Failure to demonstrate a protein coat on enamel crystallites by morphological means

Phosphotungstic acid (PTA) treatment of section of Epon-embedded enamel dissolves the crystallites and stains material postulated to be crystal-bound proteins. Alternative, capillarity forces within the channels left after crystallite removal may draw in PTA. This prediction was tested on three systems. (1) Protein free synthetic hydroxyapatite was embedded in Epon; treatment of thin sections with PTA removed most crystals, leaving empty holes outlined by stain that could not represent protein. (2) Sections of rat incisor enamel were treated with PTA and then re-embedded in Epon and sectioned at 90° to the original plane. In these sections-of-section the cut ends of dissolved crystallite profiles were coated with stain. To determine if stained protein coats can be detected in the absence of the crystallite profiles, Epon sections were partially demineralized with formic acid, re-embedded in Epon and sections-of section were PTA treated. Previously extracted crystallites left no stained coats, and only the crystallites that were not removed by formic acid left PTA-stained outlines. (3) PTA-treated sections of dogfish shark enameloid were flooded with 5-nm colloidal gold particles and sections-of-section were prepared. The presence of gold particles on the section surface and in holes previously occupied by crystallites suggested that PTA solution could also be sucked into similar holes. It is concluded that PTA outlines are not crystal-bound proteins but artefacts caused by stain lining holes left in the section when the crystallites have been extracted.