Askim Hediye Sekmen, Ismail Turkan
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 188.8.131.52), catalase (CAT; E.C 184.108.40.206), peroxidase (POX; EC220.127.116.11), ascorbate peroxidase (APX; EC 18.104.22.168) and glutathione reductase (GR; EC 22.214.171.124) (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 (EC126.96.36.199) activity was based on the method described by Herzog and Fahimi (1973).
a) Chemical Materials
- Tris-hydrochloride (Tris-HCl)
- Ethylenediamine tetraacetic acid (EDTA)
- Phenylmethanesulfonyl fluoride (PMSF)
- Dithiothreitol (DTT)
- NaH2PO4& Na2HPO4
- 3,3 -diaminobenzidine-tetra hydrochloride dihydrate (DAB)
- Citric acid
- Hydrogen peroxide (H2O2)
- Liquid nitrogen
b) Apparatus and Equipments
- pH meter
- Mortar and pestle
- Various micropipettes
- Eppendorf tubes (1.5 ml)
- Polystrene cuvette
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
- 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.
- 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.
- 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
|50 mM Na-P buffer||920 l||920 l|
|Sample||65 l||65 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.
V. Herzog, H. Fahimi, Determination of the activity of peroxidase, Anal. Biochem. 55 (1973) 554-562.