Enzymes Covalently Bound on Collagen Membranes Immobilization of Blood Clotting Factor XIII

  • Danièle C. Gautheron
  • Bruno G. Blanchy
  • Pierre R. Coulet
Part of the Advances in Experimental Medicine and Biology book series (AEMB, volume 238)


Ever since 1958 I have specialized in the field of mitochondrial research: metabolism, structure of inner mitochondrial membrane, ATP synthase. Therefore one can wonder why I started binding enzymes to collagen membranes and why we specially immobilized Factor XIII in this type of thin membranes, since we had no competence concerning blood clotting in the Laboratory. In fact, in a first approach, with J.H. Julliard, hand-made ultra-thin films of collagen were prepared in the Laboratory and used for the covalent binding of glutamate dehydrogenase by an azide method to make a simple model of interactions of glutamate dehydrogenase with inner mitochondrial membrane1. Glutamate dehydrogenase is an abundant mitochondrial enzyme that strongly interacts specifically with the inner face of the inner mitochondrial membrane, therefore from the matrix side. If the kinetic parameters of the enzyme were somewhat modified, however, the bound glutamate dehydrogenase kept its cooperative properties and regulations by the effectors ADP and GTP. This meant that the bound enzyme had kept its specific oligomeric structure and organization. It was such a success that we decided to develop the system.


Glutamate Dehydrogenase Covalent Binding Factor Xiii Adenylate Kinase Collagen Membrane 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. 1.
    J.H. Julliard, G. Godinot and D.C. Gautheron, Some modifications of the kinetic properties of bovine liver glutamate dehydrogenase covalently bound to a solid matrix of collagen, FEBS Lett, 14: 185–188 (1971).CrossRefGoogle Scholar
  2. 2.
    G.J. Pichon and B.A. Piat, Fr. Pat., 1, 596, 789 (1968).Google Scholar
  3. 3.
    G.J. Pichon and B.A. Piat, Fr. Pat., 1, 596, 790 (1968).Google Scholar
  4. 4.
    P.R. Coulet, J.H. Julliard and D.C. Gautheron, Support de protéines actives greffées, Fr. Pat., 2, 235, 133 (1973).Google Scholar
  5. 5.
    P.R. Coulet, J.H. Julliard and D.C. Gautheron, A mild method of general use for covalent coupling of enzymes to chemically activated collagen films, Biotechnol. Bioeng., 16, 1055–1068 (1974).CrossRefGoogle Scholar
  6. 6.
    B. Blanchy, C. Godinot and D.C. Gautheron, Reconstitution of ATP synthetase on a Collagen membrane that can synthetize ATP, using a pH gradient, Methods of Enzymology, 55, 742–748 (1979).CrossRefGoogle Scholar
  7. 7.
    P.R. Coulet and D.C. Gautheron, Enzyme anchoring on chemically activated collagen membranes, in: Analysis and Control of immobilized enzyme systems, D. Thomas and J.P. Kernevez ed., North Holland, p. 165–177, Amsterdam, (1976).Google Scholar
  8. 8.
    F. Paul, P.R. Coulet and D.C. Gautheron, Kinetics of collagen-bound sorbitol dehydrogenase in a Rotating-membrane reactor: opposite variations of affinity constants under diffusional limitations, Biotechnol. Bioeng., 20, 1785–1786 (1978).CrossRefGoogle Scholar
  9. 9.
    K.H.K. Lee, P.R. Coulet and D.C. Gautheron, Grafting of enzymes on collagen films using Woodward1 s reagent “K” and a water-soluble carbodiimide derivative (EDC), Biochimie, 58, 489–497 (1976).Google Scholar
  10. 10.
    L.J. Blum, P.R. Coulet and D.C. Gautheron, Collagen strip with immobilized luciferase for ATP bioluminescent determination, Biotechnol. Bioeng., 27, 232–237 (1985).CrossRefGoogle Scholar
  11. 11.
    L.J. Blum and P.R. Goulet, Bio luminescent determination of NAD(P) with immobilized bacterial luciferase and FMN oxidoreductase on collagen films, Anal. Chim. Acta, 161, 355–358 (1984).CrossRefGoogle Scholar
  12. 12.
    L.C. Clark Jr and G. Lyons, Electrode systems for continuous monitoring in cardiovascular surgery, Ann. N.Y. Acad. Sci., 102, 29–45 (1962).CrossRefGoogle Scholar
  13. 13.
    P.R. Coulet, J.J. Fombon and D.C. Gautheron, Etude, conception, réalisation d’électrodes à enzymes greffés sur films fins de collagène pour la microdétermination de glucose et d’urée, Action concertée Chim. Anal.D.G.R.S.T., 76–7–0919 et 76–7 – 0920 (1977).Google Scholar
  14. 14.
    D.R. Thévenot, R. Sternberg, P.R. Coulet, J. Laurent and D.C. Gautheron, Enzyme collagen membrane for electrochemical determination of glucose, Anal. Chem., 51, 96–100 (1979).CrossRefGoogle Scholar
  15. 15.
    C. Bertrand, P.R. Coulet and D.C. Gautheron, Multipurpose electrode with different enzyme system bound to collagen films, Anal. Chim. Acta, 126, 23–34 (1981).CrossRefGoogle Scholar
  16. 16.
    C. Bertrand, P.R. Coulet and D.C Gautheron, Enzyme electrode with collagen-immobilized cholesterol oxidase for the microdetermination of free cholesterol, Anal. Lett., 12, 1477–1488 (1979).CrossRefGoogle Scholar
  17. 17.
    G. Bardeletti, F. Séchaud and P.R. Coulet, A reliable L-lactate electrode with a new enzyme membrane for amperometric assay of lactate, Anal. Chim. Acta, 187, 47–54 (1986).CrossRefGoogle Scholar
  18. 18.
    D.C. Gautheron and P.R. Coulet, Electrodes enzymatiques, 9th Journées Nationales de Biologie, Lyon-Grenoble 15–16 Janv. 1982, in Thèmes d’Actualités Biologiques, M. Vernet, J. Croize, S. Guibaud, Y. Minaire, J. Pichot ed, p. 192–196, MEDIPHARLY, LYON (1982).Google Scholar
  19. 19.
    J.M. Brillouet, P.R. Coulet and D.C. Gautheron, Chemically activated collagen membranes for amyloglucosidase attachment. Use in a helicoidal reactor, Biotechnol. Bioeng., 19, 125–142 (1977).CrossRefGoogle Scholar
  20. 20.
    J.M. Brillouet, P.R. Coulet and D.C. Gautheron, Thin-layer flow reactor with amyloglucosidase bound to collagen membranes, Biotechnol. Bioeng., 18, 1821–1825 (1976).CrossRefGoogle Scholar
  21. 21.
    M. Laville, R. El Habib, J. Traeger, P.R. Coulet, D.C. Gautheron, K. Sanhadji, H. My, P.Y. Zech, Extracorporeal removal of anti-DNA antibodies in systemic lupus erythematosus using DNA-collagen films, Xth ESAQ Congress, Bologna (1983).Google Scholar
  22. 22.
    R. El Habib, P.R. Coulet, K. Sanhadji, D.C. Gautheron, M. Laville and J. Traeger, DNA immobilized onto an acyl-azide derivative of collagen membranes for use as immunoadsorbent, Biotechnol. Bioeng., 26, 665–669 (1984).CrossRefGoogle Scholar
  23. 23.
    P.R. Coulet, C. Godinot and D.C. Gautheron, Surface-bound aspartate aminotransferase on collagen films. Compared properties with native enzyme, Biochim. Biophys. Acta, 391, 272–281 (1975).CrossRefGoogle Scholar
  24. 24.
    M. Arrio-Dupond, P.R. Coulet and D.C. Gautheron, Coupled reaction of immobilized aspartate aminotransferase and malate dehydrogenase. A plausible model for the cellular behavior of these enzymes, Biochim. Biophys. Acta, 829, 58–68 (1985).CrossRefGoogle Scholar
  25. 25.
    M. Arrio-Dupond and P.R. Coulet, Aspartate aminotransferase immobilized on collagen films. Activity of dissociated subunits, Biochem. Biophys. Res. Commun., 89, 345–353 (1979).CrossRefGoogle Scholar
  26. 26.
    J.M. Engasser, P.R. Coulet and D.C. Gautheron, Kinetics of soluble and collagen-bound aspartate aminotransferase: Diffusional effects with a two-substrate enzymatic reaction, J. Biol. Chem., 252, 7919–7922 (1977).PubMedGoogle Scholar
  27. 27.
    P.R. Goulet, R. Sternberg and D.R. Thévenot, Electrochemical study of reactions at interfaces of glucose oxidase collagen membranes, Biochim. Biophys. Acta, 612, 317–327 (1980).CrossRefGoogle Scholar
  28. 28.
    B. Maïsterrena, G. Bardeletti and P.R. Coulet, Product distribution in diffusion-cell compartments separated by an immobilized enzyme membrane, Journal of Membrane Science, 22, 175–186 (1985).CrossRefGoogle Scholar
  29. 29.
    B. Maïsterrena, L.J. Blum, G. Bardeletti and P.R. Coulet, Vectorial product concentration obtained with a permeable immobilized enzyme membrane. A new approach to the analysis of biological transport systems, Biochemical J., 235, 693–698 (1986).CrossRefGoogle Scholar
  30. 30.
    B. Maïsterrena, L.J. Blum and P.R. Coulet, Theoretical analysis of a translocation-like model with saturable kinetics, Biochemical J., 242, 835–839 (1987).CrossRefGoogle Scholar
  31. 31.
    B. Blanchy, C. Godinot and D.C. Gautheron, Reconstitution of ATP-synthetase on a collagen membrane that can synthesize ATP using a pH gradient, Methods Enzymol., 50, 742–748 (1979).CrossRefGoogle Scholar
  32. 32.
    C. Marion, B. Roux, L. Pallota and P.R. Coulet, Study of chromatin oraganization with trypsin immobilized on collagen membranes, Biochem. Biophys. Res. Commun., 114, 1169–1175 (1983).CrossRefGoogle Scholar
  33. 33.
    B.G. Blanchy, P.R. Coulet and D.C. Gautheron, Immobilization of Factor XIII on collagen membranes, J. Biomed. Materials Res., 20, 469–479 (1986).CrossRefGoogle Scholar
  34. 34.
    L. Lorand and K. Konishi, Activation of the fibrin stabilizing factor (FSF) of plasma by thrombin, Arch. Biochem. Biophys., 105, 58–67 (1964).CrossRefGoogle Scholar
  35. 35.
    M. Matsuda, Wound healing and Factor XIII, Acta Haem. Jap., 40, 995–1002 (1977).Google Scholar
  36. 36.
    J. Benfer and H. Struck, Factor XIII and Fracture healing, Eur. Surg. Res., 9, 217–223 (1977).CrossRefGoogle Scholar
  37. 37.
    E. Beck, F. Duckert and M. Ernst, The influence of fibrin stabilizing factor on the growth of fibroblasts in vitro and wound-healing, Thromb. Diath. Haemorrh., 6, 485–491 (1961).CrossRefGoogle Scholar
  38. 38.
    A. Soria, C. Soria and C. Boulard, Fibrin stabilizing factor (Factor XIII) and collagen polymerization, Experientia, 31, 1335–1357 (1975).CrossRefGoogle Scholar
  39. 39.
    D.F. Mosher, P.E. Schad and H.K. Kleinman, Cross-linking of fibronectin to collagen by blood coagulation Factor XIIIa, J. Clin. Invest., 64, 781–787 (1979).CrossRefGoogle Scholar
  40. 40.
    H.K. Kleinman, C.M. Wilkes and G.R. Martin, Interaction of fibronectin with collagen fibrils, Biochemistry, 20, 2325–2330 (1981).CrossRefGoogle Scholar
  41. 41.
    T. Tamaki and N. Aoki, Cross-linking of α2-plasmin inhibitor and fibronectin to fibrin by fibrin-stabilizing factor, Biochim. Biophys. Acta, 661, 280–286 (1981).CrossRefGoogle Scholar
  42. 42.
    H.E. Karges and N. Heimburger, A rapid screening and quantitative assay of clotting Factor XIII, Behringwerke A.G., Laboratoriums blatter, 28, 71–78 (1978).Google Scholar
  43. 43.
    L. Lorand, O.M. Lockridge, L.K. Campbell, R. Myhrman and J. Bruner-Lorand, Transamidating enzymes. II. A continuous fluorescent method suited for automating measurements of Factor XIII in plasma, Anal. Biochem., 44, 221–231 (1971).CrossRefGoogle Scholar
  44. 44.
    G.R. Kepner and R.I. Macey, Membrane enzyme systems, molecular size determinations by radiation inactivation, Biochim. Biophys. Acta, 163, 188–203 (1968).CrossRefGoogle Scholar

Copyright information

© Plenum Press, New York 1988

Authors and Affiliations

  • Danièle C. Gautheron
    • 1
  • Bruno G. Blanchy
    • 1
  • Pierre R. Coulet
    • 1
  1. 1.Laboratoire de Biologie et Technologie des MembranesCNRS — Université Claude Bernard de LyonVilleurbanne CedexFrance

Personalised recommendations