Askim Hediye Sekmen, Ismail Turkan
This protocol outlines measurement of CAT 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 184.108.40.206), catalase (CAT; E.C 220.127.116.11), peroxidase (POX; EC18.104.22.168), ascorbate peroxidase (APX; EC 22.214.171.124) and glutathione reductase (GR; EC 126.96.36.199) (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: Peroxidase assay|
|PROTOCOL: Ascorbate peroxidase assay|
|PROTOCOL: Glutathione reductase assay|
Background – Catalase
In this protocol, CAT (EC 188.8.131.52) activity was estimated according to Bergmeyer (1970) which measures the initial rate of disappearance of H2O2at 240 nm.
a) Chemical Materials
- Tris-hydrochloride (Tris-HCl)
- Ethylenediamine tetraacetic acid (EDTA)
- Phenylmethanesulfonyl fluoride (PMSF)
- Dithiothreitol (DTT)
- NaH2PO4& Na2HPO4
- Hydrogen peroxide (H2O2)
- Liquid nitrogen
b) Apparatus and Equipments
- pH meter
- Mortar and pestle
- Various micropipettes
- Eppendorf tubes (1.5 ml)
- Quartz 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
- 50 mM Na-PO4Buffer (pH 7.0)
- 3% H2O2, 100 ml
Stock: 35% (w/v) H2O2
- Take 8.57 ml from 35% stock H2O2solution and complete to 100 ml with 50 mM Na-POH4O2buffer (pH 7.0)
- 0.1 mM EDTA, 100 ml
Dissolve 0.012g EDTA in 100 ml Na-P buffer (pH:7.8)
- 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 Na-PO4 buffer (pH 7) containing 0.1 mM EDTA
- 70 l sample
- 10 l dIHH2O2O for blank
- 10 l H2O2 Starts the reaction
|50 mM Na-P buffer||920 l||920 l|
|Sample||70 l||70 l|
- Record the decrease in H2O2concentration by reading the absorbance at 240 nm (10 seconds intervals) for 180 seconds.
(H2O2extinction coefficient (E) = 39,4 mM-1cm-1at 240 nm and 1 mmol H2O2ml-1min-1was defined as 1 unit of CAT.)
N. Bergmeyer, Methoden der enzymatischen Analyse, vol. 1. Akademie Verlag, Berlin, (1970) 636-647.