Quantitative continuous assay for hyaluronan synthase.
Analytical biochemistry
confidence
Key findings
Developed a three-enzyme coupled UV absorption assay to quantitate glucuronic acid and N-acetylglucosamine transferase activities of PmHAS; no clinical/biological endpoints.
View source on PubMed (PMID 17173853) ↗
- Sample size
- N/A
- Population
- In vitro enzyme assay (Pasteurella multocida hyaluronan synthase)
- Dosing
- N/A
- Duration
- N/A
- Route
- N/A
- Blinding
- not_reported
- Controls
- none
- Drug class
- glycosaminoglycan
Full 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 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.