Purification and Characterization of Human Serum Hyaluronidase

Alaa M Afify, M. Stern, M. Guntenhoner, R. Stern

Research output: Contribution to journalArticle

81 Citations (Scopus)

Abstract

Hyaluronidase from fresh human serum was purified to apparent homogeneity in a two-step procedure. Potent serum inhibitors of hyaluronidase activity were removed during the course of the purification. Isolation of the enzyme was expedited by the use of a newly devised ELISA-like assay. Enzyme activity was measured by following the rates of hydrolysis of hyaluronan (HA) adsorbed onto microtiter wells. Following enzymatic digestion, the remaining HA was measured using a cartilage-derived biotinylated HA-binding protein and an avidin-peroxidase reaction. Molecular sieve chromatography yielded a doublet of proteins with apparent molecular sizes of 42 and 50 kDa. The molecular size of the major band of protein obtained on sodium dodecyl sulfate-polyacrylamide gel electrophoresis under nonreducing conditions was 59 kDa. Under reducing conditions, however, the size increased to 72 kDa. The pH optimum of the enzyme was 3.7. Sodium chloride concentrations greater than 100 mM were inhibitory. Activity of the serum enzyme was further characterized with a new HA-substrate gel procedure. The serum enzyme activity is different from the liver-derived activity. The tissue source of this circulating enzyme is unknown.

Original languageEnglish (US)
Pages (from-to)434-441
Number of pages8
JournalArchives of Biochemistry and Biophysics
Volume305
Issue number2
DOIs
StatePublished - Sep 1993
Externally publishedYes

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Hyaluronoglucosaminidase
Purification
Hyaluronic Acid
Enzyme activity
Enzymes
Serum
CD44 Antigens
Avidin
Molecular sieves
Cartilage
Chromatography
Electrophoresis
Sodium Chloride
Sodium Dodecyl Sulfate
Liver
Peroxidase
Hydrolysis
Assays
Proteins
Gels

ASJC Scopus subject areas

  • Molecular Biology
  • Biophysics
  • Biochemistry

Cite this

Purification and Characterization of Human Serum Hyaluronidase. / Afify, Alaa M; Stern, M.; Guntenhoner, M.; Stern, R.

In: Archives of Biochemistry and Biophysics, Vol. 305, No. 2, 09.1993, p. 434-441.

Research output: Contribution to journalArticle

Afify, Alaa M ; Stern, M. ; Guntenhoner, M. ; Stern, R. / Purification and Characterization of Human Serum Hyaluronidase. In: Archives of Biochemistry and Biophysics. 1993 ; Vol. 305, No. 2. pp. 434-441.
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