Quantitative Continuous Assay for Hyaluronan Synthase

Krupa, Joanne C.
Shaya, David
Chi, Lianli
Linhardt, Robert J.
Cygler, Miroslaw
Withers, Stephen G.
Mort, John S.
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Biology , Chemistry and chemical biology , Chemical and biological engineering , Biomedical engineering
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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.
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.
Analytical Biochemistry, 361, 218-225
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The Linhardt Research Labs.
The Shirley Ann Jackson, Ph.D. Center for Biotechnology and Interdisciplinary Studies (CBIS)
The Linhardt Research Labs Online Collection
Rensselaer Polytechnic Institute, Troy, NY
Analytical Biochemistry
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