Quantitative Continuous Assay for Hyaluronan Synthase
Loading...
Authors
Krupa, Joanne C.
Shaya, David
Chi, Lianli
Linhardt, Robert J.
Cygler, Miroslaw
Withers, Stephen G.
Mort, John S.
Issue Date
2007-02-15
Type
Article
Language
ENG
Keywords
Biology , Chemistry and chemical biology , Chemical and biological engineering , Biomedical engineering
Alternative Title
Abstract
A 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 Xuorescently 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.
Description
Analytical Biochemistry, 361, 218-225
Note : if this item contains full text it may be a preprint, author manuscript, or a Gold OA copy that permits redistribution with a license such as CC BY. The final version is available through the publisher’s platform.
Note : if this item contains full text it may be a preprint, author manuscript, or a Gold OA copy that permits redistribution with a license such as CC BY. The final version is available through the publisher’s platform.
Full Citation
Quantitative Continuous Assay for Hyaluronan Synthase, J. C. Krupa, D. Shaya, L. Chi, R.J. Linhardt, M. Cygler, S. G. Withers, J. S. Mort, Analytical Biochemistry, 361, 218-225, 2007.
Publisher
Elsevier
Terms of Use
Journal
Volume
Issue
PubMed ID
DOI
ISSN
10960309
32697
32697