Quantitative continuous assay for hyaluronan synthase

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DOIResolve DOI: http://doi.org/10.1016/j.ab.2006.11.011
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TypeArticle
Journal titleAnalytical Biochemistry
Volume361
Issue2
Pages218225; # of pages: 8
SubjectCulture Media; Electrophoresis; Fluorescent Dyes; pha
AbstractA rapid, continuous, and convenient three-enzyme coupled UV absorption assay was developed to quantitate the glucuronic acid and N-acetylglucosamine transferase activities of hyaluronan synthase from Pasteurella multocida (PmHAS). Activity was measured by coupling the UDP produced from the PmHAS-catalyzed transfer of UDP-GlcNAc and UDP-GlcUA to a hyaluronic acid tetrasaccharide primer with the oxidation of NADH. Using a fluorescently labeled primer, the products were characterized by gel electrophoresis. Our results show that a truncated soluble form of recombinant PmHAS (residues 1-703) can catalyze the glycosyl transfers in a time- and concentration-dependent manner. The assay can be used to determine kinetic parameters, inhibition constants, and mechanistic aspects of this enzyme. In addition, it can be used to quantify PmHAS during purification of the enzyme from culture media
Publication date
LanguageEnglish
AffiliationNRC Biotechnology Research Institute; National Research Council Canada
Peer reviewedNo
NRC number47565
NPARC number3540042
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Record identifierdaee6f39-e43b-4946-b4e5-c68ed0d8a509
Record created2009-03-01
Record modified2016-05-09
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