title: Isoenzyme-specific differences in the degradation of hyaluronic acid by mammalian-type hyaluronidases.
abstract: 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.
authors.affiliation: Institute of Pharmacy, Faculty of Chemistry and Pharmacy, University of Regensburg, Universitätsstrasse 31, 93053 Regensburg, Germany.
authors.forename: Edith S A
authors.initials: ES
authors.lastname: Hofinger
authors.forename: Julia
authors.initials: J
authors.lastname: Hoechstetter
authors.forename: Martin
authors.initials: M
authors.lastname: Oettl
authors.forename: Günther
authors.initials: G
authors.lastname: Bernhardt
authors.forename: Armin
authors.initials: A
authors.lastname: Buschauer
mesh_terms: D000818:Animals; D002417:Cattle; D002460:Cell Line; D004330:Drosophila; D019075:Electrophoresis, Capillary; D006801:Humans; D006820:Hyaluronic Acid / Q000378:metabolism*; D006821:Hyaluronoglucosaminidase / Q000145:classification* / Q000502:physiology*; D006863:Hydrogen-Ion Concentration; D007527:Isoenzymes / Q000145:classification / Q000502:physiology; D008297:Male; D011994:Recombinant Proteins / Q000145:classification / Q000378:metabolism; D013379:Substrate Specificity; D013737:Testis / Q000201:enzymology
chemical_list: D007527:Isoenzymes; D011994:Recombinant Proteins; D006820:Hyaluronic Acid; D006821:Hyaluronoglucosaminidase