The binding of salivary proteins and its effect on the demineralization of hydroxyapatite

Abstract

Histatins, statherin and proline-rich proteins (PRPs) are important members of the human saliva proteome. They are components of the acquired enamel pellicle (AEP) which provides protection to the teeth and perform other vital functions in the oral cavity such as preventing spontaneous precipitation of calcium phosphate salts (statherin) and exhibiting fungicidal activity (histatins). The aims of the study were to (1) isolate histatins, statherin and acidic PRPs from parotid secretion, (2) develop a novel functional assay for the measurement of hydroxyapatite demineralization by modifying pre-existing methods and (3) test the binding of mixed PS proteins and purified salivary proteins (histatin 1, statherin and PRP1) to HA and the subsequent effect on demineralization.

The steps in the histatin, statherin and PRP purification process include zinc precipitation of parotid saliva, ion exchange chromatography, gel filtration chromatography and high performance liquid chromatography (HPLC). Protein purity was assessed by polyacrylamide gel electrophoresis (PAGE). The functional properties of the proteins were examined in two ways: Binding of mixed and purified proteins to ceramic hydroxyapatite (HA) powder was monitored by PAGE and HPLC, and demineralization of HA exposed to acidic conditions after incubation with mixed and purified proteins was determined in spectrophotometric assays for free calcium and phosphate. Histatins and PRPs were successfully isolated to high levels of purity from human parotid saliva (HPS). Salivary protein binding experiments showed that the order of the three proteins studied in terms of their apparent affinity for HA is: statherin > histatin 1 > PRP1. The linearized Langmuir adsorption plots for histatin 1 and statherin fit well, showing R2 values of 0.9880 and 0.9975, respectively. However, the adsorption isotherm for PRP1 did not exhibit linear characteristics (R2 value of 0.61). Functional assays performed to measure the release of calcium and phosphate ions showed variable levels of protection afforded to HA by preincubation with the three proteins. On a molar basis, histatin 1 provided the most protection against demineralization. The in vitro data generated add to a more complete understanding of the functional characteristics of histatins, statherin and PRP1 and thereby provide insights into their potential capacity to protect enamel in the oral environment.