Chemical Modification of Collagen and the Effects on Enzyme-Binding: Mechanistic Considerations

  • Jack R. Giacin
  • Seymour G. Gilbert
Part of the Advances in Experimental Medicine and Biology book series (AEMB, volume 86A)


The effect of structural modification on the enzyme-binding capacity of collagen has been studied using β-galactosidase (E. COLI K12 immobilized to collagen membrane by the impregnation procedure. The apparent steady-state activities of the resultant collagen-enzyme complexes were determined as a means of evaluating the enzyme-binding capacity of the modified collagen. in addition, the amount of enzymic protein bound to the collagen support was determined by the tryptophan content of the complex.

The tertiary structure of the collagen matrix was modified by cross-linking with the difunctional reagent, glutaraldehyde, and by aging in the dry state. Such structural modifications were found to markedly reduce the enzyme (β-galactosidase) binding capacity of collagen films. The enzyme-binding capacity of the cross-linked collagen membrane was completely restored by proteolytic enzyme treatment of the aged film but only partly so for the glutaraldehyde treated films. Proteolytic enzymes used to treat a dispersion of collagen microfibrils prior to casting into a membrane also resulted in an increase in enzyme-binding. The effect of structural modification of collagen on enzyme-binding and the locus of enzyme attachment are discussed.


Collagen Molecule Collagen Membrane Collagen Film Enzyme Binding Tryptophan Content 
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. Arnold, H., and Pette, D. (1968). Binding of Glycolytic Enzymes to Structure Proteins of the Muscle. European J. Biochem. 6:163.CrossRefGoogle Scholar
  2. Barndt, R. L., Leeder, S. G., Giacin, J. R. and Kleyn, D. H. (1975). Sanitation of a biocatalytic reactor used for the hydrolysis of acid whey. J. Food Sci. 40:291.CrossRefGoogle Scholar
  3. Bartenov, G. M. and Zuyev, Y. S. (1968). Strength and failure of viscoelastic materials. Pergamon Press, New York.Google Scholar
  4. Bernath, F. R. and Vieth, W. R. (1974). Collagen as a carrier for enzymes. Materials Science and Process Engineering Aspects of Enzyme Engineering.Google Scholar
  5. Bowes, J. H. and Kenton, R. H. (1949). The effect of deamination and esterification on the reactivity of collagen. J. Biochem. 44:142.Google Scholar
  6. Brandau, H. and Pette, D. (1966). Enzyme localization and enzyme activity, II. Localization of enzymes of energy metabolism in cross-striated muscle. Enzyml. Biol. Clin. 6:123.Google Scholar
  7. Cartmell, E, and Fowles, G. W. Z. (1956), Valency and Molecular Structure. Butterworths Scientific Publications. London, England.Google Scholar
  8. Chien, J. C. W. and Chang, E. P. (1973). Influence of telopeptide on the morphology and physicomechanical properties of reconstituted collagen. Biopolymers, 12: 2045.CrossRefGoogle Scholar
  9. Coulet, P. R., Julliard, J. H. and Gautheron, D. C. (1974). A mild method of general use for covalent coupling of enzymes to chemically activated collagen films. Biotech. and Bioengr. 16:1055.CrossRefGoogle Scholar
  10. Coulet, P. R. Godinot, C. and Gautheron, D. C. (1975). Surface-Bound Aspartate Aminotransferase on Collagen films. Compared Properties with Native Enzyme. Biochemica et Biophsica Acta. 391:272.Google Scholar
  11. Drake, M. P., Davison, P. E., Bumps, S. and Schmitt, F. O. (1966). Action of proteolytic enzymes on tropo-collagen and insoluble collagen. Biochemistry 5(1), 301PubMedCrossRefGoogle Scholar
  12. Eskamani, A. (1972). Characterization of lactase immobilized on collagen. Ph. D. thesis, Rutgers University, New Brunswick, NJ.Google Scholar
  13. Eskamani, A., Chase, T., Jr., Frendenberger, J. and Gilbert, S. G. (1974). Determination of protein immobilized on solid support by tryptophan content. Anal. Biochem. 57:421PubMedCrossRefGoogle Scholar
  14. Gaitondi, M. K., and Dovey, T. (1970). A rapid and direct method for the quantitative determination of tryptophan in the intact protein. Biochem. J. 117:907.Google Scholar
  15. Giacin, J. R., Jakubowski, J., Leeder, J. G., Gilbert, S. G. and Kleyn, D. H. (1974). Characterization of lactase immobilized on collagen. Conversion of whey lactose by soluble and immobilized lactase. J. Food Sci. 39:751.CrossRefGoogle Scholar
  16. Gilbert, S. G. (1973). Enzyme Engineering User’s Conference. December 7, 1973. Rutgers Inter-disciplinary Enzyme Technology Group. Rutgers University, New Brunswick, N. J.Google Scholar
  17. Goldman, R., Goldstein, L., and Katchalski, E. (1971). Biochemical Aspects of Reactions on Solid Supports. G. R, Stark, Ed. Academic Press, New York.Google Scholar
  18. Goldman, R., (1973). Enzyme membrane model systems and their implication in biological research. Biochimie, 55:953.Google Scholar
  19. Green, D. E., Murer, E., Hultin, H. O., Richardson, S. H., Salmon, B., Brierley, G. P. and Baum, H. (1965). Association of integrated metabolic pathways with membranes. I. Glycolytic enzymes of the red corpuscle and yeast. Arch. Biochem, Biophys. 112:635.CrossRefGoogle Scholar
  20. Grossberg, A. L and Pressman, D. (1963). Effect of Ace-tylation on the Active Site of Several Antihapter Antibodies: Further Evidence for the Presence of Tyrosine in Each Site. Biochemistry, 2:90.PubMedCrossRefGoogle Scholar
  21. Hoare, D. G. and Koshland, D. E. (1966). Procedure for the Selective Modification of Carboxyl Groups in Proteins. J. Am. Chem. Soc. 88(9):2057Google Scholar
  22. Hodge, A. J. and Petraska, J. A. (1963). Aspects of Protein Structure. G. N. Ramachandran, Ed. Academic Press, New York.Google Scholar
  23. Hsieh, F., Davidson, B., and Vieth, W. R. (1976). Modeling of continual mass transfer level effects in an enzyme-membrane reactor system; with a direct approach to the identification of intrinsic rate parameters. J. Applied Chemistry and Biotechnology. In Press.Google Scholar
  24. Julliard, J. H. Godinot, C. and Gautheron, D. A. (1971). Some modifications of the kinetic properties of bovine liver glutamate dehydrogenase (NAD(P)) Co-valently bound to a solid matrix of collagen. (FEBS Letters, 14:185.Google Scholar
  25. Klapper, M. H. and Klotz, I. M. (1972). Acylation with dicarbonylic Acid Anydrides. Methods in Enzymology, C. H. W. Hirs, Ed. Volume XXV, Academic Press, New York.Google Scholar
  26. La Bella, F. S. (1971). Cross-links in elastin and collagen. Biophysical properties of the skin. H. R. Eldin, Ed. Wiley Interscience, New York.Google Scholar
  27. Lee, K. H., Coulet, P. R., Gautheron, D. C. (1976). Grafting of enzymes on collagen films using Woodward’s reagent “K” and a water soluble carbodil-mide derivative. Biochimie, 58:489.PubMedCrossRefGoogle Scholar
  28. Lieberman, E. R. (1971). Studies on the permeation of gases through collagen films. Ph. D. Thesis, Rutgers University, New Brunswick, N. J.Google Scholar
  29. Lieberman, E. R., Gilbert, S. G. and Shrinivasa, V., (1972) The use of gas permeability as a molecular probe for the study of cross-linked collagen structures. Trans. N. Y. Acad. Sci. 34:694.PubMedCrossRefGoogle Scholar
  30. Lieberman, E, R. and Gilbert, S. G, (1973). Gas permeation of collagen films as affected by cross-linkages, moisture and plasticizer content. J. Polymer Sci.: Symposium No. 41:33.CrossRefGoogle Scholar
  31. Lin, P. M. (1975). Modification of collagen for enzyme Immobilization. Ph. D. Thesis, Rutgers University, New Brunswick, N J.Google Scholar
  32. Lin, P. M., Giacin, J. R., Leeder, J. G. and Gilbert, S. G, (1976). Chemical and enzymatic modification of collagen: the effect of cross-linking on the enzyme-binding capacity of collagen. J. Food Sci., 41: 1056.CrossRefGoogle Scholar
  33. Luo, K. M., Giacin, J. R. and Gilbert, S. G. (1976). Chemical modification of collagen for enzyme immobilization. Presented 36th Annual IFT Meeting, Anaheim, California, June 7-9.Google Scholar
  34. Piez, K. A., Lewis, M. S. Martin, G. R. and Gross, J. (1961). Subunits of the collagen molecule. Biochem, Biophys. Acta. 53:596.CrossRefGoogle Scholar
  35. Piez, K. A. (1967), Treatise on Collagen, Vol. I, Chemistry of Collagen, G. N. Ramachandran, Ed., Academic Press, New York.Google Scholar
  36. Ramachandran, G. N. (1967). Treatise on Collagen, Vol. I, Structure of collagen at the molecular level, G. N. Ramachandran, Ed., Academic Press, New York.Google Scholar
  37. Richards, F. M. and Knowles, J. P. (1968). Glutaraidehyde as a protein cross-linking reagent. J. Mol. Biol. 37:231.PubMedCrossRefGoogle Scholar
  38. Rubin, A. L., Drake, M. P., Davison, P. E., Pfahl, D., Speakman, P. T. and Schmitt, F. O. (1965). Effects of pepsin treatment on the interaction properties of tropocollagen macromolecules. Biochem. 4:181.CrossRefGoogle Scholar
  39. Rubin, A. L. and Stangel, K. H. (1969). Biomaterials, L Stark and G. Agarual, Ed., Plenum Press, New YorkGoogle Scholar
  40. Silman, I. H., Alba-Weissenberg, M. and Kachalski, E. (1966). Some water-insoluble papain derivatives. Biopolymers, 4: 441.PubMedCrossRefGoogle Scholar
  41. Stark, G. R. (1965). Reaction of cyanate with functional groups of proteins. III. Reactions with amino and carboxyl groups. Biochemistry, 4:1030.PubMedCrossRefGoogle Scholar
  42. Stark, M., Rauterberg, J. and Kuhn, K. (1971) Evidence for a non-helical region at the carboxyl terminus of the collagen molecule. FEBS letters, 13:101.PubMedCrossRefGoogle Scholar
  43. Stevens, F. S. (1966). The depolymerizing action of pepsin on collagen. Molecular weights of the component polypeptide chains. Biochim. Biophys. Acta. 130:190.CrossRefGoogle Scholar
  44. Tanzer, M. L. (1973). Cross linking of collagen. Science 180:54.CrossRefGoogle Scholar
  45. Venkatasubramanian, K., Saini, R. and Vieth, W. R. (1974). On the mechanisms of enzyme and whole microbial cell attachment to collagen, J. Ferm. Technol. 52:268.Google Scholar
  46. Verzar, F. (1964). Aging of the collagen fiber. In “International Review of Connective Tissue Research”. Vol. 2: Academic Press, New York.Google Scholar
  47. Veis, A., Anesey, J. and Massell, S. (1967). A limited microfibril model for the three-dimensional arrangement within collagen fibrils. Nature, London, 215:931.PubMedCrossRefGoogle Scholar
  48. Vieth, W. R., Gilbert S. G. and Wang, S. S. (1972a). Urea hydrolysis on collagen urease complex membrane.Google Scholar
  49. Vieth, W. R., Gilbert, S. G. and Wang, S. S. (1972b). Performance of collagen-invertase complex membrane in a biocatalytic module. Trans. New York Academy of Sci. 34:454.CrossRefGoogle Scholar

Copyright information

© Plenum Press, New York 1977

Authors and Affiliations

  • Jack R. Giacin
    • 1
  • Seymour G. Gilbert
    • 1
  1. 1.Food Science DepartmentCook College, Rutgers UniversityNew BrunswickUSA

Personalised recommendations