Developmental changes in the pH of enamel fluid and its effects on matrix-resident proteinases

The objectives of this study were to measure pH in developing enamel at progressively older (more mature) stages of amelogenesis in vivo, and then to formulate synthetic enamel fluid mixtures that approximated these pH values for in vitro studies. The ultimate goal was to characterize the molecular weights of proteinases visualized by enzymograms incubated in synthetic enamel fluid using gelatin and casein as substrates. For most experiments, the proteinases were extracted en masse from small freeze-dried enamel strips directly into a non-reducing sample preparation buffer. In some experiments, we pre-treated the enamel strips with acetic acid to determine if this common method for demineralization and protein extraction caused any changes in the activity levels of the enamel proteinases. In other experiments, we first soaked enamel strips in synthetic enamel fluid to determine solubility of the proteinases within an aqueous phase. The results indicated that the pH of developing enamel remained fairly constant near pH 7.23 across the secretory stage, but it was generally more acidic (6.93) and fluctuated in focal areas between mildly acidic (6.2-6.8) and near-neutral (7.2) conditions across the maturation stage. The pH then slowly rose to near 7.35 when the enamel was almost mature (hard). The acidic conditions were generally inhibitory to most enamel proteinases, but there were some caseinase activities in mid-maturation-stage enamel near 23-30 kDa which appeared to be activated by weakly acidic conditions (pH 6.28). Pre-treatment of enamel samples with 0.5 M acetic acid markedly altered the overall profile of enamel proteinases, causing activation of some latent proteinase activities and permanent inhibition of other activities. Most proteinases in whole homogenates were insoluble in synthetic enamel fluid. This suggests that they may be tightly bound, directly or indirectly, to matrix proteins or mineral components in situ.