Age-related de-phosphorylation of proteins in dentin: a biological tool for assessment of protein age

Mature tooth dentin has essentially no metabolic activity, and thus post-tr anslational modifications accumulate with aging in this tissue. In the pres ent paper, we have studied age-related covalent changes: of human dentin pr oteins. Dentin phosphoproteins (PP) were extracted and purified using ion e xchange chromatography. Collagen was purified by CNBr cleavage and acetic a cid extraction. The amino acid composition of the resultant protein prepara tions was determined by HPLC after post-column derivatization. Likewise the extent of aspartic acid (Asp) racemization was determined in total dentin, dentin collagen and PP. Collagen only displayed small, insignificant chang es in amino acid composition and racemization with age. In contrast, PP exh ibited significant age-related changes in amino acid composition, cross-lin king and racemization. Thus the rate of Asp racemization in PP was 500-fold that found in collagen. Moreover, the phosphoserine (Ser(P)) content in hu man PP decreases dramatically with age, resulting in almost complete dephos phorylation over a lift: span. The loss of Ser(P) was accompanied by an inc reased content of the bifunctional cross-link histidinoalanine consistent w ith a B-elimination pathway. The relative Ser(P) content was highly correla ted with dentin age (r(2) = 0.96). The Ser(P) contents may thus potentially be applied in forensic investigations to deduce human age. The possible ro le of covalent modifications of the protein matrix in the degradation of mi neralized tissue and its implications fur the age-related decline of tissue functionality is discussed.