Manual for Oxidative Stress Studies in plantsisca.co.in/BIO_SCI/lab_manual/ISBN...

Manual for

Oxidative Stress Studies

in plants

By

Monika Dalal

Manual for

Oxidative Stress Studies

in plants

By

Monika Dalal

National Research Centre on Plant Biotechnology

Pusa campus, New Delhi-12

2014

International E - Publication

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International E - Publication 427, Palhar Nagar, RAPTC, VIP-Road, Indore-452005 (MP) INDIA

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2014

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of the publisher.

ISBN: 978-93-84648-04-6

International Science Congress Association

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Manual for oxidative stress studies in plants iii

Preface

Reactive oxygen species (ROS) are routinely produced during various physiological processes of

the plants. Due to their highly reactive nature, ROS cause oxidative damage to membrane lipids,

proteins, DNA and RNA. To protect the cells from oxidative damage, cells have evolved anti-

oxidant defense which neutralizes, scavanges or dismutates ROS. Under normal physiological

conditions, a redox homeostasis is maintained in the cells. However, under stress conditions such

as abiotic or biotic stresses, there is an increased production of reactive oxygen species which

causes unrestricted oxidative damage to the cells (oxidative stress). Hence ROS were initially

considered as unwanted toxic by-products of aerobic metabolism of plants. However, the studies

in the past two decades revealed that reactive oxygen species (ROS) play a significant role in

plant growth, development, and biotic and abiotic stress responses. Therefore most of the plant

biologists working on plant growth and development and stress responses often measure ROS

and anti-oxidant enzymes. This manual covers methods for oxidative stress components most

commonly used and accepted by the National and International scientific community. Hence this

manual will be useful as ready reckoner for the students and scientists working in this area.

Date: 16.7.14

Place: New Delhi (Monika Dalal)



Manual for oxidative stress studies in plants iv

Contents

1 Introduction 1-4

2. Measurement of oxidative stress

2.1 Spectrophotometric method for hydrogen peroxide estimation 6

2.2 In situ detection of hydrogen peroxide 7

2.3 In situ detection of superoxide 8

2.4 NADPH oxidase assay 10

2.5 Measurement of lipid peroxidation 12

2.6 Conductivity test 13

2.7 Cell viability assay 15

3 Antioxidant Estimation

3.1 Ascorbic acid 17

3.2 Glutathione 18

4 Antioxidant enzyme assays

4.1 Preparation of enzyme extract 22

4.2 Protein estimation 22

4.3 Spectrophotometric assays for antioxidant enzymes

4.3.1 Superoxide dismutase 24

4.3.2 Ascorbate peroxidase 27

4.3.3 Glutathione reductase 28

4.3.4 Catalase 30

4.3.5 Peroxidase 31

5 Isozyme profile and in-gel activity assay of antioxidant enzymes

5.1 Preparation of sample 33

5.2 Native PAGE 33

5.3 Superoxide dismutase 36

5.4 Ascorbate peroxidase 37

5.5 Glutathione reductase 38

5.6 Peroxidase 38

5.7 Catalase 39

5.8 NADPH-oxidase 40

6 Calculation of enzyme activity 41

7 About the author 42



Manual for oxidative stress studies in plants 1

O2 + e- O2

∙ -

O2

+ e-

H2O2+ e-

2H +

+ e-

H +

.OH+ e-

H +H2OO2

∙-

Over all reaction : O2 + 4e- + 4H+ 2H2O

1. Introduction

The origin of oxidative stress is associated with the evolution of oxygen (O2) evolving

photosynthetic organisms about 2.7 billion years ago. The atmospheric oxygen in its ground-

state has two unpaired electrons with parallel spins. Hence, oxygen is usually non-reactive to

organic molecules which have paired electrons with opposite spins. However, partial reduction

or monovalent reduction of oxygen which involves transfer of single electron leads to generation

of active or reactive oxygen species/intermediates (AOS or ROS/ ROIs). These ROS result from

the excitation of O2 to form singlet oxygen (1O2) and transfer of one, two or three electrons to O2

to form a superoxide radical (O2.-), hydrogen peroxide (H2O2) or a hydroxyl radical (HO

.)

respectively. ROS are highly reactive species and cause unrestricted oxidation of various cellular

components and macromolecules. Oxidative stress can be defined as any condition in which

cellular redox homeostasis of the cells is disrupted. An imbalance in which the redox steady

state of the cell is altered in the direction of pro-oxidant can result in generation of ROS.

Sources of ROS in plant cell

ROS are routinely generated in the plant cells due to electron transport in chloroplast and

mitochondria. Superoxide radicals are generated when electrons are misdirected or donated to

oxygen.

In chloroplast, superoxide is produced through Mehler reaction, a process of photoreduction of

oxygen to produce superoxide by the electron acceptors of PSI. The tetravalent reduction of

oxygen to water in mitochondria also leads to generation of various active oxygen species.

Membrane associated NADPH oxidase is one of the potent system for generation of superoxide.

Enzymes such as xanthine oxidase, aldehyde oxidase, lipoxygenase, and other flavin




2O2∙ - + 2H+ 2H2O + O2

O2∙ -+Fe3+ + O2Fe

2+

+Fe2+ +Fe3+H2O2.OH OH-+

O2 + 3Chl∗ 1O2 + Chl

dehydrogenases are also capable of generating superoxide as a catalytic by-product. The

dismutation of superoxide via enzymatic or spontaneous reaction leads to the formation of H2O2

in cells.

H2O2 is also the by-product of β-oxidation of fatty acids and peroxisomal photorespiration

reactions. In presence of transition metal such as Fe3+

and Cu2+

, superoxide can undergo H2O2

dependent reduction to produce hydroxyl radical.

Chloroplasts are the primary source of singlet oxygen. Up on illumination, chlorophyll acts as

photosensitizer and transfers excitation energy to oxygen to produce singlet oxygen. Cell wall

peroxidases are also the source of superoxide and H2O2 in plants.

Under normal growth conditions, the production of ROS in the cells is low. However under

stress condition such as abiotic stress, pathogenesis, the cellular homeostasis is disturbed which

leads to enhanced production of ROS. ROS cause peroxidation of membrane lipids, protein

oxidation, enzyme inhibition, DNA and RNA damage.

Antioxidative defense

To protect cells from oxidative attack, organisms have evolved defense mechanisms constituted

by enzymatic and non-enzymatic components. The enzymatic antioxidant defense includes

enzymes that are capable of removing, neutralizing, or scavenging free radicals. These enzymes

are superoxide dismutase (SOD), catalase (CAT), peroxidase (POX), glutathione reductase (GR)

and ascorbate peroxidase (APX). The non enzymatic components include glutathione, ascorbate,

α- tocopherol, flavonoids, etc.




H2O2

H2O

APX

ASC

MDHA DHA

DHA reductase

NADPH

NADP

MDHA reductase

GSSG

2GSH

GR

NADPH

NADP

2 O2∙- + 2 H+ O2 + H2O2

SOD

2H2O2 H2O + O2Catalase

PeroxidaseH2O2 2H2O + RRH2+

SOD dismutates superoxide radical to H2O2 and oxygen. H2O2 gets disposed by catalase and

peroxidase.

Ascorbate peroxidase, glutathione reductase, and dehydroascorbate reductase constitute a

metabolic cycle to remove H2O2 which is known as Halliwell- Asada Pathway.

Tocopherol and carotenoids are effective quenchers of singlet oxygen. Ascorbate and glutathione

act as both antioxidants and as substrates in enzyme catalysed detoxification reactions.

Role of ROS in plant

Initially ROS were considered as toxic by - product of aerobic metabolism. However the studies

have revealed that reactive oxygen species (ROS) play a significant role in plant growth,

development, and biotic and abiotic stress responses. During pathogen attack, membrane bound

NADPH oxidase, cell wall peroxidases and apoplastic amine oxidases produce ROS which

activates other pathogen defense responses in plants. The studies on plant NADPH oxidases

which are also known as respiratory burst oxidase homologues (RBOHs) have revealed role of

ROS in the growth of root hairs and pollen tubes, cell wall loosening during germination, fruit

ripening, and hypocotyl elongation. During abiotic stress, role of AtRBOH in ABA induced

stomatal closure has been shown.




Suggested reading

• Apel K, Hirt H (2004) Reactive oxygen species: metabolism, oxidative stress, and signal

transduction. Annu Rev Plant Biol 55: 373-399

• Gapper C, Dolan L (2006) Control of plant development by reactive oxygen species.

Plant Physiol 141: 341–345

• Kwak JM, Nguyen V, Schroeder JI (2006) The role of reactive oxygen species in

hormonal responses. Plant Physiol 141: 323–329

• Moller IM, Jensen PE, Hansson A (2007) Oxidative modifications to cellular components

in plants. Annu Rev Plant Biol 58: 459-481

• Torres M A, Jones JDG, Dangl JL (2006) Reactive oxygen species signaling in response

to pathogens. Plant Physiol 141: 373–378




2. Measurement of Oxidative Stress

Oxidative stress can be measured by determining the concentration of reactive oxygen species

(ROS) that is superoxide (O2.-), hydroxyl radical (.OH) and hydrogen peroxide (H2O2) in the

cells. Among these, superoxide and hydroxyl radical are unstable in aqueous solution and hence

direct determination of these free radicals is very difficult in solution at room temperature. The

best option for characterizing free radicals is through Electron Paramagnetic Resonance (EPR)

spectroscopy. In EPR, spin traps are used where a nitrone or nitroso compound e.g. 5,5-

dimethyl-1-pyrroline N-oxide (DMPO), reacts with a target free radical to form a stable and

distinguishable free radical to be detected by EPR spectroscopy.

Hydrogen peroxide is relatively stable which allows direct measurement of its concentration.

There are several methods available for its detection in biological systems. These include

methods based on horseradish peroxidase, chemiluminescence based method, and detection

based on dyes (Xylenol orange, Amplex red dye). Recently, deprotection reaction-based probes

that fluoresce on H2O2-specific removal of a boronate group have been developed for selective

measurement of H2O2 in cells. Furthermore, organelle-targetable fluorescent probes has been

devised which can be genetically targeted to various subcellular organelles, and help in

estimation of H2O2 in the subcellular comparments where it is produced (Rhee et al. 2010).

There are indirect methods which are most commonly used for measuring the oxidative stress in

cells. These indirect methods are based on measurement of rate of production or removal of the

ROS and measurement of damage (of the target) by the ROS. As EPR is a highly specialized

instrument not available in all the labs, the EPR method has not been covered in this manual. All

other methods which can be convenient done in most of the labs are described here.

Reference

Rhee SG, Chang TS, Jeong W, Kang D (2010) Methods for detection and measurement of

hydrogen peroxide inside and outside of cells. Mol Cells 29: 539-549




H2O2 +Fe 2+ → Fe 3+ + HO• + OH−

Fe 3+ + XO → (Fe 3+ : XO)

2.1 Spectrophotometric method for Hydrogen Peroxide estimation

The spectrophotometric measurement of H2O2 is based on method given by Gay et al.

(1999), and Gay and Gebicki (2000).

Principle

The assay is based on the oxidation of ferrous ions (Fe 2+

) to ferric ions (Fe 3+

) by peroxides

e.g. H2O2, under acidic conditions. The Fe3+

forms a complex with xylenol orange (3, 3’-

bis [N, N-di (Carboxymethyl)-aminomethyl]-o-cresolsulfone-phthalein, disodium salt, XO)

resulting in a purple colored complex. The increase in the absorbance is recorded at 560

nm. Presence of sorbitol enhances the color intensity and sensitivity of the assay by

increasing the concentration of Fe3+

which produces colored complex with Xylenol

Orange.

Solutions

• Solution A: 25 mM ammonium ferrous (II) sulfate in 2.5 M H2SO4

• Solution B: 100 mM sorbitol, 125 μM xylenol orange in water. Store the solutions at 4°C.

Procedure

Assay solution (AS): Mix 1 volume of solution A with 100 volumes of solution B.

1 Assay solution (AS) 1 ml

2 Enzyme extract Up to 0.1 ml

Total volume 1.100 ml

Mix and incubate assay reactions for 30-45 minutes in dark at room temperature. Record

the absorbance at 560 nm. Plot standard curve of H2O2 using linear regression analysis and

calculate the sample H2O2 concentration.




Standard curve of hydrogen peroxide

Serially dilute a 30% (8.8M) hydrogen peroxide stock solution to generate concentration

range of 0-100μM. H2O2 dilutions for standards should be prepared fresh from the main

stock. To generate standard curve replace enzyme extract with serial diluted H2O2

standards in the above mentioned table.

Note: High Hydrogen peroxide can bleach color from the dye, resulting in a lower

absorbance value. Therefore it is advised to use serial dilution of extract to find optimum

range.

References

• Gay C, Collins J, Gebicki J (1999) Hydroperoxide assay with the ferric-xylenol orange

complex. Anal Biochem 273: 149–155

• Gay C, Gebicki JM (2000) A critical evaluation of the effect of sorbitol on the ferric-

xylenol orange hydroperoxide assay. Anal Biochem 284: 217-220

2.2 In situ detection of hydrogen peroxide

The histochemical staining of H2O2 is described according to Thordal-Christensen et al.

(1997) and Bindschedler et al. (2006).

Principle

3,3′-Diaminobenzidine (DAB) is oxidized by hydrogen peroxide in the presence of

peroxidases to generate a dark brown precipitate. This precipitate reveals the presence and

distribution of hydrogen peroxide in plant cells.

Solutions

• 3,3′-Diaminobenzidine (DAB) solution:

a) Make 1 mg ml−1

DAB with 0.05% v/v Tween 20 in 50 mM Tris acetate buffer

(pH 5.0)

OR




b) Dissolve 50 mg DAB in 45 ml distilled H2O (final conc. 1 mg ml−1

). Adjust the

pH to 3.0 with 0.2 M HCl (to dissolve DAB). Add 25 μl Tween 20 (0.05% v/v)

and 2.5 ml 200 mM sodium phosphate buffer to DAB solution. The pH will be

around 7.0.

• 90% Ethanol

• Bleaching solution: Mix ethanol, acetic acid and glycerol in the ratio of 3:1:1respectively.

Procedure

• Take leaf sample, if leaves are bigger in size, leaf discs may be used.

• Gently vacuum infiltrate with DAB solution and store in dark for 8-24 h.

• Remove the chlorophyll by incubating the leaves in 90% ethanol at 70 °C until complete

removal of chlorophyll. Alternatively, the leaves can be placed in bleaching solution and

heated at 90 - 95°C in water bath for 15 min until complete removal of chlorophyll.

• The brown precipitate formed by reaction of DAB with the hydrogen peroxide can be

directly visualized. Samples can be stored and examined in 70% glycerol.

• For control reaction, DAB solution is supplemented with 10 mM ascorbic acid (a

scavenger of H2O2) before infiltration.

Reference

• Thordal-Christensen H, Zhang Z, Wei Y, Collinge DB (1997) Subcellular localization of

H2O2 in plants. H2O2 accumulation in papillae and hypersensitive response during the

barley—powdery mildew interaction. Plant J 11: 1187-1194

• Bindschedler LV, Dewdney J, Blee KA, Stone JM, Asai T, Plotnikov J, Denoux C, Hayes

T, Gerrish C, Davies DR, Ausubel FM, Bolwell GP (2006) Peroxidase-dependent

apoplastic oxidative burst in Arabidopsis required for pathogen resistance. Plant J 47:

851-63

2.3 In situ detection of superoxide

In situ O2. -

is estimated based on the nitroblue tetrazolium (NBT) staining as described by

Jabs et al. (1996) and Dutilleu et al. (2003).




Principle

Superoxide radical reduces the NBT resulting in the formation of blue color formazan.

Solutions

• NBT solution: 0.5 mg ml-1 NBT prepared in 10 mM sodium phosphate buffer (pH 7.8).

• Superoxide dismutase (10 units ml-1)

• 90% ethanol

Procedure

1. Take leaf sample, if leaves are bigger in size, leaf discs may be used.

2. Gently vacuum infiltrate with NBT solution and store in dark for 1-2 h at room

temperature.

3. Incubating the leaves in 90% ethanol at 70 °C until complete removal of chlorophyll.

4. The blue color precipitate formed by reaction of NBT with the superoxide can be

directly visualized. Samples can be stored and examined in 70% glycerol.

5. For control, superoxide dismutase (10 units ml-1

) is added to the staining medium before

infiltration.

6. To inhibit the activity of SOD, inhibitors such as 1 mM diethyldithiocabamate (DDC) or

5 mM KCN can be added to the NBT solution.

7. To study the effects of NADPH – oxidase mediated superoxide generation; the leaf

discs can be pre-incubated with 10 mM imidazole or 50µM diphenyleneiodonium (DPI)

for 30 min prior to the incubation with NBT to inhibit NADPH-oxidase activity.

References

• Jabs, T, Dietrich RA, Dangl JL (1996) Initiation of runaway cell death in an

Arabidopsis mutant by extracellular superoxide. Science 27: 1853–1856

• Dutilleul C, Garmier M, Noctor G, Mathieu C, Chetrit Philippe, Foyer CH, de Paepe R

(2003) Leaf mitochondria modulate whole cell redox homeostasis, set antioxidant

capacity, and determine stress resistance through altered signaling and diurnal

regulation. Plant Cell 15: 1212-1226




NADPH + 2O2 O2-. + NADP+ + H+

NADPH oxidase

2.4 NADPH oxidase assay

NADPH oxidase is a membrane-bound enzyme that catalyzes the oxidation of NADPH

resulting in generation of superoxide. NADPH dependent superoxide (O2.-) generation in

membrane fraction is determined according to Van Gestelen et al. (1997).

Principle

Assay is based on the measurement of rate of SOD-inhibitable reduction of NBT using

NADPH as electron donor. NBT is reduced to blue color formazan by superoxide which

can be detected at 530 nm. The NBT reduction rates are linear with time up to 10 to 15 min

and are linearly dependent on the protein concentration (10-50 µg) in the sample.

Solutions

• Extraction medium: 50 mM Tris-HCL pH 7.5, 250 mM sucrose, 1 mm EDTA, 1 mM

ascorbic acid and 0.6% polyvinyl pyrrolidone.

• Resuspension buffer : 5 mM Potassium phosphate buffer, pH 7.8, 250 mM sucrose and

3 mM KCl

• 150 mM Tris-HCL buffer (pH 7.4)

• 750 mM sucrose

• 3 mM NADPH

• 3 mM Nitroblue tetrazolim chloride (NBT)

• 0.3 mM Diphenylene iodonium chloride (DPI)

Preparation of membrane fraction

1. Grind fresh tissue (0.5 g) in pre chilled mortar and pestle with 5 ml of ice-cold

extraction buffer.

2. Filter the homogenate through 4 layers of cheese cloth and transfer the filtrate to fresh

tubes on ice.

3. Centrifuge at 10,000 g for 15 min at 4°C. Take the supernatant.




4. Centrifuge the supernantant at 50,000 g for 10 minutes at 4°C to pellet the total

membrane fraction.

5. Discard supernatant and resuspend the pellet in 1 ml ice-cold resuspension buffer. This

membrane fraction is used for assay. All the steps of membrane preparation are carried

out at 4°C.

Procedure

The 3 ml reaction mixture consists of:

S.No. Stock solution Final

concentration

Volume for

reaction

1 150 mM Tris-HCL buffer (pH 7.4 ) 50 mM 1 ml

2 750 mM sucrose 250 mM 1 ml

3 3 mM NBT 0.1 mM 0.1 ml

4 Membrane fraction 10-50 μg protein Up to 0.8 ml

5 3 mM NADPH 0.1 mM 0.1 ml

Pre-incubated the reaction mix (containing 50mM Tris, 250mM sucrose, 0.1 mM NBT and

membrane fraction) for 1 min. The reaction is started by addition of 0.1 ml of NADPH and

change in absorbance is recorded at 530 nm up to 5 min.

Blank

• A reaction without NADPH to account for reduction of NBT in absence of NADPH.

Controls

• A reaction with 50 units ml-l SOD is added to measure the NBT reduction in the

presence SOD.

• A reaction is done in presence of 10 μM DPI (0.1 ml of 0.3 mM DPI) to inhibit NADPH

O2.- dependent generation.




Calculations

The selective reduction of NBT by superoxide is calculated from the difference in the NBT

reduction rate in the presence and absence of 50 units of SOD. The enzyme activity is

expressed as ∆ A530 nm per min per mg protein. Another method of measurement is to

calculate the difference in monoformazan concentrations (and hence superoxide

generation) using an extinction coefficient of 12.8 mM-l cm-l.

Reference

• Van Gestelen P, Asard H, Caubergs RJ (1997) Solubilization and separation of a plant

plasma membrane NADPH-O2- Synthase from other NAD(P)H oxidoreductases. Plant

Physiol 115: 543-550

2.5 Measurement of lipid peroxidation

Lipid peroxidation is oxidative degradation of lipid-fatty acids by reactive oxygen species

and hence it is considered as one of the measure of oxidative stress in the cells. The

method for lipid peroxidation estimation described by Heath and Packer (1968) is given as

follows.

Principle

Oxidative degradation of lipid-fatty acids by reactive oxygen species increases the

concentration of lipid hydroperoxides and aldehydes in the cells. These lipid

hydroperoxides and aldehydes reacts with 2-thiobarbituric acid (TBA) hence called as

Thiobarbituric acid reactive substances (TBARS). The TBARS content is measured in

terms of malondialdehyde (MDA) which results from decomposition of the unstable

peroxides of polyunsaturated fatty acids. MDA reacts with (TBA) resulting in the

formation of a red colored complex with absorbance maxima at 532 nm.

Solutions

• 0.1 % Trichloroacetic acid (TCA)

• Thiobarbituric acid reagent: 5% TBA in 20% TCA




Extraction

Leaf sample (0.1 g) is homogenized in 5 ml 0.1 % trichloroacetic acid (TCA). The

homogenate is centrifuged at 12 000 g for 10 min and supernatant is used for the

estimation of MDA content.

Procedure

1. To 1.0 ml aliquot of the supernatant, add 4.0 ml of Thiobarbituric acid reagent.

2. Heat the mixture for 30 min at 95° C in a water bath and immediately cool in an ice

bath.

3. Centrifuge the cool samples at 10 000 g for 10 min.

4. Record the absorbance of the clear supernatant at 532 nm and for non specific

absorbance at 600 nm. The value of non specific absorbance is subtracted from the

values recorded at 532 nm.

Control

• A sample blank containing the sample plus the TCA solution without TBA should be

taken for individual sample to account for non specific reactions.

The MDA content can be calculated either from a standard curve of MDA (0- 20µM range)

or based on the extinction coefficient of MDA 155 mM-1

cm-1

. For MDA standard, replace

sample with the dilutions of MDA. The MDA content is represented as nmols per g dry or

fresh weight.

Reference

• Heath, RL, Packer L (1968) Photoperoxidation in isolated chloroplast. I. Kinetics and

stoichiometry of fatty acid peroxidation. Arch Biochem Biophys 125: 189-198

2.6 Conductivity test

Oxidative stress induces lipid peroxidation and membrane damage. When the stress level is

high, it affects the membrane function and causes leakage of cellular ions and other

molecules. The change in permeability of membranes or injury can be readily quantified by




measuring cellular electrolyte leakage from affected leaf tissue into an aqueous medium.

Electrolyte leakage from affected leaf tissue is commonly used to measure the stress-

induced damage to the cells. The method described by Shanahan et al. (1990) is as

follows.

Procedure

1. Take leaf samples and cut into 10 mm leaf discs or pieces.

2. Place the leaf pieces in standard glass vials that can accommodate a conductivity

electrode.

3. Wash the sample for 2-3 times with de-ionized water.

4. Keep at least 5 replicates for each treatment (i. e. control and stress).

5. Add 20 ml of deionized water to each vial so that a leaf material is completely

submerged.

6. Incubate the vials at 10 °C for 24h.

7. After incubation take out the vials and allow the temperature to come to room

temperature.

8. Measure the conductivity of the medium by inserting a conductivity electrode into

each vial.

9. Cap all the vials and autoclave for 15 min to kill all tissues.

10. Allow the contents of the vials to cool to room temperature.

11. Measure the conductivity of all samples. Calculate the cell membrane stability and

membrane injury as follows

Formula

CMS % = {[1-(T1/T2)]/ [1-(C1/C2)]} x 100

Membrane injury % = 100 - CMS %

C1 and T1 are the initial conductivity readings of control (C) and stressed (T) sample after

10 °C incubation.




C2 and T2 are the final conductivity readings of control and stressed sample after

autoclaving. (CMS, cell membrane stability)

Reference

• Shanahan JF, Edwards IB, Quick JS, Fenwick RJ (1990) Membrane thermostability and

heat tolerance of spring wheat. Crop Sci 30: 247-251

2.7 Cell viability assay

Oxidative stress increases the reactive oxygen species which results in lipid peroxidation

and membrane damage ultimately leading to cell death. In case of biotic stresses, cell death

is associated with oxidative burst and hypersensitive response (HR). Thus cell viability is

an important variable in host-pathogen interaction.

Principle

The cell viability test also known as TTC reduction test is based on the ability of the tissue

to reduce 2,3,4-Triphenyl tetrazolium chloride (TTC). The live tissue will reduce the TTC

due to the activity of various dehydrogenases while dead tissue will not have the ability to

reduce TTC. The method described by Chen et al. (1982) is as follows.

Solutions

• 0.08% TTC in sodium phosphate buffer (50 mM, pH 7.5)

• 95% ethanol

Procedure

1. Take the leaf samples and cut into 10 mm leaf discs or pieces. Take samples with equal

fresh weight (e.g. 200 mg) for quantitative assay.

2. Transfer the leaf samples in glass vials and add 5 ml of 0.08 % TTC in sodium

phosphate buffer (50 mM, pH 7.5) and vacuum infiltrate the samples.

3. Incubate the samples for 10-12h in dark at room temperature.

4. Rinse the leaf samples with distilled water.




5. Place the samples in 5 ml of 95% ethanol. Evaporate the ethanol by heating the samples

in boiling water bath. This step extracts the red color formazan from the leaf samples.

6. Let the samples cool to room temperature. Add 10 ml of 95% ethanol and vortex.

7. Record the absorbance at 485nm.

The results can be presented as relative units or as percentage of control. In case of

qualitative analysis, do not extract the formazan with ethanol. Wash the cells with distill

water (step 4) and visualize the formazan in the tissue or cells.

Note: In place of TTC, other tetrazolium dyes e.g. 1% MTT 3-[4,5-dimethyltiazol-2-yl]-

2,5-diphenyltetrazolium bromide] can be used. Record the absorbance at 570 nm.

Reference

• Chen HH, Shen ZY, Li PH (1982) Adaptability of crop plants to high temperature

stress. Crop Sci 22: 719-725




2O 2. - + 2H+ 2 H2O2 + dehydroascorbate

Ascorbate

Tocopheroxyl radical + Ascorbate Tocopherol + Monodehydroascorbate

Non enzymatic action

Enzymatic action

H2O2 + 2 ascorbate 2H2O + 2 monodehydroascorbateAscorbate peroxidase

3. Antioxidant Estimation

3.1 Ascorbic acid estimation

Ascorbic acid is one of the major antioxidant metabolites in plant cells. It is responsible for

the non-enzymatic as well as enzymatic scavenging of reactive oxygen species. In non-

enzymatic mode of action, ascorbate reacts with superoxide radical or the tocopheroxyl

radical. In enzymatic mode, it scavenges H2O2 with the help of ascorbate peroxidase

enzyme and gets converted to monodehydroascorbic acid and/or dehydroascorbic acid. The

oxidized forms are recycled back to ascorbic acid by monodehydroascorbate reductase and

dehydroascorbate reductase using reducing equivalents from NADPH or glutathione,

respectively.

Principle

The method described by Mukherjee and Choudhuri (1983) gives total ascorbic acid

content in the cells. The estimation is based on the reduction of dinitrophenylhydrazine (in

acidic medium) by ascorbic acid to phenyl hydrazone which results in the formation of

pink coloured complex.

Solutions

• 6 % Trichloroacetic acid or 5 % metaphosphoric acid in 10 % acetic acid solution

• 2 % Dinitrophenylhydrazine: 2 g dinitrophenylhydrazine is dissolved in distilled water

made acidic by adding few drops of analytical grade concentrated hydrochloric acid,

and than volume is made up to 100 ml




• Thiourea solution (10 % thiourea in 70 % ethanol)

Extraction

Fresh leaf sample (0.5 g) is extracted with 10 ml of 6 % trichloroacetic acid or 5%

metaphosphoric acid in10 % acetic acid solution. Homogenate is centrifuged at 5000 g at 4

°C. Supernatant is used for estimation of total ascorbic acid.

Procedure

1. Mix 4 ml of the extract with 2 ml of 2 % dinitrophenylhydrazine (in acidic medium)

followed by the addition of 1 drop of 10 % thiourea (in 70 % ethanol).

2. The mixture is incubated at 37 °C for 3 h or in boiling water bath (100 °C) for 15 min.

3. Cool the reaction mixture by keeping in ice bath, and add 5 ml of 80 % (v/v) H2SO4 to

the mixture (kept on an ice bath).

4. Incubate for 30 min.

5. Record the absorbance at 530 nm.

6. For preparation of blank, 5 ml of dinitrophenylhydrazine reagent is replaced by 5 ml of

80 % (v/v) H2SO4.

Calculation

The concentration of ascorbic acid in the sample is calculated from a standard curve of

known concentration of ascorbic acid in 6 % Trichloroacetic acid or 5% metaphosphoric

acid in10 % acetic acid solution. For standard curve, replace sample with diluted ascorbate

solutions.

Reference

• Mukherjee SP, Choudhari MA (1983) Implications of water stress-induced changes in

the levels of endogenous ascorbic acid and hydrogen peroxide in Vigna seedlings.

Physiol Plant 58: 166-170

3.2 Glutathione estimation

Glutathione is an important antioxidant. It is a tripeptide of glutamic acid, cycteine, and

glycine. On oxidation, the sulphur of cysteine forms a thiyl radical that reacts with a




second oxidised glutathione forming a disulphide bond (GSSG). GSH reacts with singlet

oxygen, superoxide and hydroxyl radicals and functions as a free radical scavenger. In

enzymatic reactions, GSH acts as reducing agent that recycles oxidized ascorbic acid to

reduced form with the help of enzyme dehydroascorbate reductase (DHAR). The oxidized

form (GSSG) gets recycled to reduced form (GSH) by glutathione reductase enzyme.

Principle

GSH and GSSG are assayed according to the method of Smith (1985). Glutathione (GSH)

reacts with 5, 5’-Dithiobis (2-nitrobenzoic acid) (DTNB) to generate a colored compound

(5-mercapto-2-nitrobenzoic acid) and glutathione disulfide (GSSG). The GSSG is

enzymatically reduced to GSH by glutathione reductase used in the assay which then re-

enters the cycle. The colored compound is taken as the measure of total glutathione

concentration which can be determined by taking the absorbance at 412 nm.

Solutions

• 5% sulfosalicylic acid or 5% metaphosphoric acid

• 0.5 M Phosphate buffer (pH 7.5)

• 0.1 M Phosphate buffer (pH 7.5) with 5 mM EDTA

• 2-Vinylpyridine

• 2 mM NADPH

• 6 mM 5,5-dithiobis-(2-nitrobenzoic acid) (DTNB)

• Glutatione reductase (GR): Dilute with 0.1 M phosphate buffer (pH 7.5) to get 1 units ml

-1

Extraction

The leaf sample (1 g) is homogenized in 10 ml of ice cold 5% metaphosphoric acid. The

homogenate is centrifuged at 18 000 g for 15 min at 4 °C. The supernatant is used for the

glutathione pool.

Pre-treatment of the sample

The same supernatant is used for estimation of both GSSG and total glutathione pool

(GSH+GSSG). Take two aliquots of 1 ml each. Neutralize both the aliquot with 1.5 ml of

0.5 M phosphate buffer (pH 7.5). Treat one neutralized aliquot with 0.2 ml of

2‐vinylpyridine to mask GSH. This aliquot is used for GSSG estimation (aliquot A). Add

0.2 ml of water to the second neutralized aliquot (aliquot B) which is utilized for the total




glutathione pool (GSH+GSSG) assay. The contents of the tube are mixed or vortexed until

an emulsion formed. The tube is then incubated for 60 min at room temperature. This mix

is used as extract for the GSSG and total glutathione assay.

GSSG aliquot (A)

1 Supernatant 1 ml

2 0.5 M phosphate

buffer (pH 7.5)

1.5 ml

3 2‐vinylpyridine 0.2 ml

Procedure

The 1 ml reaction mix is prepared as follows

Stock conc. Volume for the reaction

0.1 M phosphate buffer, pH 7.5 with

5mM EDTA

0.5 ml

2 mM NADPH 0.1 ml

6 mM DTNB 0.2 ml

1 Unit GR 0.1 ml

Extract (neutralized aliquot-A or B) 0.1 ml

The reaction is started by adding of the extract (0.1ml) or GSH (for standard) and change

in absorbance is monitored at 412 nm every 1 min interval up to 5 min. A reaction blank is

prepared without sample.

Total glutathione aliquot (B)

1 Supernatant 1 ml

2 0.5 M phosphate

buffer (pH 7.5)

1.5 ml

3 Distilled water 0.2 ml




Calculation

The standard curve of GSH is prepared in the concentration range of 50-400 ng. GSH in

the sample can be estimated as the difference between the amount of total glutathione and

GSSG.

Reference

• Smith IK (1985) Stimulation of glutathione synthesis in photorespiring plants by

catalase inhibitors. Plant Physiol 79: 1044-1047




4. Antioxidant enzyme Assays

4.1 Preparation of enzyme extract

The tissue for enzyme assays should be either freshly harvested or weighed amount can be

frozen in liquid nitrogen and stored at -80 °C till further use. In case of stored samples, the

frozen sample should not be allowed to thaw till the time of extraction. For this, the

samples should be taken out of -80 °C and immediately kept in liquid nitrogen. For

preparing the extract, homogenize a pre-weighed sample (200 mg) in liquid nitrogen and

add the homogenized powder in ice cold 1.5 ml of 50 mM Sodium phosphate buffer (pH

7.0) containing 2 mM EDTA, 5 mM β - mercaptoethanol (β - ME) and 4% PVP-40.

Extraction procedure for all enzymes (superoxide dismutase, ascorbate peroxidase,

glutathione reductase, peroxidase, and catalase) is same except that for ascorbate

peroxidase for which the extraction buffer also contains 5 mM ascorbate. Centrifuge the

homogenate at 30 000 g for 30 min at 4 °C. The supernatant can be dispensed in aliquots

and stored at -80 °C till further analysis.

4.2 Protein estimation

For estimation of protein in the extracts follow the given procedure.

To an aliquot of 0.1 ml supernatant, add 0.1 ml of 20 % Trichloroacetic acid (TCA)

solution which will precipitate the protein. Incubate the precipitated protein for 1-2 h at -

20 °C followed by centrifugation at 10,000 g for 10 min at 4°C. Dissolve the pellet in 0.5

ml of 1N sodium hydroxide solution. An aliquot from this is taken for protein estimation.

Protein estimation procedure

Protein estimation by Lowry et al. (1951) is described as follows:

Solutions:

• Solution A: 2 % Sodium carbonate in 0.1 N Sodium hydroxide

• Solution B: 1 % Copper sulphate solution

• Solution C: 2 % Sodium –Potassium tartarate solution




• Solution D: (Alkaline copper solution) 1ml of reagent (B) and 1 ml of reagent (C) in

100 ml of reagent (A)

• Solution E: 1N Folin – Ciocalteu reagent

Procedure

1. To an aliquot of TCA processed sample (made up to 1 ml), add 5 ml of solution (D).

2. After 10 min incubation at room temperature, add 0.5 ml of solution (E).

3. Mix the mixture immediately by vortexing.

4. Incubate for 30 min at room temperature and record the absorbance at 610 nm.

5. The reaction mix without protein serves as blank.

6. Protein concentration is calculated from standard curve of Bovine Serum Albumin

(BSA).




2 O2∙- + 2 H+ O2 + H2O2

SOD

4.3 Spectrophotometric assays for antioxidant enzymes

4.3.1 Superoxide dismutase (SOD, EC: 1.15.1.1)

For SOD activity measurement, method described by Beauchamp and Fridovich (1971) is

given as follows.

Principle

SOD catalyze the dismutation of superoxide radical (O2∙ -

) to hydrogen peroxide (H2O2).

The SOD activity assay is based on the ability of superoxide dismutase to inhibit the

reduction of nitro-blue tetrazolium (NBT) by superoxide. In this method, illumination of

riboflavin in the presence of O2 and electron donor like methionine generates superoxide

anions. In absence of superoxide dismutase activity, superoxide radical (O2.-) interacts

with NBT thereby reducing the yellow tetrazolium to a blue precipitate (Formazan). The

control reaction which is done without any enzyme will show maximum blue color. In

presence of enzyme the intensity of color decreases with increase in enzyme activity. The

reduction of NBT by superoxide radicals to blue coloured formazan is measured at 560 nm.

In the reaction, factors such as intensity, distance and duration of illumination and the

reaction temperature create variability. Hence calibration for these parameters is a must for

the individual laboratories.

Solutions

• 50 mM Sodium phosphate buffer, pH 7.8

• 0.12 mM Riboflavin (store cold in a dark bottle)

• 1.72 mM Nitroblue tetrazolium (NBT) (store at 4 °C)

• 201 mM Methionine

• 1% Triton X-100




Assay solution

From stock solutions, an assay solution is prepared as given below

• 27 ml Sodium phosphate buffer

• 1ml NBT solution

• 1.5 ml of Methionine solution

• 0.75 ml of Triton X-100 solution

Procedure

1. Assay mix 1 ml

2. enzyme extract Up to 0.1 ml

3. riboflavin 0.03 ml

Place the tubes in a light box providing uniform light intensity. A foil-lined box with an

internally mounted 40 W fluorescent bulb would be ideal. Incubate the tubes for 8 minutes

under illumination. The reaction is stopped by switching off the illumination. Record the

absorbance of the reactions at 560 nm.

The different isoforms of SOD viz. Cu/Zn-SOD, Fe-SOD, and Mn-SOD can be

distinguished on the basis of their sensitivity to cyanide and hydrogen peroxide. The Cu/Zn

SOD is inhibited by cyanide while H2O2 inhibits both Cu/Zn-SOD and Fe-SOD whereas

Mn-SOD is unaffected. For this, add 3 mM of KCN or 5 mM H2O2 in the reaction mixture.

Controls

• A reaction without enzyme exposed to light along with tests gives maximum blue color.

This is considered as 100 % NBT reduction or no inhibition (v).

• A reaction without enzyme but not exposed to light serves as blank for the

spectrophotometer readings.




Formula

One unit of enzyme activity is defined as the amount of enzyme that inhibits NBT

reduction by 50%.

The activity is calculated as Unit = [v/V-1]

Where v and V are absorbance in the absence and presence of enzyme extract,

respectively. The activity is represented as units per mg protein (U mg -1 protein).

There are two ways to estimate the 50 % inhibition.

1. A series of reactions with different volumes of the (same) enzyme extract need to be

prepared e.g. 0.01 to 0.1 ml. Determine percent inhibition of NBT reduction by plotting

the percent inhibition versus amount of enzyme in test reactions. Determine the amount

of enzyme resulting in one half of maximum inhibition.

2. Use one concentration or volume of enzyme and calculate the enzyme activity based on

standard curve generated from pure enzyme (SOD) activity.

Reference

• Beauchamp C, Fridovich I (1971) Superoxide dismutase: improved assay and an assay

applicable to acrylamide gels. Anal Biochem 44: 276-287




H2O2

H2O

APX

ASC

MDHA DHA

DHA reductase

NADPH

NADP

MDHA reductase

4.3.2 Ascorbate Peroxidase (APX, EC: 1.11.1.11)

For APX activity measurement method described by Nakano and Asada (1981) is given as

follows.

Principle

Ascorbate peroxidase (APX) reduces hydrogen peroxide to water with the help of ascorbic

acid, which is oxidized to mono-dehydroascorbic acid (MDHA). Mono-dehydroascorbic

acid is spontaneously metabolized to dehydroascorbate (DHA). MDHA and DHA are

converted to ascorbate by the enzymes MDHA reductase and DHA reductase respectively.

There are chloroplastic and cytosolic isozymes of ascorbate peroxidase which show some

differences in their enzymatic properties.

The assay is based on the decrease in absorbance at 290 nm, due to oxidation of ascorbic

acid to mono-dehydroascorbic acid and dehydroascorbic acid (Nakano and Asada, 1981).

Preparation of enzyme extract

Extraction is same as described in section 4.1, except that 5 mM ascorbate is added in the

extraction buffer.

Solutions

• Solution A: Sodium phosphate buffer (100 mM, pH 7.0) containing 0.6 mM ascorbate

and 0.12 mM EDTA

• Solution B: 1.5 mM H2O2 solution




Procedure

1 Solution A 1.5 ml


3 Solution B 0.2 ml

4 Distilled water 1 ml

Total volume 3 ml

The reaction is started by the addition of solution B and the decrease in absorbance is

recorded at 290 nm (in UV-VIS spectrophotometer) at interval of 15 sec up to 60 seconds.

A reaction without the solution B serves as blank as blank.

The enzyme activity is can be calculated by two methods

1. Find the initial and final concentration of ascorbic acid using linear regression analysis

of the standard curve with known concentrations of ascorbic acid. Calculate the

concentration of ascorbic acid oxidized (initial reading – final reading = quantity of

ascorbic acid oxidized) per min per mg protein.

2. Based on extinction coefficient of ascorbate 2.8 mM-1 cm-1. Present results as nmol per

min per mg protein (nmol H2O2 min-1

mg-1

protein), taking into consideration that 1.0

mol of ascorbate is required for the reduction of 1.0 mol of H2O2.

Reference

• Nakano Y, Asada K (1981) Hydrogen peroxide is scavenged by ascorbate specific

peroxidase in spinach chloroplasts. Plant Cell Physiol 22: 867-880

4.3.3 Glutathione reductase (GR, EC: 1.6.4.2)

For GR activity measurement, method described by Schaedle and Bassham, (1977) is

given as follows.

Principle

Glutathione reductase catalyze the reduction of oxidized glutathione (glutathione

disulphide) (GSSG) to reduced glutathione (GSH) by using NADPH as reductant.




GSSG

2GSH

GR

NADPH

NADPDHA

ASC

DHA reductase


Refer to section 4.1 for preparation of enzyme extract.

Solutions

• Solution A: Phosphate buffer (100 mM, pH 7.5) containing 7.2 mM MgCl2

• Solution B: 3.6 mM NADPH

• Solution C: 15 mM oxidized glutathione (GSSG)

Procedure

1 Solution A 1.5 ml

2 Solution B 0.1 ml

3 Solution C 0.1 ml


5 Distilled water 1 ml

Total volume 3 ml

The reaction is started by adding solution B. The decrease in absorbance is recorded at 340

nm at interval of 60 sec up to 5 min. The reaction mix without solution B (NADPH) serves

as blank. Enzyme activity is calculated based on extinction coefficient of NADPH 6.22

mM-1

cm-1

. The enzyme activity is expressed as nmol of NADPH per min per mg protein.




Catalase2H2O2 2 H2O + O2

Reference

• Schaedle M, JA Bassham (1977) Chloroplast glutathione reductase. Plant Physiol 59:

1011-1012

4.3.4 Catalase (CAT) (EC: 1.11.1.6)

The catalase activity described by Aebi (1984) is given as follows.

Principle

Catalase catalyzes the reduction of hydrogen peroxide to water and molecular oxygen.

Compared to ascorbate peroxidase it is considered a less efficient system of H2O2

scavenging due to its higher Km value for H2O2 than APX. It is also localized in

mitochondria and peroxisomes, and absent in chloroplast, one of the important site of H2O2

generation.

Catalase assay is based on the absorbance of H2O2 at 240 nm (UV-range). A decrease in

the absorbance is recorded over a time period.



Assay solutions

• Solution A: Sodium phosphate buffer (100 mM, pH 7.0)

• Solution B: 30 mM H2O2 solution

Procedure

1 Solution A 1.5 ml


3 30 mM H2O2 1 ml

4 Distill water 0.4 ml

Total volume 3 ml




RH2 + H2O2 2 H2O + R Peroxidase

The reaction is started by addition of 1 ml of 30 mM H2O2. The decrease in absorbance is

recorded for 1 min at 15 sec interval at 240 nm. The reaction mix in which H2O2 solution is

replaced by phosphate buffer serves as blank.

The enzyme activity is can be calculated by two methods

1) Calculate the initial and final concentration of H2O2 using linear regression analysis of

the standard curve with known concentrations of hydrogen peroxide and calculate the

concentration of H2O2 reduced per min per mg protein.

2) Based on extinction coefficient of H2O2 39.4 mM-1

cm-1

.

Reference:

• Aebi H (1984) Catalase in vitro. Methods Enzymol 105: 121-126

4.3.5 Peroxidase (POD) (EC: 1.11.1.7)

For POD activity measurement, method described by Chance and Maehly (1955) is given

as follows.

Principle

Peroxidase also referred as non-specific peroxidase or guaiacol-peroxidase catalyses the

reduction of hydrogen peroxide with a concurrent oxidation of guaiacol to a colored tetra-

guaiacol. The increase in absorbance is recorded at 470 nm.



Assay solutions

• Solution A: Sodium phosphate buffer (100 mM, pH 7.0)

• Solution B: 10 mM H2O2 solution

• Solution C: 20 mM guaiacol




Procedure

1 Solution A 1.5 ml


3 Solution B 0.120 ml

4 Solution C 0.480 ml

Total volume 3 ml

The reaction is started by addition of solution C (20 mM guaiacol). The increase in

absorbance is recorded for 3 min at 1 min interval at 470 nm. The reaction mix without

enzyme serves as blank.

Enzyme activity is calculated as per extinction coefficient of its oxidation product, tetra-

guaiacol 26.6 mM-1

cm-1

. Enzyme activity is expressed as μmol tetra-guaiacol formed per

min per mg protein.

Reference

• Chance B, Maehly AC (1955) Assay of catalase and peroxidase. Methods enzymol 2:

764-775




5. Isozyme profile and in-gel activity assay for anti-

oxidant enzymes

5.1 Preparation of sample

The enzyme extract preparation is same as that described for spectrophotometric assays,

except for NADPH oxidase where membrane fraction (as described for spectrophotometric

assay of NADPH oxidase) is used.

5.2 Native PAGE for isozyme profile and in-gel activity

Native polyacrylamide gel electrophoresis (Native PAGE) separates proteins primarily by

their charge-to-mass ratio and in their native conformations. The native electrophoresis can

be done with Laemmli protocol (Laemmli, 1970) by omitting the SDS and reducing agents

such as DTT from the standard Laemmli protocol. Care should be taken not to include the

reducing agent in the sample buffer and do not heat the sample. This non-reducing and

non-denaturing separation technique maintains protein secondary and tertiary structure and

allows for the detection of biological activity.

Stock solutions for Native PAGE

Resolving gel buffer: 1.5 M Tris-Cl Buffer (pH 8.8)

Tris - 18.15 g

ddH2O - 75 ml

Adjust the pH to 8.8 with HCl and make up to 100 ml with ddH2O. Solution can be stored

up to 3 months at 4 °C.

Stacking gel buffer: 0.5 M Tris-Cl, pH 6.8

Tris - 06.0 g

ddH2O - 75 ml

Adjust the pH to 6.8 with HCl and make up to 100 ml with ddH2O. Solution can be stored

up to 3 months at 4 °C.




Acrylamide/Bis-acrylamide stock solution (30%/0.8% w/v)

Acrylamide - 30.0 g

Bis-acrylamide - 0.8g

Distilled water - made up to 100ml

Store in dark bottle at 4 °C

APS solution: 10 % (w/v) ammonium persulfate (APS)

Ammonium persulphate - 0.1g

Distilled water - 1.0 ml

APS solution should be made fresh.

Sample loading buffer (2X): (0.125 M Tris-Cl, 20% v/v glycerol, 0.02% bromophenol

blue, pH 6.8, 10 ml)

Solution B (stacking gel buffer) - 2.5 ml

Glycerol - 2.0 ml

Bromophenol blue - 2.0 mg

ddH2O - made up to 10.0 ml

Store in 0.5 ml aliquots at -20 °C for up to 6 months.

Tank buffer or electrode buffer: (0.025 M Tris, 0.192 M glycine, pH 8.3)

Tris - 3.028 g

Glycine - 14.413 g

ddH2O - made up to 1L

It is not necessary to check the pH. Store at room temperature for up to 1 month.




Resolving gel: (10ml volumes is sufficient for 1 gel in mini apparatus)

Resolving gel Final gel concentration

Gel Percentage 7% 10% 12%

Acrylamide solution 2.33 ml 3.33 ml 4 ml

Resolving gel buffer (pH8.8) 2.5 ml 2.5 ml 2.5 ml

ddH2O 5.115 ml 4.115 ml 3.445

Ammonium persulphate

solution∗

0.05 ml

(50 µl)

0.05 ml

(50 µl)

0.05 ml

(50 µl)

TEMED∗ 0.005 ml

(5 µl)

0.005 ml

(5 µl)

0.005 ml

(5 µl)

Total volume 10 ml 10 ml 10 ml

∗∗∗∗ APS and TEMED should be added in the last and should be mixed immediately and

poured for casting the gel.

Stacking gel: (5 ml volumes is sufficient for 1 gel in mini apparatus)

∗∗∗∗ APS and TEMED should be added in the last and should be mixed immediately and

poured for casting gel.

Stacking gel Final gel

concentration

Gel Percentage 4%

Acrylamide solution 0.67 ml

stacking gel buffer (pH 6.8) 1.25 ml

ddH2O 3.05 ml

Ammonium persulphate solution∗ 0.025 ml (25 µl)

TEMED∗ 0.005 ml (5 µl)

Total volume 5 ml




Electrophoresis

Estimate the protein concentration of the samples. For loading, calculate the volume of

enzyme extract required for loading of equal concentration of protein. Take 50-200µg of

protein and based on volume of the sample mix equal volume of sample loading buffer.

The samples should be maintained at 4 °C to avoid inactivation and degradation of

proteins. After loading the sample, the initial run can be done at 70 volts and after the

sample reaches resolving gel the volts can be increased to 120.

Note: Different enzymes may required different resolving gel percentage (%). Gel running

conditions for all enzyme activity gels is same except for APX which requires 2 mM

ascorbate in the running buffer and a pre-run of 30 min prior to sample loading.

Reference

• Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of

bacteriophage T4. Nature 227: 680-685

5.3 Superoxide dismutase isozymes and in-gel activity

SOD isoforms can be resolved in 10 % native gel. The gels are stained according to

method described by Beauchamp and Fridovich (1971). After the run, soak the gel in 50

mM sodium phosphate buffer containing 0.24 mM NBT and 28 µM riboflavin for 20 min

in dark. After incubation, transfer the gel in 28 mM TEMED solution and expose to light

source (box with 40 W fluorescent bulbs) at room temperature. The stained native activity

gels have a light to dark purple background with clear (achromatic) bands representing the

area where SOD isozymes are present.

A representative gel for SOD isozyme profile from wheat leaf




For differentiation of the isoforms (Cu-Zn SOD, Fe-SOD and Mn-SOD), the gels are

incubated with 3 mM KCN or 5 mM H2O2 for 30 min prior to NBT staining. Incubation in

3 mM potassium cyanide inhibits Cu-Zn SOD, 5 mM H2O2 inhibits both Cu-Zn SOD and

Fe-SOD while Mn -SOD is not affected by both the inhibitors. The gels can be kept in

distilled water and stored at 4°C

Reference

• Beauchamp C, Fridovich I (1971) Superoxide dismutase: improved assay and an assay

applicable to acrylamide gels. Anal Biochem 44: 276-287

5.4 Ascorbate peroxidase isozymes and in-gel activity

APX isoforms are resolved in 10 % native gel. Before loading the sample, remember to

add 2 mM ascorbate in the running buffer and pre-run the gel for 30 min. The staining

procedure given by Mittler and Zilinskas (1993) is described as follows. After run,

equilibrate the gels with sodium phosphate buffer (50 mM, pH 7.0) containing 2 mM

ascorbate for 30 min. Transfer the gel to a solution of sodium phosphate buffer (50 mM,

pH 7.0) containing 4 mM ascorbate and 2 mM H2O2 for 20 min. Wash the gels with

sodium phosphate buffer (50 mM, pH 7.0) for 1 min. Visualize the isoforms by

submerging the gel in solution of sodium phosphate buffer (50 mM, pH 7.0) containing 28

mM TEMED and 2.45 mM NBT. Allow the reaction to continue for 10 min and stop the

reaction by washing the gel with distilled water. As this staining is also based on NBT, the

stained native activity gels have a light to dark purple background with clear (achromatic)

bands representing the area where APX isozymes are present. The gels can be kept in

distilled water and stored at 4°C.

A representative gel for APX isozyme profile from leaf of different genotypes of wheat




Reference

• Mittler R, Zilinskas BA (1993) Detection of ascorbate peroxidase activity in native gels

by inhibition of the ascorbatedependent reduction of nitroblue tetrazolium. Anal

Biochem 212: 540-546

5.5 Glutathione reductase isozymes and in-gel activity

The GR isoforms are resolved in 10 % native gel. The isoforms are detected according to

Anderson et al. (1990). Incubate the gel in 50 mM Tris-Cl (pH 7.0) containing 0.24 mM of

(3-[4, 5-Dimethyl thiazol – 2, yl] -2, 5 –diphenyl –tetrazolium bromide (MTT), 0.34 mM

of 2, 6 – dichlorophenolindophenol, 3.6 mM GSSG and 0.4 mM NADPH for 1h. The gel is

de-stained with distilled water. For accuracy, the duplicate gels can be assayed for GR

activity with and without GSSG. The gels can be kept in distilled water and stored at 4°C.

A representative gel for GR isozyme profile from wheat leaf

Reference

• Anderson JV, Hess JL, Chevone BI (1990) Purification, characterization, and

immunological properties of two isoforms of glutathione reductase from Eastern white

pine needles. Plant Physiol 94: 1402–1409

5.6 Peroxidase isozymes and in-gel activity

The POD isoforms are resolved in 7 % native gel. The isoforms are stained according to Rao et

al. (1996). The gel is incubated in sodium acetate buffer (200 mM, pH 5.0) containing 2 mM

benzidine. The isoforms are visualized by addition of 3 mM H2O2. The gels can be kept in





A representative gel for POD isozyme profile from leaf of different genotypes of wheat

Reference

• Rao MV, Paliyath G, Ormrod DP (1996) Ultraviolet B and ozone induced biochemical

changes in antioxidant enzymes of Arabidopsis thaliana. Plant Physiol 110: 125-136

5.7 Catalase isozymes and in-gel activity

Catalase staining is done following the method of Woodbury et al. (1971). The isoforms

are resolved in 7 % native gel. Rinse the gel in 3 changes of distilled water for 15 min

each. Incubate the gels in 0.003 % H2O2 for 10 min and give a brief rinse with distilled

water. The CAT isoforms are detected by incubating the gel in 1 % ferric chloride and 1%

potassium ferricyanide solution for 10 min. Rinse the gel in distilled water and store. The

stained activity gels will have a light to dark green appearance with clear (achromatic)

bands representing the area where CAT isozymes are present. The gels can be kept in


A representative gel for CAT activity gel from wheat leaf

Reference

• Woodbury W, Spencer AK, Stahman MA (1971) An improved procedure using

ferricyanide for detecting catalase isozymes. Anal Biochem 44: 301–305




5.8 NADPH-oxidase in-gel activity

NADPH oxidase isoenzymes separation and staining is done as per the method of Lopez-

Huertas et al. (1999). Load 100 to 200 μg protein (membrane fraction as described for

spectrophotometric estmation of NADPH oxidase, section 2.4) per sample (depending on

activity level). Tha staining is based on reduction of NBT by superoxide resulting in the

formation of blue color formazan. Resolve the protein in 7.5 % native gel. Pre-incubated

the gels in the dark for 20 min in a solution containing 50 mM Tris-HCl buffer (pH 7.4)

and 2 mM NBT. Transfer and incubate the gel in 100 ml of 1 mM NADPH solution. The

appearance of blue formazan bands is indicative of NADPH oxidase activity. As controls,

inhibitors such as 50 mM DPI (inhibits NADPH oxidase) may be added in the pre-

incubation (NBT) solution. The reaction is stopped by immersion of the gels in distilled

water. Gels can be stored in distilled water at 4 oC.

Reference

• Lopez-Huertas E, Corpas JF, Sandalio ML, del Rio AL (1999) Characterization of

membrane polypeptides from pea leaf peroxisomes involved in superoxide radical

generation. Biochem J 337: 531-536




Rate of change of absorbance / min

Extinction coefficient (in μM-1) ∗= X µmol ml-1 min-1

6. CALCULATION OF ENZYME ACTIVITY

The enzyme activities are presented as

1. Units of substrate used or product formed per unit sample weight per unit time (μmol or

nmol g-1

fresh wt. or dry weight min-1

). In case of abiotic stress experiments in which

there is changes in tissue water content, activity should be presented on dry weight basis.

Or

2. Units of substrate used or product formed per unit weight of protein per unit time (μmol

or nmol min-1

mg-1

protein)

Units of substrate used or product formed can be calculated either by referring to a standard

curve prepared from known concentration of substrate or product or purified enzyme per se.

or by multiplying with its extinction coefficient (ε) reported in literature.

Formula for calculation based on extinction coefficient:

∗If path length is 1cm

Calculate and correct for the dilution factor and weight of the sample and to calculate specific

activity, divide the value obtained by the amount of protein present in the sample.




7. About the author

Monika Dalal is a Senior Scientist at National Research Centre on Plant

Biotechnology (ICAR). She is Ph. D. in Life Sciences from Devi Ahilya

University, Indore (MP). She did her Post Doctorate from Indian Institute of

Science, Bangalore, and Department of Plant Sciences, University of Arizona,

USA. Currently she is working on ‘Root biology under drought stress

conditions in wheat’.

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