Study on Interaction between Broad Bean Phenolic Compounds and Proteins using Fluorescence Method

Protein-polyphenol association may affect polyphenols absorption/bioavailability from gastrointestinal tract and finally modulate their biological activities. Fluorescence spectroscopy has been widely used to investigate the interactions of drugs and proteins. Measuring of quenching of intrinsic fluorescence of proteins can be also applied to study binding affinity of phenolic compounds to proteins. The aim of our study was to investigate interactions between phenolic compounds and proteins (isolated from broad bean seeds) using fluorescence quenching method. Phenolic compounds were extracted from broad bean coats and separated chromatographically into low molecular weight (LMW) fraction and tannins. Total phenolic contents of low molecular weight and tannin fractions were 203.6 and 740.5 mg of catechin equivalents per mg, respectively. Protocatechuic, gallic, p-hydroxybenzoic, p-coumaric acids and epicatechin were identified as predominant constituents of low molecular weight fraction using RP-HPLC method. Column chromatography on Sephadex G-200 gel was used for purification of 11S globulins from broad bean seeds. Fluorometric titration was applied to measure quenching of 11S globulin fluorescence by phenolic compounds. Titration curves depict dose dependent decrease of fluorescence intensity of protein both for LMW fraction and tannins. However, tannin fraction was more effective quencher of protein fluorescence than low molecular weight fraction. Emission spectra of 11S globulin at λex 282 nm showing quenching effect of phenolic compounds were recorded. Additionally, binding constants were calculated to characterise binding affinity of p-hydroxybenzoic acid and epicatechin to 11S protein using Stern-Volmer plot. The results were corrected both for inner filter effect and phenolic compounds intrinsic fluorescence.