Measurement of Enzyme Activities and Optimization of Continuous and Discontinuous Assays

Camille Bénard; Yves Gibon

doi:10.1002/cppb.20003

Measurement of Enzyme Activities and Optimization of Continuous and Discontinuous Assays

Curr Protoc Plant Biol. 2016 Mar;1(2):247-262. doi: 10.1002/cppb.20003.

Authors

Camille Bénard¹, Yves Gibon¹

Affiliation

¹ UMR1332 Biologie du Fruit et Pathologie and Plateforme Métabolome, Centre de Génomique Fonctionnelle Bordeaux, INRA-Bordeaux and Bordeaux University, Villenave d'Ornon, France.

PMID: 30775869
DOI: 10.1002/cppb.20003

Abstract

The measurement of enzyme activities represents an important step towards the understanding of biological networks. Continuous or discontinuous assays can be used, as well as highly sensitive assays, depending on the abundance of the enzymes under study. To exemplify such methods, two protocols for phosphoenolpyruvate carboxylase activity (EC 4.1.1.31) in plant extracts are given. For this, an extraction protocol is also described. Then, an optimization protocol for enzyme assays using enzymatic, chemical, or biological standards is proposed. This protocol evaluates in one run the optimal extract dilution, the recovery of a standard, and the technical error in a given matrix. The interest of using biological standard in routine measurements is highlighted. © 2016 by John Wiley & Sons, Inc.

Keywords: PEPC; continuous assay; cycling assay; discontinuous assay; enzyme activity; optimization; recovery.