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

Abstract

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 (Kb) 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 Kb values were much smaller, on the order of 103 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.