Measurement of Enzyme Activities and Optimization of Continuous and Discontinuous Assays

Camille Bénard1, Yves Gibon1

1 UMR1332 Biologie du Fruit et Pathologie and Plateforme Métabolome, Centre de Génomique Fonctionnelle Bordeaux, INRA‐Bordeaux and Bordeaux University, Villenave d'Ornon, France
Publication Name:  Current Protocols in Plant Biology
Unit Number:   
DOI:  10.1002/cppb.20003
Online Posting Date:  June, 2016
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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 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: continuous assay; cycling assay; discontinuous assay; enzyme activity; optimization; PEPC; recovery

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Table of Contents

  • Introduction
  • Basic Protocol 1: Continuous Assay of Phosphoenolpyruvate Carboxylase Activity
  • Alternate Protocol 1: Discontinuous Assay of Phosphoenolpyruvate Carboxylase Activity
  • Support Protocol 1: Extraction of Soluble Enzymes
  • Basic Protocol 2: Optimization of Enzyme Assays Using a Standard
  • Reagents and Solutions
  • Commentary
  • Figures
  • Tables
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Basic Protocol 1: Continuous Assay of Phosphoenolpyruvate Carboxylase Activity

  • Pre‐assay buffer X5 stock solution (see recipe)
  • Malate dehydrogenase (MDH) 100−1 in 200 mM Tricine/KOH, pH 8, 10 mM MgCl 2 stock solution (store at −80°C)
  • Lactate dehydrogenase (LDH), 10−1 in 200 mM Tricine/KOH pH 8, 10 mM MgCl 2 (prepare fresh)
  • Phosphoenolpyruvate (PEP), 20 mM (prepare fresh)
  • 60 mM NADH in 60 mM NaOH stock solution (store at −80°C)
  • Extraction buffer (see the protocol 3Support Protocol)
  • Ice
  • Crude enzyme extract (see the protocol 3Support Protocol)
  • 15‐ml Falcon tubes or similar
  • Hand pipets (single and 8‐ or 12‐channel), 0.5‐10 μl, 10‐100 μl, and 50‐1,200 μl ranges
  • Flat‐bottom polystyrene microplate
  • Vortex mixer and microplate shaker
  • Centrifuge with microplate adapter
  • Microplate reader, filter‐based or equipped with a monochromator, enabling measurements at 340 nm (e.g., SAFAS MP96, Monaco)

Alternate Protocol 1: Discontinuous Assay of Phosphoenolpyruvate Carboxylase Activity

  Additional Materials (also see protocol 1)
  • 20 mM PEP (prepare fresh)
  • MDH, 100−1 in 200 mM Tricine/KOH, pH 8, 10 mM MgCl 2 stock solution (store at −20°C)
  • NADH, 5 mM in 5 mM NaOH stock solution (store at −20°C); heat 10 min at 95°C just before use
  • Crude enzyme extract and extraction buffer (see the protocol 3Support Protocol)
  • Ice
  • Stopping solution (see recipe)
  • 0.5 M NaOH stock solution
  • Tricine/KOH, 1 M, pH 9, stock solution (store at −20°C)
  • 10 mM 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT) stock solution (store at −20°C)
  • 4 mM Phenazine ethosulfate (PES) stock solution (store at −20°C)
  • 200 mM EDTA stock solution
  • 50% Ethanol
  • Alcohol dehydrogenase (ADH) 2000−1 in 200 mM Tricine/KOH, pH 9, stock solution (store at −20°C)
  • Standards: 0, 12.5, 25, 50, and 100 μM oxaloacetate in the extraction buffer, prepare freshly
  • Microplate heating block (capable of heating to up to 95°C)
  • Microplate reader, filter‐based or equipped with a monochromator, enabling measurements at 570 nm (e.g., SAFAS MP96, Monaco)
  • Microplate sealing tape

Support Protocol 1: Extraction of Soluble Enzymes

  Additional Materials (also see protocol 1)
  • Pre‐extraction buffer X10 (see recipe)
  • 10 % (v/v) Triton‐X100 stock solution
  • Glycerol
  • 2 mM leupeptin (100×) stock solution (store at −80°C)
  • 50 mM dithiothreitol (100×) stock solution (store at −80°C)
  • 100 mM phenylmethylsulfonyl fluoride (PMSF; 100×) stock solution in isopropanol (store up to 1 month at 4°C; protect from light)
  • Ice
  • Biological material ground to homogeneity under cryogenic conditions
  • Liquid nitrogen
  • Hand pipets (single and 8‐channel), 0.5 to 10‐μl, 5 to 100‐μl, 50 to 1,200‐μl ranges
  • 15‐ml Falcon tubes
  • V‐bottomed polyethylene microplates
  • 1.1‐ml Micronic tubes or similar

Basic Protocol 2: Optimization of Enzyme Assays Using a Standard

  Additional Materials (also see protocol 1)
  • Crude enzyme extracts (see the protocol 3Support Protocol): use three technical replicates (three aliquots extracted in parallel) and six blanks (pseudo extracts, i.e. extraction buffer alone)
  • Standard (reference enzyme with known activity, chemical standard or biological standard): use six technical replicates in order to achieve robust measurements while exploiting every well on the microplate. It would not be possible to use six replicates for each extract dilution, but it can be considered that lower replication is compensated at least in part by the use of a dilution series
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Literature Cited

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Internet Resources
  Web site giving access to the most comprehensive database about enzyme properties.
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