Glycoconjugate Journal

, Volume 25, Issue 2, pp 101–109 | Cite as

Isoenzyme-specific differences in the degradation of hyaluronic acid by mammalian-type hyaluronidases

  • Edith S. A. Hofinger
  • Julia Hoechstetter
  • Martin Oettl
  • Günther Bernhardt
  • Armin BuschauerEmail author


Bovine testicular hyaluronidase (BTH) has been used as a spreading factor for many years and was primarily characterized by its enzymatic activity. As recombinant human hyaluronidases are now available the bovine preparations can be replaced by the human enzymes. However, data on the pH-dependent activity of hyaluronidases reported in literature are inconsistent in part or even contradictory. Detection of the pH-dependent activity of PH-20 type hyaluronidases, i.e. recombinant human PH-20 (rhPH-20) and BTH, showed a shift of the pH optimum from acidic pH values in a colorimetric activity assay to higher pH values in a turbidimetric activity assay. Contrarily, recombinant human Hyal-1 (rhHyal-1) and bee venom hyaluronidase (BVH) exhibited nearly identical pH profiles in both commonly used types of activity assays. Analysis of the hyaluronic acid (HA) degradation products by capillary zone electrophoresis showed that hyaluronan was catabolized by rhHyal-1 continuously into HA oligosaccharides. BTH and, to a less extent, rhPH-20 exhibited a different mode of action: at acidic pH (pH 4.5) HA was degraded as described for rhHyal-1, while at elevated pH (pH 5.5) small oligosaccharides were produced in addition to HA fragments of medium molecular weight, thus explaining the pH-dependent discrepancies in the activity assays. Our results suggest a sub-classification of mammalian-type hyaluronidases into a PH-20/BTH and a Hyal-1/BVH subtype. As the biological effects of HA fragments are reported to depend on the size of the molecules it can be speculated that different pH values at the site of hyaluronan degradation may result in different biological responses.


Bovine testicular hyaluronidase Human Hyal-1 Human PH-20 Bee venom hyaluronidase Capillary zone electrophoresis 



bovine serum albumin


bovine testicular hyaluronidase


bee venom hyaluronidase


capillary zone electrophoresis




glucuronic acid


hyaluronic acid


hyaluronic acid disaccharide unit


recombinant human Hyal-1


recombinant human PH-20


size exclusion chromatography



The authors thank S. Bollwein for technical assistance and S. Weitensteiner for assistance. Support of this work by the Graduate Training Program (Graduiertenkolleg) GRK 760 “Medicinal Chemistry: Molecular Recognition—Ligand–Receptor Interactions” of the Deutsche Forschungsgemeinschaft is gratefully acknowledged.


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Copyright information

© Springer Science+Business Media, LLC 2007

Authors and Affiliations

  • Edith S. A. Hofinger
    • 1
  • Julia Hoechstetter
    • 1
  • Martin Oettl
    • 1
  • Günther Bernhardt
    • 1
  • Armin Buschauer
    • 1
    Email author
  1. 1.Institute of Pharmacy, Faculty of Chemistry and PharmacyUniversity of RegensburgRegensburgGermany

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