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Peroxidase assay

Askim Hediye Sekmen , Ismail Turkan
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Askim Hediye Sekmen

Overview

This protocol outlines measurement of POX activity in plant tissue by spectrophotomeric assay.

Background - Antioxidant enzymes

Plants, being aerobic organisms, utilize molecular O2as a terminal electron acceptor. As a reduction, highly reactive intermediates, reactive species (ROS), are produced. ROS such as singlet oxygen (O21), superoxide (O2-.) and hydrogen peroxide (H2O2) are normal products of metabolism and are produced in all cellular compartments within a variety of processes. In general, they are maintained at constant basal levels in healthy cells. However, they can destroy normal metabolism through oxidative damage of lipids, proteins, and nucleic acids when they are produced in excess as a result of oxidative stress (Gill and Tuteja, 2010). To overcome oxidative stress, together with non-enzymatic antioxidant molecules (ascorbate, glutathione,  α-tocopherol etc.), plants detoxify ROS by up-regulating antioxidative enzymes like superoxide dismutase (SOD; EC 1.15.1.1), catalase (CAT; E.C 1.11.1.6), peroxidase (POX; EC1.11.1.7), ascorbate peroxidase (APX; EC 1.11.1.11) and glutathione reductase (GR; EC 1.6.4.2) (Turkan and Demiral, 2009). SOD provide the first line of defense against the toxic effects of elevated levels of ROS. The SODs converts O2-.to H2O2. The hydrogen peroxide produced is then scavenged by catalase and a variety of peroxidases. Catalase dismutates H2O2into water and molecular oxygen, whereas POX decomposes H2O2by oxidation of co-substrates such as phenolic compounds and/or antioxidants. APX is involved in scavenging of H2O2in water-water and ASH-GSH cycles and utilizes ASH as the electron donor. GR is a potential enzyme of the ASH-GSH cycle and plays an essential role in defense system against ROS (Gill and Tuteja, 2010; Ahmad et al., 2010). This protocol is one of a number of ANTIOXIDANT ENZYME PROTOCOLS

PROTOCOL: Superoxide dismutase assay
PROTOCOL: Catalase assay
PROTOCOL: Ascorbate peroxidase assay
PROTOCOL: Glutathione reductase assay

Background - Peroxidase

In this protocol, POX (EC1.11.1.7) activity was based on the method described by Herzog and Fahimi (1973).

Materials/Equipment

a) Chemical Materials

  • Tris-hydrochloride (Tris-HCl)
  • Ethylenediamine tetraacetic acid (EDTA)
  • TritonX-100
  • Phenylmethanesulfonyl fluoride (PMSF)
  • Dithiothreitol (DTT)
  • NaH2PO4& Na2HPO4
  • Gelatine
  • 3,3′-diaminobenzidine-tetra hydrochloride dihydrate (DAB)
  • Citric acid
  • Na2HPO4.12H2O
  • Hydrogen peroxide (H2O2)
  • Liquid nitrogen

b) Apparatus and Equipments

  • pH meter
  • Mortar and pestle
  • Various micropipettes
  • Eppendorf tubes (1.5 ml)
  • Spectrophotometer
  • Centrifuge
  • Polystrene cuvette

Units, terms, definitions

Procedure

a) Solutions

Extraction Buffer 50 mM Tris-HCl (MW: 157.60 g/mol) 0.1 mM EDTA (MW: 292.2 gr/mol) 0.2% TritonX-100 1 mM PMSF (MW: 174.19 gr/mol) 2 mM DTT (MW: 154.25 g/mol) Total Volume: 100 ml

  • 50 mM Tris-HCl (pH 7.8),100 ml

    Weigh 0.788 g Tris-HCl, dissolve in 100 ml dIH2O and adjust pH 7.8 by 1 N KOH.
  • 0.1 mM EDTA (292.2 gr/mol), 100 ml

    0.00292 g EDTA in 100 ml homogenization buffer
  • 0.2% TritonX-100, 100 ml

    200 µl TritonX100 in 100 ml homogenization buffer
  • 1mM PMSF (174.19 gr/mol),100 ml

    0.01742 g PMSF in 100 ml homogenization buffer
  • 2 mM DTT (154.25 gr/mol), 100 ml

    0.031 g DTT in 100 ml homogenization buffer

Dissolve EDTA, TritonX-100, PMSF, DTT in 80 ml of Tris-HCl buffer (pH 7.8) and complete the volume to 100 ml with Tris-HCl buffer. Assay Solutions Solution I

  • 0.1% (w/v) gelatine

    Weigh 0.1 gr gelatine, dissolve in 50 ml warm dIH2O.
  • 1.68 mM DAB (214.27 gr/mol)

    After solution containing gelatine is cooled, add 0.0036 gr DAB

Solution II

  • 55 mM Citric acid (192.13 gr/mol)
  • 88 mM Na2HPO4.12H2O (358.14 g/mol)

    Dissolve 1.06 g citric acid and 3.16 gr Na2HPO4.12H2O in 40 ml dIH2O. Then adjust to 50ml (pH: 4.4).

Mix the Sol I and Sol II (reaction mix)

  • 0.6% H2O2

    Stock: 30% (w/v) H2O2

    Take 200 µl from 30% stock H2O2solution and complete to 10 ml.

b) Methods

Extraction

  • Grind 0.5 g tissue in a cold mortar and pestle using liquid nitrogen and suspend in 1.5 ml of homogenization buffer solution.
  • Centrifuge the suspension at 14 000 rpm for 30 min at 4 ºC
  • Take the supernatant for the enzyme assay.

Assay Medium

  • 920 μl reaction mix
  • 65 μl enzyme extract containing 100 μg soluble protein (determined by Bradford method)
  • 15 μl dIH2O for blank
  • 15 μl H2O2→ Starts the reaction
  Blank Sample
50 mM Na-P buffer 920 μl 920 μl
ddH2O 15 μl -
Sample 65 μl 65 μl
H2O2 - 15 μl
  • The increase in the absorbance at 465 nm was followed for 180 seconds .
  • A unit of POX activity was defined as mmol H2O2decomposed ml-1min-1.

Other resources

Notes and troubleshooting tips

Links to resources and suppliers

Literature references

V. Herzog, H. Fahimi, Determination of the activity of peroxidase, Anal. Biochem. 55 (1973) 554–562.

Health, safety & hazardous waste disposal considerations

 


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Page last modified on Wednesday 04 of May, 2011 09:38:51 EST by Admin36802 points . (Version 3)