The distribution of aspartic acid residues in bovine dentine phosphoprotein.

Recently, Weiner and Hood (Science 190:987989, 1975) reported the use of an acid hydrolysis procedure in the study of the aspartic acid linkages of the soluble protein of the organic matrix of mollusk shells. Peptide bonds on both sides of aspartic acid residues have been shown to be particularly labile to hydrolysis by dilute acetic or hydrochloric acids (SCHULTZ et al, Biochemistry 1:694-698, 1962; LEACH, in Proc. Internat. Wool Textile Res. Conf., Pt I, CSIRO, Melbourne, Australia, p. C185, 1955). By following the release of aspartic acid and other amino acids, particularly serine and glycine, these workers were able to conclude that the repeating sequence (ASP-Y) is a common occurrence in mollusk shell proteins. This report deals with the application of this dilute acid hydrolysis to a very similar protein derived from bovine dentin matrix. Phosphoprotein was isolated from unerupted bovine molars by EDTA extraction and separation on hydroxyapatite columns (NAWROT et al, Biochemistry 15:3445-3449, 1976). This protein has been shown to contain 7% phosphorus with 34 of its amino acids as serine and aspartic acid. The liberation of aspartic acid and other amino acids by 0.25 M acetic acid in vacuo at 110 C was followed using the amino acid analyzer. The figure gives the results at various time intervals from 18-120 hours for the release of aspartic acid, serine, and all amino acids. At 120 hours, 47% of all possible amino acid residues were released. Aspartic acid and serine residues combined made up 90% of the residues which were released after 120 hours. Based on the aspartic acid content of the protein estimated by complete acid hydrolysis in HCl X 4.6 x 10-1 pmole'mg', the acetic acid released 80% of the total aspartic acid. Only small amounts of other amino acids (< 2 X 10-2 jzmolemg-1) were released during this time. In order to account for the release of serine and amino acids other than aspartic acid, approximately half of the total aspartic acid must occur in sequences of the type (ASP-Y)n, where