Determination of driving forces for bovine serum albumin-Ponceau4R binding using surface plasmon resonance and fluorescence spectroscopy: A comparative study.

Ponceau 4R (P4R) and bovine serum albumin (BSA) may interact changing food properties. We compared fluorescence spectroscopy and surface plasmon resonance (SPR) for studying, in vitro , the interactions between BSA and P4R at pH 7.4 and 3.5 in different temperatures. Fluorescence data pointed to the formation of a complex where P4R was bound on site I or II of BSA, with a stoichiometry around one and a binding constant (K b ) ranging from 1.37 × 10 5 to 20.15 × 10 6 L mol −1 . The complex formation at both pH was enthalpically driven (standard enthalpy change, ΔH° F = −60.69 and −63.06 kJ mol −1 , for pH 7.4 and 3.5, respectively). Using SPR, we also found the formation of 1:1 BSA-P4R complexes, but the calculated K b values were much smaller, on the order of 10 3 L mol −1 . Again, we found that the formation of BSA-P4R complex was driven by enthalpy decreasing; however the standard enthalpy change was less negative than that found by fluorescence (ΔH° SPR = −15.05 and −40.55 kJ mol −1 , at pH 7.4 and 3.5, respectively). Our results show that these distinct techniques provided different thermodynamic binding parameters for the BSA-P4R interaction, especially regarding ΔH° values, indicating that BSA-P4R binding was a multisite phenomenon, and that sites far from tryptophan residues were the main responsible by electrostatic interaction. Thus, this work clearly shows the importance of using complementary techniques for a complete thermodynamic characterization of complexes formed between azo-colorants and proteins; which is directly related to physicochemical properties of systems containing both molecules together. [ABSTRACT FROM AUTHOR]