Methods: Whole salivary proteins from human volunteers were adsorbed to synthetic hydroxyapatite powder, human enamel powder and actual human enamel surfaces and challenged with 10mM acetic acid. Demineralisation was determined by spectrophotometric assay of phosphate released into the acid. Adsorbed proteins were characterised by SDS-PAGE. Salivary proteins were fractionated by size exclusion and/or hydroxyapatite chromatography and the protective properties of the fractions investigated as described above.
Results: Whole salivary proteins offered no significant protection when adsorbed to synthetic hydroxyapatite powder but reduced demineralisation by ~12% (p<0.01) when adsorbed to human enamel powder. Even greater protection was achieved using human enamel surfaces with a ~43% reduction in demineralisation (p<0.0001) but repeated acid challenges gradually desorbed the protective proteins which corresponded to a loss of protection. Hydroxyapatite chromatography of whole saliva revealed that a subset of proteins comprising ~15% of the total protein were protective. Further fractionation of this subset is underway to identify the specific protective species. Treatment with chaotrophic agents abolishes the protective effects suggesting protection is dependent on protein conformation or complex formation.
Conclusions: The results emphasise the importance of using natural enamel surfaces when investigating the protective effect of salivary proteins. The protective species in saliva comprise a minor subset of the total proteins that are protective in their native conformation. A better understanding of how salivary proteins protect against demineralisation will aid the design of therapeutic saliva substitutes and potentially allow for caries/erosion susceptible individuals to be identified based on unfavourable polymorphisms of their salivary proteome.