Characterisation of human salivary proteins inhibiting the acid demineralisation of powdered hydroxyapatite substrates and natural enamel surfaces

The adsorption of salivary proteins to enamel is critical for the protection against acid demineralisation but it is unclear which proteins are involved in protection or the mechanism by which they act. The aim was to investigate salivary protein adsorption and protection using powdered synthetic hydroxyapatite, powdered enamel and natural enamel as substrates and to characterise the specific salivary proteins providing protection. Human whole salivary proteins and its purified fractions were adsorbed to the various substrates and challenged with acid. Adsorbed proteins were characterised by SDS-PAGE and their protective efficacy determined by spectrophotometric assay of phosphate released into the acid during dissolution. A significantly higher degree of protection was provided by salivary proteins when adsorbed to natural enamel surfaces. It was found that the protective species in saliva reside in a small subset comprising 15% of the total protein and efficacy is dependent on conformation. The desorption of salivary proteins during an acid challenge correlated with loss of protection and it was hypothesised that the proteins desorbed during the challenge may be the protective species. Protein S100-A8 and S100 were identified as members of these putative protective proteins. Many proteins (including protein S100-A8 and S100) remained adsorbed to enamel even after 19 acid challenges but they were not protective. Size exclusion chromatography under non-denaturing conditions resulted in co-elution of two proteins identified as α-amylase and cystatin. Their adsorption
behaviour was dependent on the actual substrate used. This thesis demonstrates that the use of powdered substrates may not be ideal substitutes for natural enamel surfaces when investigating salivary protein adsorption and the effect of salivary proteins on enamel demineralisation. This is an important consideration as correctly identifying the protective species and understanding the mechanism of protection will inform the development of prophylactic/therapeutic peptides for clinical use in dry mouth cases.