In Vitro Enzymatic Hydroxylation of Prolyl Residues in the al&B2 Fragment of Rat Collagen*

Two 36-amino acid-containing peptides (al-CBZ), isolated from rat skin and tendon collagen by cleavage of the al chain with CNBr, were subjected to enzymatic hydroxylation with collagen proline hydroxylase. The peptides dithered initially only in that the ratio of hydroxyproline to proline was considerably lower in the tendon peptide, although the imino acid content of the two peptides was the same. In vitro hydroxylation produced a 75 % increase in the hydroxyproline content of the tendon peptide but only an 8% increase in hydroxyproline in the shin peptide. These findings provide direct confirmation of the suggestion, made in an earlier study, that intracellular factors other than the sequences of collagen chains in shin and tendon account for the differences in the degree of hydroxylation of susceptible prolyl residues. Intact collagen preparations from various sources were similarly shown to be underhydroxylated. The distribution of newly formed hydroxyproline in (rl-CB2 was investigated by analysis of chymotryptic fragments. Hydroxylation was largely limited to one of these fragments as predicted if hydroxylation occurred only at position 3 of the collagen triplet Gly-X-Y, i.e. at prolyl residues followed by glycine. This finding is in agreement with sequence analyses of vertebrate collagens and with enzymatic studies using synthetic peptides.