Proteins in Enamel Tuft of Porcine Mature Teeth

Abstract

The purpose of this study was to determine the protein components of enamel tuft of porcine mature enamel and to investigate its formation processes. During demineralization of mature enamel with 0.5mol/l acetic acid, the insoluble structure, which includes the enamel tuft, along with the surface of the dentin was collected with tweezers. Since the collected enamel tuft still contained minerals, it was further demineralized with acetic acid and divided into soluble and insoluble samples. The freeze-dried samples were analyzed by SDS electrophoresis, mass spectrometry and Western blotting. From silver staining following electrophoresis, many protein bands in the enamel tuft were observed after demineralization with acetic acid. Mass spectrometry and Western blotting identified these bands to be mainly blood proteins (albumin, α-2-HS-glycoprotein and hemoglobin) and a small amount of enamel proteins (amelogenin, enamelin and sheath protein); degraded fragments of the blood proteins were also found. Therefore, the blood proteins reside in the enamel tuft itself, and do not contaminate the sample during preparation. The origin of albumin found in the enamel tuft was further investigated by examining several immature enamels using Western blotting. Albumin was only found in the highly mineralized enamel layer at the enamel-dentin junction, and not at the secretory, transition and maturation stages of the enamel and dentin. These results suggest that blood invades the enamel at the initiation stage, which corresponds to the highly mineralized enamel layer, and that the blood proteins cannot be degraded by enamel proteinases, allowing them to condense in the enamel tuft during crystallization.