Note on Tooth Enamelins Immuno-Identification of Two Non-Amelogenin Proteins of Developing Bovine Enamel Isolated by Affinity Chromatography

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Abstract
Affinity chromatography of “Enamelin Extracts” of developing bovine molar enamel on CNBr activated Sepharose 4B to which polyclonal antibodies of whole bovine serum and fetuin were cross-linked, revealed that at most, only 1–2% of the proteins in the extracts were not bound to the columns. The approximately 98% or more of the proteins in such extracts were bound to the resin and were eluted in the position of the serum proteins and fetuin. The small amount of protein which was not bound to the affinity column and which was eluted very early, was subjected to SDS-PAGE and immunostained with polyclonal antibodies to two non-amelogenin proteins isolated and identified previously (∼26kDa and 22kDa). The antibody to the ∼50kDa protein immunostained strongly with only one protein band of ∼26kDa. The antibody to the ∼22kDa band reacted strongly with one protein band of ∼22kDa, weakly with an ∼100kDa and very weakly with several lower molecular weight bands, suggesting either aggregation and degredation of the 22kDa component, or more likely that the ∼22kDa and lower molecular weight proteins are all derived from the ∼100kDa component. There was no immuno-crossreactivity of the 22kDa and 26kDa antibodies and none of the components eluted in the early first fraction reacted with polyclonal antibodies to amelogenins or to amelogenin peptides. The analytical data confirms previous results from these and other laboratories that the proteins extracted from developing enamel, using the technique described, to solubilize a class of non-amelogenin proteins termed “enamelins,” actually consist almost wholly of albumin and other serum and salivary proteins and contain only very small amounts of several, non-amelogenin components.