Protocol

Authors

Askim Hediye Sekmen, Ismail Turkan

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, -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

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 ^∘C
• Take the supernatant for the enzyme assay.

Assay Medium

• 920 l reaction mix
• 65 l enzyme extract containing 100 g soluble protein (determined by Bradford method)
• 15 l dIH₂O for blank
• 15 l H₂O₂ Starts the reaction

	Blank	Sample
50 mM Na-P buffer	920 l	920 l
ddH2O	15 l	–
Sample	65 l	65 l
H2O2	–	15 l

• 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.

Literature references

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