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Histidinoalanine is a naturally occurring cross-linking amino acid found in mammalian bone, dentin, cartilage and aorta, and also in the extracellular fluid of bivalve clams. The exact components from which this amino acid derives have not been characterized in the case of bone. In this study, the origin of histidinoalanine in bone was investigated in order to clarify the possible role of the matrix component containing this amino acid in bone mineralization. An extract of bovine bone powder with 0.5 M EDTA/1M NaCl was desalted and separated by calcium-induced precipitation. The supernatant fraction was chromatographed successively on Sepharose CL-6B, hydroxyapatite, DEAE cellulose, and reverse-phase high-performance liquid chromatography columns. The fraction with the highest affinity for hydroxyapatite was found to contain the highest concentration of histidinoalanine. A phosphoprotein with a molecular weight of about 24 K that contained 1.2 mol of histidinoalanine per molecule was isolated. The amino acid composition of this fraction showed a high content of acidic amino acids and serine, at least 42% of which was phosphorylated. These results suggested a possible formation mechanism of histidinoalanine that includes the beta-elimination of phosphoserine, producing a dehydroalanine residue and an addition of histidine on it. It was concluded that this low molecular weight phosphoprotein is one of the major sources of histidinoalanine in bovine bone.

Trypsin digestion and successive calcium-induced precipitation of the insoluble bovine dentin matrix effectively separated the collagen and phosphoprotein fractions which were firmly associated together in this material. Amino acid analysis by four different systems revealed that the lysinoalanine and histidinoalanine, which had been previously reported to occur in the human dentin collagen, were concentrated in the phosphoprotein fraction but were not present in the collagen fraction. Furthermore, it was found that the free-type phosphoprotein which was isolated from EDTA extract of dentin powder also contained both "cross-linking" amino acids. The results indicated the both "cross-links" distributed within the dentin phosphoprotein and were not likely to contribute the unique stability of dentin collagen.