MULTIPLE INTERACTIONS BETWEEN POLYPHENOLS AND A SALIVARY PROLINE-RICHPROTEIN REPEAT RESULT IN COMPLEXATION AND PRECIPITATION

Polyphenols (tannins) in the diet not only precipitate oral proteins, producing an astringent sensation, but also interact with dietary prot eins and digestive enzymes in the gut, resulting in a variety of antin utritive and toxic effects. Salivary proline-rich proteins (PRPs), whi ch are secreted into the oral cavity, form complexes with and precipit ate dietary polyphenols, and thus, they constitute the primary mammali an defense directed against ingested tannins. In order to characterize the interaction, NMR studies were performed which involved titrating a series of polyphenols into a synthetic 19-residue PRP fragment. The results show that the predominant mode of association is a hydrophobic stacking of the polyphenol ring against the pro-S face of proline and that the first proline residue of a Pro-Pro sequence is a particularl y favored binding site. Measurement of dissociation constants indicate s that the larger and more complex polyphenols interact more strongly with the PRP fragment; the order of binding affinity was determined as procyanidin dimer B-2 > pentagalloylglucose > trigalloylglucose >> pr oanthocyanidin monomer (-)epicatechin approximate to propyl gallate. S maller polyphenols can bind with one phenolic ring stacked against eac h proline residue, whereas larger polyphenols occupy two or three cons ecutive prolines. The more complex polyphenols interact with the PRP f ragment in a multidentate fashion; moreover, they self-associate or st ack when bound. Thus, a model is proposed in which multiple polyphenol /polyphenol and polyphenol/PRP interactions act cooperatively to achie ve precipitation.