Nonocollagenous Proteins of Dentin. Isolation and Partial Characterization of Rat Dentin Proteins and Proteoglycans Using a Three-step Preparative Method

The purpose of this study was to develop a method for fractionation of dentin proteins and proteoglycans into pools. The sequential procedure consisted of: (1) addition of 1.0 M CaCl2 to solutions of EDTA extracts of rat dentin in the presence of protease inhibitors to form a CaCl2 precipitate (Fraction I), (2) dialysis of the resultant supernatant against 0.1 M formic acid to form an acid precipitate (Fraction II), and (3) passage of the 0.1 M formic acid supernatant over a Sephadex G-50 column to obtain a high molecular weight, excluded peak (Fraction III) and a lower molecular weight, included peak (Fraction IV). Each of the four fractions was characterized by amino acid analysis, slab gel electrophoresis and ion-exchange chromatography on DEAE-cellulose. Fraction I contained almost exclusively phosphoproteins while Fraction II consisted of several acidic proteins, albumin, proteoglycans and a protein with a relatively low level of organic phosphate. A unique glycoprotein with an apparent Mr = 95,000 was found in Fraction III along with smaller amounts of other proteins, including albumin and a phosphoprotein with a relatively low level of organic phosphate. Fraction IV contained several low molecular weight, gamma-carboxyglutamate-containing proteins similar to those found in bone. The data show that the method selectively fractionates the proteins and proteoglycans of rat dentin. Furthermore the method is rapid and allows preparative steps to be performed in the presence of protease inhibitors. This new procedure should be a useful step in the comprehensive isolation of dentin proteins in experiments designed to study their detailed chemical nature and metabolism.