Development of a Competition Assay for the Evaluation of the Binding of Human Parotid Salivary Proteins to Dietary Complex Phenols and Tannins Using a Peroxidase-Labeled Tannin

Abstract

Proline-rich proteins (PRP) are secreted by herbivores, including humans, in their parotid saliva into the oral cavity. These PRP have a high affinity for dietary complex phenols and tannins (CPT). The stable complexes formed may then pass through the digestive tract and are excreted. It is postulated that such complexes may modulate both the antinutritional effects and beneficial properties of CPTs. A novel competitive tannin−salivary protein binding assay is described that allows the detailed investigation of binding of tannins to salivary proteins. The assay is based on the competition between an enzyme-labeled tannin [(−)-epigallocatechin conjugated to horseradish peroxidase via a linker molecule] and a test tannin to bind to parotid salivary protein immobilized in the wells of a polystyrene microtiter plate. The binding affinities of a series of flavan-3-ol monomers from tea and a commercial tannic acid preparation were determined. Major differences in the binding affinities of the compounds tested to the salivary proteins were observed. A relationship between the level of tannin galloylation and the affinity of binding to parotid salivary protein is demonstrated. Keywords: Proline-rich proteins; parotid saliva; polyphenol complexation; tannin; flavan-3-ol