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Salivary Proteins Interact with Dietary Constituents to Modulate Tooth Staining

Salivary Research Unit, King’s College London, Floor 17, Guy’s Tower, London SE1 9RT, UK; and
¹ GlaxoSmithKline R & D, St George’s Avenue, Weybridge, Surrey KT13 ODE, UK;

View larger version (51K): [in a new window]   Figure 1. SDS-PAGE of parotid salivary proteins in 16% tricine electrophoresis gels (Invitrogen, Paisley, UK). Resolved proteins were stained (with 0.2% Coomassie Brilliant Blue R250 in 25% methanol, 10% acetic acid) in gels or transferred onto nitrocellulose for antibody binding (see online Appendix). (A) Interaction of parotid salivary proteins (S) with tea (T). Increasing amounts of tea (1, 10, and 50 µL) were added to saliva (50 µL), and interacting salivary proteins were depleted from the electrophoretic profile. Ten µL of sample (equivalent to approximately 25 µg of salivary protein) were added to sample wells. Arrows indicate some protein bands showing a noticeable depletion. Preliminary identities, assigned on the basis of electrophoretic mobility and staining characteristics, are proline-rich protein (A) and histatin (B). The mobilities of standard proteins (M), with relative molecular weights ranging from 210 down to 3 kDa, are shown. (B) The salivary proteins binding to hydroxyapatite (HA) from a sample of parotid saliva (S) under assay conditions (1 hr at 37°C). The presence of statherin binding to hydroxyapatite is shown by a Western blot (W) of the proteins eluted from hydroxyapatite, probed with an anti-statherin antibody. (C) Histatin (Hp) and proline-rich protein (Pp) enriched fractions from saliva and the profiles of enriched proteins binding to hydroxyapatite under assay conditions. For comparison with the enriched histatin fraction, a sample of synthetic histatin 5 (Hs Sigma Chem. Co. Ltd.) is shown.

View larger version (14K): [in a new window]   Figure 2. Folin phenol assay of epigallocatechin (egc). (A) A standard curve of increasing amounts of epigallocatechin up to 25 µg in solution. (B) Increasing amounts of epigallocatechin (up to 25 µg) were incubated with 5 mg of hydroxyapatite, and the bound epigallocatechin was assayed. Approximately 0.25 µg of epigallocatechin bound to hydroxyapatite from an aqueous solution containing 50 µg of epigallocatechin. Values are means ± standard deviation of 6 assays.

View larger version (24K): [in a new window]   Figure 3. Anthocyanin assay. (A) Standard curve of increasing amounts of anthocyanin in solution measured in the presence of 1 M HCl at 525 nm. Mean ± SD of 5 assays. Eight µg of anthocyanin gave an absorbance reading of approximately 1.00, and therefore this amount was used in the hydroxyapatite binding assay (Fig. 3B). (B) Anthocyanin (AN) and parotid saliva (S) sequentially or simultaneously bound to hydroxyapatite. Mean ± SD of 6 assays. Saliva first, then anthocyanin sequentially (S/AN); anthocyanin first, then saliva sequentially (AN/S); saliva and anthocyanin simultaneously (S+AN). Background absorbance due to saliva has been subtracted from the results. aS/AN and S+AN were statistically significantly greater than anthocyanin alone, p < 0.05 by paired t test. (C,D) Anthocyanin and salivary fractions sequentially or simultaneously bound to hydroxyapatite. Total amount of histatin (H) fraction in solution added to hydroxyapatite was 0.42 µg or 1.7 µg. Proline-rich protein (P) fraction in solution added to hydroxyapatite was 25 µg or 100 µg. Total amount of anthocyanin added to hydroxyapatite was 8 µg. Histatin first, then anthocyanin sequentially (H/AN); histatin and anthocyanin simultaneously (H+AN); anthocyanin only (AN). Proline-rich proteins first, then anthocyanin sequentially (P/AN); proline-rich proteins and anthocyanin simultaneously (P+AN); anthocyanin only (AN). Mean ± SD of 6 assays. Background absorbance due to histatins and proline-rich proteins has been subtracted from the results. Binding of anthocyanin was statistically different by ANOVA (p < 0.05) in the presence of histatins or proline-rich proteins. bAt 1.7 µg protein, H/AN and H+AN were statistically significantly lower than anthocyanin alone (p < 0.05, paired t test). aP/AN and P+AN were statistically significantly greater than anthocyanin alone (p < 0.05, paired t test).

View larger version (11K): [in a new window]   Figure 4. Assay of black tea. (A) Standard curve with increasing amounts of black tea in solution measured in the presence of 1 M HCl at 415 nm. Mean ± SD of 5 assays. The amount of black tea polyphenols is unknown, but the figures shown are based on the volume of tea stock solution (2 g in 100 mL water) and published figures suggesting 1 mg polyphenol per mL tea (Bravo, 1999). (B) Assay of black tea and salivary protein added sequentially to hydroxyapatite. Total amounts of protein added were: parotid saliva only (S), 400 µg; histatin (H) fraction, 1.7 µg; and proline-rich protein (P) fraction, 100 µg. Mean ± SD of 3 assays. Saliva first, then tea sequentially (S/T); histatin first, then tea sequentially (H/T); proline-rich proteins first, then tea sequentially (P/T). aS/T and P/T were statistically significantly greater than black tea alone (p < 0.05, paired t test).