In Vitro Assays for Measuring Protein Histidine Phosphatase Activity

  • Brandon S. McCullough
  • Amy M. BarriosEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 2077)


In order to obtain a detailed kinetic characterization, identify inhibitors, and elucidate the biological roles of an enzyme, it is advantageous to have a facile, sensitive enzyme assay protocol. Here we present a brief overview of the techniques available to monitor histidine phosphatase activity and provide protocols for measuring the activity and inhibition of PHPT1 in vitro using the fluorescent probe 6,8-difluoro-4-methylumbelliferyl phosphate (DiFMUP). This assay uses small quantities of commercially available materials, making its use feasible for most laboratories.

Key words

In vitro enzyme assay Enzyme kinetics Dephosphorylation Phosphohistidine Phosphatase activity Fluorogenic substrate 



This work was supported by a Teva Pharmaceuticals Mark A. Goshko Memorial Grant award (56426-TEV) and an NSF award (CHE 1308766) to A.M.B.


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© Springer Science+Business Media, LLC, part of Springer Nature 2020

Authors and Affiliations

  1. 1.Department of Medicinal ChemistryUniversity of UtahSalt Lake CityUSA

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