Elsevier

Analytical Biochemistry

Volume 110, Issue 2, 15 January 1981, Pages 360-368
Analytical Biochemistry

Proteolytic enzymes as probes for the triple-helical conformation of procollagen

https://doi.org/10.1016/0003-2697(81)90204-9Get rights and content

Abstract

Type I procollagen was thermally denatured and partially refolded by cooling to 20°C. The partially refolded protein was then used as a model system for testing proteolytic enzymes as probes for quantitative assay of fully aligned triple-helical molecules. Pepsin and chymotrypsin both digested fully denatured procollagen. However, digestion times of greater than 60 min were required, even with a large molar excess of the proteinases. These enzymes therefore are only useful for examining the folding of procollagen under conditions in which the process occurs at a slow rate. In contrast, trypsin cleaved the collagen domain of denatured procollagen within 2 min. Trypsin did not efficiently remove the precursor specific peptides, and therefore a mixture of chymotrypsin and trypsin was employed as an appropriate proteolytic probe for triple-helical conformation.

References (34)

  • W. Traub et al.

    Advan. Prot. Chem

    (1971)
  • P. Bornstein et al.

    The Protein

    (1979)
  • P. Dehm et al.

    Biochim. Biophys. Acta

    (1972)
  • J. King et al.

    J. Mol. Biol

    (1971)
  • F.W. Studier

    J. Mol. Biol

    (1973)
  • S.A. Jimenez et al.

    J. Biol. Chem

    (1973)
  • J. Engel

    Arch. Biochem. Biophys

    (1962)
  • J. Uitto et al.

    Biochim. Biophys. Acta

    (1972)
  • S.A. Jimenez et al.

    Arch. Biochem. Biophys

    (1974)
  • J. Uitto et al.

    Biochem. Biophys. Res. Commun

    (1973)
  • W.W-Y. Kao et al.

    J. Biol. Chem

    (1977)
  • J. Vuust et al.

    J. Biol. Chem

    (1972)
  • A.G. Brownell et al.

    J. Biol. Chem

    (1976)
  • A.L. Rubin et al.

    Science

    (1963)
  • K. Kühn et al.

    Biochem. Zeitschr

    (1966)
  • D.L. Layman et al.
  • D.J. Prockop et al.
  • Cited by (262)

    • Collagen Gly missense mutations: Effect of residue identity on collagen structure and integrin binding

      2018, Journal of Structural Biology
      Citation Excerpt :

      The DSC melting profiles for G502A, G502S and G502V mutants also exhibited a second lower-stability thermal transition, consistent with the CD results (Fig. S1). The tightly packed triple helix confers resistance to general proteases (Bruckner and Prockop, 1981; Yu et al., 2014), and the recombinant constructs with Gly missense mutants were treated with trypsin to assess disruption of the native triple helix. After a 15-min digestion at 20 °C, the control protein VCL-Int remained resistant to trypsin digestion, as demonstrated by its intact size on SDS-PAGE (Fig. S2A).

    View all citing articles on Scopus
    View full text