 2-phenylchromone molecules in HSA (b) Microenvironment of site 1, subdomain IIA of HSA (c) Microenvironment of site 2, subdomain IIIA of HSA.

Caruso and co-workers [57] used fluorescence spectroscopy to describe the interaction between isovitexin and HSA. The authors calculated the contribution of enthalpy and entropy by van't Hoff equation and concluded that the process was enthalpically driven. The complete description of the system was reached with molecular docking, as one can see in Figure 8. The oxygen atoms of Ala191 and Pro339 form hydrogen bonds with isovitexin, while neutral polar side chain residues like Gln221, Asn295, Tyr341, and Tyr452 are mainly taking part in electrostatic interactions (charge neutralization).

In this last article, the authors used the Scatchard method, which has been recently used to calculate the number of binding sites and to verify allosterism. With the Scatchard method, the authors concluded that there was one binding site for isovitexin in HSA, which was in agreement with the results obtained by the authors when they used the binding equilibrium model.

In another article, Caruso and co-workers [58] used a similar methodology in order to describe the interaction between 2-phenylchromone and HSA. In this case, the contributions of enthalpy and entropy calculated by van't Hoff equation showed that the process was entropically driven. With the Scatchard method, the authors concluded that there were two cooperative binding sites for 2-phenylchromone in HSA with two different binding constants. Note that this type of information could not be obtained from Stern-Volmer or double-logarithm models; only the Scatchard method can offer information about the system without the use of any binding model a priori.

Also in this study, molecular docking showed that 2-phenylchromone binds at subdomain IIA (Site 1) and subdomain IIIA (Site 2). As shown in Figure 9, at Site 1 amino acids Lys199, Leu (219, 234, 238, and 260), Ala291, and Glu292 are residues involved in the interactions with the molecule. At Site 2 amino acids Leu387, Tyr411, Leu453, Arg485, and Phe488 compose the microenvironment of interaction.

## 6. Conclusion

This chapter presents the real possibilities of combinations, between physical experimental techniques with computational tools which can contribute significantly to the advancement of the proposal of new drugs within the point of view of molecular action. Moreover, it has been demonstrated how to carry out such work,

Molecular Mechanism of Flavonoids Using Fluorescence Spectroscopy and Computational Tools DOI: http://dx.doi.org/10.5772/intechopen.84480

combining the knowledge between fluorescence spectroscopy and molecular docking. These techniques may be added to pharmacology, pharmacokinetics, and pharmacognosy research fields, which represent the cradle of the search for new medicinal products and design.
