Conversion of proline to hydroxyproline and its incorporation into collagen.

Most, if not all, of the hydroxyproline in collagen appears to be derived from proline (1, 2). It has been proposed by several investigators (2-7), on indirect evidence, that it is a bound form of proline which is hydroxylated and incorporated into collagen. Recent data (8-11) indicate an analogous situation in the hydroxylation of lysine and its incorporation into collagen. The type of compounds which could possibly serve as substrates for the formation of bound hydroxyproline might be (a) those that contain within the same molecule proline destined for hydroxylation as well as the proline destined for collagen-proline, (b) those that contain within the same molecule either proline destined for collagen-hydroxyproline or proline destined for collagen-proline, but not both concurrently. Examples of the first type might be (al) a precollagen macromolecule with an excess of proline and lacking hydrosyproline, or (~2) a single completed peptide strand of tropocollagen or subunit thereof with an excess of proline and lacking hydrosyproline. Within our present understanding of protein synthesis, examples of the second might be (bl) prolyl adenylate, or (&) prolyl soluble ribonucleic acid. To gain more information about the hydroxylation of proline, the specific activities of proline and hydroxyproline from “soluble collagen” (extracted in cold 0.45 M sodium chloride (12)) were compared during a 24-hour period after administration of uniformly labeled L-proline-Cl4 to guinea pigs. Variables such as the reproducibility of individual specific activity determinations, purity of collagen samples, and adsorption of free labeled proline by isolated collagen were evaluated. The results favor hypothesis (a*) as the bound form of proline which is hydroxylated.