A spectrophotometric assay of D-glucuronate based on Escherichia coli uronate isomerase and mannonate dehydrogenase.
(2004) Protein Expression and Purification — Vol. 37, n° 2, p. 352-360 (2004)
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- Abstract
- Escherichia coli uronate isomerase and mannonate dehydrogenase were overexpressed in E. coli BL21(DE3)pLysS cells and purified to near-homogeneity. The kinetic properties of the two enzymes were investigated. The isomerase was found to be inhibited by EDTA and to be stimulated by Zn(2+), Co(2+), and Mn(2+), but not by Mg(2+) or Ca(2+). Both enzymes were used to develop a sensitive spectrophotometric assay, in which D-glucuronate is converted to D-mannonate with concomitant oxidation of NADH to NAD(+). The sensitivity of this assay permits the detection of less than 1 nmol D-glucuronate. This assay can also be used to determine the concentration of beta-glucuronides and glucuronate 1-phosphate after enzymatic hydrolysis of these compounds with beta-glucuronidase or alkaline phosphatase.
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Linster, C., & Van Schaftingen, E. (2004). A spectrophotometric assay of D-glucuronate based on Escherichia coli uronate isomerase and mannonate dehydrogenase. Protein Expression and Purification, 37(2), 352-360. https://doi.org/10.1016/j.pep.2004.06.015 (Original work published 2004)