Interaction of colon-derived polyphenol metabolites with bovine serum albumin

Following ingestion, polyphenols entering the large intestine undergo extensive metabolism by colonic microbiota and metabolites of the parent compounds enter the circulatory system. It is of limited relevance attempting to unravel the mechanism of action of intact parent polyphenols. Their colonic metabolites are more plausible candidates for investigation of putative biological activity. The aim of this work was investigating the interaction between BSA and polyphenol metabolites using tryptophan fluorescence quenching. Fluorescence spectra of BSA were recorded before and after the addition of selected metabolites, under physiological conditions (37°C and pH 7.4), in the range of 300-550 nm at an excitation wavelength of 294 nm. Calculation of KSV demonstrated that, for all the tested metabolites (with the exception of 3-hydroxyphenylacetic and 3,4-dihydroxyphenylacetic acids), the type of quenching was static, suggesting the formation of a complex between the molecules and the fluorophore (tryptophan). Urolithins had the lowest KD value and bound BSA with higher affinity with respect to the other tested compounds. The binding stoichiometry was 1:1 for all the tested metabolites, suggesting the existence of a single binding site in the protein. Competitive binding experiments with quercetin showed a displacement of urolithins from the complex, demonstrating that site I (the hydrophobic binding pocket located in the subdomain IIA of BSA, which is known to bind a variety of ligands) is likely to be the preferred binding site for these molecules. The affinity of BSA for urolithins lets hypothesise that these phenolic metabolites could circulate in vivo as protected by plasma proteins. In the presence of solid tumors there is a reduction in tumor venous blood pH below 7, as a consequence of lactate accumulation. This acidic environment may cause the release of urolithins due to pH-induced decreased affinity, and these phenolic metabolites can exert local anticancer effects.