Purification of (14C) protocollagen and its hydroxylation by prolyl-hydroxylase. Academic Article uri icon


  • Protocollagen, the unhydroxylated precursor form of collagen, was purified from matrix-free cells of embryonic chick tendon and examined for its ability to serve as a substrate for prolyl-hydroxylase. The cells were incubated in the presence of [14C]proline and 0.3 mM ,-dipyridyl, an inhibitor of prolyl-hydroxylase, and [14C]-protocollagen was purified by extraction in 0.1 N acetic acid, ammonium sulfate precipitation, and limited proteolytic digestion. The enzymic digestion with either pepsin or -chymotrypsin was carried out at 15 under conditions in which the NH2-terminal extensions on the [14C]protocollagen were removed and contaminating proteins were digested but the helical portion of the molecule remained intact. The enzyme-modified protocollagen was pure by polyacrylamide gel electrophoresis in sodium dodecyl sulfate and was comprised of polypeptide chains which were similar to the 1 and 2 chains of collagen except that they contained no hydroxylated proline or lysine. The enzyme-modified [14C]protocollagen served as a substrate for the synthesis of hydroxyproline by prolyl-hydroxylase at 25, 30, or 37 but not at 15 or 20. There was a sharp change in the ability of the enzyme-modified [14C]protocollagen to serve as a substrate for prolyl hydroxylase at about 28, a temperature close to the Tm for the thermal transition of enzyme-modified protocollagen. If the enzyme-modified protocollagen was first heat denatured, it was readily hydroxylated by prolyl hydroxylase at 15. The results demonstrated that hydroxylation of prolyl residues by prolyl-hydroxylase was dependent upon the structure of the prolyl-containing substrate. Since ultracentrifugation indicated that the substrate was not grossly aggregated at 15 and neutral pH, the results suggested that a helical conformation of the substrate in itself prevented its hydroxylation. The Km for the hydroxylation of the random-coil form of unmodified [14C]protocollagen at 37 was found to be 2 mM and the turnover number for prolyl-hydroxylase expressed on the basis of moles of hydroxy[14C]proline per mole of enzyme was 4 sec1. 1973, American Chemical Society. All rights reserved.

published proceedings

  • Biochemistry

author list (cited authors)

  • Berg, R. A., & Prockop, D. J.

citation count

  • 113

complete list of authors

  • Berg, RA||Prockop, DJ

publication date

  • August 1973