pH Behavior of Biological Hydroxyapatite Surfaces, Apparent pK Values, and the Effect of Fluoride

Objectives: Recent chemical force titrations (Zhang et al. Analyt. Chem. 2000) using phosphate functionalised AFM cantilever tips revealed an apparent pK for phosphate at the surface of self assembled monolayers (SAMs) of 4.5 compared with 7.2 in solution. Since the modulation of growth and dissolution of phosphate rich biological apatite crystals depends on surface interactions with proteins, hydroxyapatite crystals from dental enamel were similarly investigated using carboxyl functionalised AFM tips. Methods: Apatite crystals were isolated from developing rat enamel and deproteinated by extraction in phosphate buffer pH 7 followed by 4M urea. The binding force between carboxylated tips and crystals was measured as a function of pH. Results: Above pH 9 little binding occurred due to mutual repulsion of anionic charges on tip and crystal. As pH decreased, binding increased, probably reflecting greater hydrogen bonding between, protonated carboxyl tip (pK 8) and protonated crystal surfaces peaking at about pH 6.5, possibly the apparent pK for surface phosphate. Below 6.5, binding became erratic, probably due to attachment of ions to the tip and their removal from the surface consistent with dissolution of the apatite. Exposure of crystals to fluoride before and during titrations shifted this peak down to about pH 5.5 i.e. fluoride rendered protonation more difficult. Conclusions: Maximum hydrogen bonding occurred between apatitic surfaces and fully protonated carboxyl SAMs at pH ~6.5, lower than solution phosphate pK 7.2. The fluoride induced shift down to ~5.5 may be partly due to pH and fluoride effects on the Helmholz double layer. It also likely reflects more stable binding in the crystal surface of counter cations, to phosphate, i.e. sodium or calcium, rendering their replacement by protons more difficult. In both cases hydrogen bonding of e.g. proteins to such surfaces may be greatest at pH values approaching 6.5. Supported,MRC.UK. Grant. G990 1098