Protocol

Author

Ismail Turkan

Overview

This protocol outlines measurement of GR 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: Peroxidase assay

PROTOCOL: Ascorbate peroxidase assay

Background – glutathione reductase

In this protocol, GR (EC 1.6.4.2) activity was measured according to Foyer and Halliwell 1976.

Materials/Equipment

a) Chemical Materials

• TritonX-100
• Phenylmethanesulfonyl fluoride (PMSF)
• Dithiothreitol (DTT)
• NaH₂PO₄& Na₂HPO₄
• -Nicotinamide adenine dinucleotide phosphate, reduced tetra salt (NADPH.Na₄)
• Oxidized glutathione (GSSG)
• Liquid nitrogen

b) Apparatus and Equipments

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

Procedure

a) Solutions

Extraction Buffer 50 mM Tris-HCl (pH:7.8) 0.1 mM EDTA 0.2% TritonX100 1 mM PMSF 2 mM DTT Total Volume: 100 ml

• 50 mM Tris-HCl (pH 7.8), 100 ml
• 0.1 mM EDTA (292.2 gr/mol), 100 ml

  0.00292 g EDTA in 100 ml extraction buffer

• 0.2% TritonX-100, 100 ml

  200 μl TritonX100 in 100 ml extraction buffer

• 1mM PMSF (174,19 gr/mol), 100 ml

  0.01742 g PMSF in 100 ml extraction buffer

• 2 mM DTT (154,25 gr/mol), 100 ml

  0.031 g DTT in 100 ml extraction buffer

Dissolve EDTA, TritonX100, 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 25 mM Na-PO₄buffer 5 mM GSSG (MW: 612.63 g/mol) 1.2 mM NADPH.Na₄(MW: 833.35 g/mol)

• 25 mM Na-PO₄buffer, pH 7.8
• 5 mM GSSG, 10 ml (Prepare before use.)

  Weigh 0.0306 g GSSG, dissolve in 10 ml 25 mM Na-PO₄buffer.

• 1.2 mM NADPH. Na₄, 10 ml (Prepare before use)

  Weigh 0.01 g NADPH. Na₄, dissolve in 10 ml 25 mM Na-PO₄buffer.

b) Methods

Extraction

• Grind 0.5g of tissue with a cold mortar and pestle using liquid nitrogen, and suspend in 1.5 ml of homogenization buffer.
• Centrifuge the suspension at 14000 rpm for 30 min at 4 ^∘C.
• Take the supernatant for the enzyme assay.

Assay Medium (Total Vol = 1 ml)

• 700 l Na-PO₄buffer
• 100 l 5 mM GSSG
• 100 l sample
• 100 l 1.2 mM NADPH.Na₄

	Blank	Sample
25 mM Na-P (pH 7.8)	800 l	700 l
GSSG	100 l	100 l
NADPH.Na4	100 l	100 l
Sample	–	100 l

• Record oxidation of NADPH by reading the absorbance at 340 nm continuously for 180 seconds.
• Correction is made for auto-oxidation of NADPH without using enzyme extract.

(Extinction co-efficient of NADPH (E) = 6.2 mM^-1cm^-1at 340 nm).

Literature references

Foyer and Halliwell, 1976 C.H. Foyer and B. Halliwell, The presence of glutathione and glutathione reductase in chloroplasts: a proposed role in ascorbic acid metabolism, Planta 133 (1976), pp. 21-25.