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Anal Biochem. 2008 Jun 15;377(2):251-8. doi: 10.1016/j.ab.2008.03.018. Epub 2008 Mar 15.

A comparison of sugar indicators enables a universal high-throughput sugar-1-phosphate nucleotidyltransferase assay.

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  • 1Department of Biochemistry, University of Wisconsin, Madison, WI 53706, USA.

Abstract

A systematic comparison of six sugar indicators for their sensitivity, specificity, cross-reactivity, and suitability in the context of crude lysates revealed para-hydroxybenzoic acid hydrazide (pHBH) to be best suited for application in a plate-based phosphatase-assisted universal sugar-1-phosphate nucleotidyltransferase assay. The addition of a general phosphatase to nucleotidyltransferase reaction aliquots enabled the conversion of remaining sugar-1-phosphate to free sugar, the concentration of which could be rapidly assessed via the pHBH assay. The assay was validated using the model glucose-1-phosphate thymidylyltransferase from Salmonella enterica (RmlA) and compared favorably with a previously reported HPLC assay. This coupled discontinuous assay is quantitative, high throughput, and robust; relies only on commercially available enzymes and reagents; does not require chromatography, specialized detectors (e.g., mass or evaporative light scattering detectors), or radioisotopes; and is capable of detecting less than 5 nmol of sugar-1-phosphate. It is anticipated that this high-throughput assay system will greatly facilitate nucleotidyltransferase mechanistic and directed evolution/engineering studies.

PMID:
18387352
[PubMed - indexed for MEDLINE]
PMCID:
PMC2442561
Free PMC Article
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