Skip to main content
SpringerLink
Log in

Large scale preparation and characterization of mucopolysaccharase contamination free heparinase

  • Original Article
  • Published:
Applied Biochemistry and Biotechnology Aims and scope Submit manuscript

Abstract

By a combination of hydroxylapatite chromatography and negative adsorption on QAE-Sephadex at pH 8.3, heparinase (E.C.4.2.2.7) can be successfully isolated from all the other mucopolysaccharase contaminants present inFlavobacterium heparinum. Hydroxylapatite isolates heparinase primarily from chondroitinases, hyaluronidase, and most glycuronidases. QAE-Sephadex chromatography at pH 8.3 further separates heparinase from heparitinases, sulfatases, and the remaining glycuronidases. The heparinase preparation thus obtained contains no statistically significant levels of other contaminating mucopolysaccharases except for heparitinases that are present at an apparent maximum level of 3.4%. Owing to the presence of a crossreaction of heparinase on heparitin sulfate at conditions employed for the assay of heparitinase, the heparitinase level of 3.4% could be misleading because of the action of heparinase on heparitin sulfate. Characterization of this heparinase preparation shows that the enzyme has an optimum salt concentration of 0.08M NaCl, an optimum pH of 6.5, an activation energy of 5 kcal/mol, and a Km of 7.95 x 10-6 M. These parameters are almost identical to those displayed by a homogeneous heparinase preparation. The method described here is suitable for scale-up purposes using batch Chromatographic procedures.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

References

  1. Korn, E. D. and Payza, A. N. (1956),J. Biol. Chem. 223, 859–864.

    CAS  Google Scholar 

  2. Dietrich, C. P. (1969a),Biochemistry 8, 2089–2094.

    Article  CAS  Google Scholar 

  3. Hovingh, P. M. and Linker, A. (1970),J. Biol. Chem. 245, 6170–6175.

    CAS  Google Scholar 

  4. Yamagata, T., Saito, H., Habuchi, O., and Suzuki, S. (1968),J. Biol. Chem. 243, 1523–1535.

    CAS  Google Scholar 

  5. Michelacci, Y. M. and Dietrich, C. P. (1974),Biochem. Biophys. Res. Commun. 56, 973–980.

    Article  CAS  Google Scholar 

  6. Michelacci, Y. M. and Dietrich, C. P. (1976),J. Biol. Chem. 251, 1154–1158.

    CAS  Google Scholar 

  7. Lloyd, A. G., Law, B. A., Fowler, L. J., and Emberg, G. (1968),Biochem. J. 110, 54p.

    Google Scholar 

  8. Dietrich, C. P. (1969b),Biochem. J. 111, 91–95.

    CAS  Google Scholar 

  9. Linker, A. and Hovingh, P. (1965),J. Biol. Chem. 240, 3724–3728.

    CAS  Google Scholar 

  10. Warnick, C. T. and Linker, A. (1970),Fed. Proc. 29, 675.

    Google Scholar 

  11. Silva, M. and Dietrich, C. P. (1975),J. Biol. Chem. 250, 6841–6846.

    CAS  Google Scholar 

  12. Linker, A. and Hovingh, P. (1979),Heparin: Structure, Cellular Functions and Clinical Application, McDuffie, N. M., ed., Academic, NY, pp. 3–24.

    Google Scholar 

  13. Oik, Y., Kimata, K., Shinomura, K., and Suzuki, S. (1980),Biochem. J. 191, 193–202.

    Google Scholar 

  14. Toole, B. P. (1981),Cell Biology of Extracellular Matrix, Hay, E. D., ed., Plenum, NY, pp. 259–294.

    Google Scholar 

  15. Langer, R., Linhardt, R., Hoffberg, S., Larsen, A. K., Cooney, C. L., Tapper, D., and Klein, M. (1982),Science 217, 261–263.

    Article  CAS  Google Scholar 

  16. Linker, A. and Hovingh, P. (1972a),Meth. Enzymol. 28, 902–911.

    Article  Google Scholar 

  17. Dietrich, C. P., Silva, M. E., and Michelacci, Y. M. (1973),J. Biol. Chem. 248, 6408–6415.

    CAS  Google Scholar 

  18. Ototani, N., Kikuchi, M., and Yosizawa, Z. (1981),Carbohydr. Res. 88, 291–303.

    Article  CAS  Google Scholar 

  19. Yang, V. C, Linhardt, R. J., Bernstein, H., Cooney, C. L., and Langer, R. (1985a),J. Biol. Chem. 260, 1849–1857.

    CAS  Google Scholar 

  20. Grant, A. C, Linhardt, R. J., Fitzgerald, G. L., Park, J. J., and Langer, R. (1984),Anal. Biochem. 137, 25–32.

    Article  CAS  Google Scholar 

  21. Lowry, O. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951),J. Biol. Chem. 193, 265–275.

    CAS  Google Scholar 

  22. Bradford, M. M. (1976),Anal. Biochem. 72, 248–254.

    Article  CAS  Google Scholar 

  23. Linhardt, R. J., Fitzgerald, G. L., Cooney, C. L., and Langer, R. (1982),Biochim. Biophys. Acta 702, 192–203.

    Google Scholar 

  24. Linker A. and Hovingh, P. (1972b),Biochemistry 11, 563–568.

    Article  CAS  Google Scholar 

  25. Cerbelaud, E. (1983), M.S. Dissertation, Massachusetts Institute of Technology, pp. 86–88.

  26. Galliher, P. M., Cooney, C. L., Langer, R. S., and Linhardt, R. J. (1981),Appl. Environ. Micro. 41, 360–365.

    CAS  Google Scholar 

  27. Yang, V. C. and Langer, R. (1985b),Anal. Biochem. 147, 148–155.

    Article  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Department of Applied Biological Sciences, Massachusetts Institute of Technology, 02139, Cambridge, Massachusetts

    Victor C. Yang, Howard Bernstein, Charles L. Cooney & Robert Langer

  2. College of Pharmacy, The University of Michigan, 48109-1065, Ann Arbor, Michigan

    Victor C. Yang

  3. Department of Chemical Engineering, Massachusetts Institute of Technology, 02139, Cambridge, MA

    Howard Bernstein & Charles L. Cooney

  4. Department of Surgery, Children’s Hospital Medical Center, 02115, Boston, Massachusetts

    Robert Langer

Authors
  1. Victor C. Yang
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Howard Bernstein
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Charles L. Cooney
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Robert Langer
    View author publications

    You can also search for this author in PubMed Google Scholar

Rights and permissions

Reprints and permissions

About this article

Cite this article

Yang, V.C., Bernstein, H., Cooney, C.L. et al. Large scale preparation and characterization of mucopolysaccharase contamination free heparinase. Appl Biochem Biotechnol 16, 35–50 (1987). https://doi.org/10.1007/BF02798354

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF02798354

Index entries

Search

Navigation