Chapter 16

Spectrophotometric Assays for Antioxidant Enzymes

in Plants
Sathya Elavarthi and Bjorn Martin

Abstract
Reactive oxygen species (ROS) are formed in biological systems as part of normal metabolism. Adverse
environmental factors like drought stress result in increased levels of ROS that are detrimental to the plant
(1, 2). To avoid damage caused by these excess ROS, plants have developed elaborate mechanisms to
manage them at sustainable levels. Enzymes play an important role in lowering the ROS levels and helping
avoid oxidative stress. Superoxide dismutase, catalase, ascorbate peroxidase, and glutathione reductase
play a vital role in combating oxidative stress. Measuring these enzyme activities spectrophotometrically
provides researchers an easy and precise way to study and understand an important part of the defense
against oxidative stress. In this chapter we provide details of the assays we used to determine the enzyme
activities spectrophotometrically. Antioxidant enzyme responses to moderate water-deficit stress were
studied. All enzyme assays were conducted using wheat leaf tissue.

Key words: Antioxidant enzymes, ascorbate peroxidase, catalase, glutathione reductase, oxidative
stress, superoxide dismutase.

1. Introduction

Plants are frequently exposed to oxidative stresses that are caused

by both biotic and abiotic factors. Under stress, enormous
amounts of reactive oxygen species (ROS) are produced that can
cause peroxidation, resulting in damage to cell membranes, pro-
tein oxidation, enzyme inhibition, and strand breakage in nucleic
acids (3). Plants have developed elaborate mechanisms to detoxify
these ROS and in these mechanisms, enzymes play a very impor-
tant role (4). Major ROS scavenging enzymes in plants include

R. Sunkar (ed.), Plant Stress Tolerance, Methods in Molecular Biology 639,

DOI 10.1007/978-1-60761-702-0_16, © Springer Science+Business Media, LLC 2010

273
274 Elavarthi and Martin

superoxide dismutase (SOD), catalase (CAT), ascorbate peroxi-

dase (APX), and glutathione reductase (GR) (5).
Transgenic wheat lines containing a bacterial mtlD gene
for mannitol biosynthesis were used in this study (6). Manni-
tol has been shown to protect plants from oxidative stress dam-
age by scavenging hydroxyl radicals (7). The antioxidant enzyme
activities of transgenic lines containing the enzyme mannitol-
1-phosphate dehydrogenase (mtlD) targeted to cytosol (TA2
lines) and chloroplasts (TA5 lines) along with a transgenic con-
trol (pahc20) and wild type (Bobwhite) were determined under
well-watered and water-deficit conditions. Moderate water-deficit
stress was imposed on the plants over a 30-day period starting
at the jointing stage of the plants and the antioxidant enzyme
responses to stress were studied (Fig. 16.1). The stress levels
were controlled by measuring volumetric water content of the
pots using time domain reflectometry (TDR).

Fig. 16.1. Superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX), and glutathione reductase (GR)
activities were determined on wheat leaf samples from both well-watered and stressed plants. Samples were collected
from Bobwhite (wild type), pahc20 (transgenic control), 2-115, 2-118 [mannitol-1-phosphate dehydrogenase (mtlD) tar-
geted into cytosol], and 5-104, 5-108 (mtlD targeted into chloroplast) after 30 days of stress. The enzyme activities
were determined spectrophotometrically and expressed as units SOD per gram fresh weight (SOD), millimoles H2 O2 per
minute per gram fresh weight (CAT), micromoles ascorbate per minute per gram fresh weight (APX), and millimoles TNB
per minute per gram fresh weight (GR).
 Antioxidant Enzyme Assays 275

1.1. Superoxide 1. SODs are metalloproteins that catalyze the dismutation reac-
Dismutase (SOD) tion of two superoxide free radicals to one molecule of O2
and one molecule of H2 O2 .
2. SODs occur in different isoforms with different metal co-
factors.
3. SODs are considered the first line of defense against damage
by the superoxide radical (8).
4. Superoxide free radicals are generated in vitro, especially in
the presence of bright light.
5. Nitroblue tetrazolium (NBT), which is a yellow compound,
is reduced to blue monoformazon by the superoxide radical.
6. SOD activity is quantified based on the competitive inhi-
bition of NBT reduction by the superoxide radical. At
near-neutral pH, the dismutation rate of superoxide radicals
increases 1,000-fold in the presence of SOD.

1.2. Catalase (CAT) 1. CAT is a heme protein that catalyzes the decomposition of
H2 O2 into H2 O and O2 .
2. CAT is present in the cytosol and in peroxisomes.
3. Rapid decomposition of H2 O2 in the cell reduces the like-
lihood of formation of the highly reactive hydroxyl radical
through metal-catalyzed Fenton and Haber-Weiss reactions.
4. The decomposition of H2 O2 in the presence of CAT is fol-
lowed as a decrease in absorbance at 240 nm.
5. CAT activity is measured as the decrease in absorbance per
unit time.

1.3. Ascorbate 1. Ascorbate-specific peroxidases play an important role in

Peroxidase (APX) decomposition of H2 O2 generated both in the chloroplasts
and in the cytosol.
2. APXs are distributed in stroma, thylakoids, microbodies,
cytosol, and mitochondria (9).
3. Ascorbate acts as the electron donor for the decomposition
of H2 O2 in the cell.
4. The enzyme activity is assayed by measuring the rate of
decrease in absorbance of ascorbate at 290 nm.

1.4. Glutathione 1. GR is a NADPH-dependent enzyme that catalyzes the

Reductase (GR) reduction of oxidized glutathione (GSSG) to reduced glu-
tathione (GSH) (10).
2. GR helps to maintain a high ratio of GSH/GSSG as part of
the ascorbate–glutathione cycle, thus playing an important
role in cell metabolism (11).
276 Elavarthi and Martin

3. The activity of GR is measured by following the reduction

of 5,5 -dithio-bis-(2-nitrobenzoic acid) (DTNB) to 2-nitro-
5-thiobenzoic acid (TNB) by GSH.
4. The increase in absorbance per unit time at 412 nm due
to the formation of TNB is determined using a spectropho-
tometer.

2. Materials

2.1. Hardware and 1. A UV/Vis spectrophotometer.

Crude Leaf Extract 2. Quartz/clear plastic disposable cuvettes.
3. Liquid nitrogen.
4. Mortar and pestle.
5. Refrigerator for storing buffers.
6. Fresh leaves.
7. Potassium phosphate buffer (0.2 M, pH 7.0) containing
0.1 mM EDTA.

2.2. Superoxide 1. Potassium phosphate buffer (50 mM, pH 7.8) with 2 mM

Dismutase (SOD) EDTA (see Notes 1–4).
2. L -Methionine (30 mg/mL).
3. NBT hydrochloride (1.41 g/mL).
4. Triton-X100 (1%, w/v).
5. Riboflavin (0.044 g/mL).
6. Pure SOD from Sigma.

2.3. Catalase (CAT) 1. Potassium phosphate buffer (50 mM, pH 7.0) (see
Notes 1–4).
2. H2 O2 solution (30 mM).

2.4. Ascorbate 1. Potassium phosphate buffer (50 mM, pH 7.0) (see

Peroxidase (APX) Notes 1–4).
2. Ascorbic acid solution (100 mM).
3. H2 O2 solution (100 mM).

2.5. Glutathione 1. Potassium phosphate buffer (50 mM, pH 7.8) with 2 mM

Reductase (GR) EDTA (see Notes 1–4).
2. DTNB solution (3 mM).
3. NADPH solution (10 mM).
4. GSSG solution (10 mM).
 Antioxidant Enzyme Assays 277

3. Methods

3.1. Crude Leaf 1. Fresh leaf tissue was collected from stressed and well-watered
Extract for plants of both transgenic and control lines.
Antioxidant Enzyme 2. Approximately 200 mg of leaf tissue was weighed and
Assays ground to a fine powder in liquid nitrogen using a pre-
cooled mortar and pestle.
3. The exact weight of each powdered sample was determined
before it was thoroughly homogenized in 1.2 mL of 0.2 M
potassium phosphate buffer (pH 7.8 with 0.1 mM EDTA).
4. The samples were centrifuged at 15,000×g for 20 min at
4◦ C.
5. The supernatant was removed, the pellet resuspended in 0.8
mL of the same buffer, and the suspension centrifuged for
another 15 min at 15,000×g.
6. The combined supernatants were stored on ice and used
to determine different antioxidant enzyme activities (see
Notes 5 and 6).

3.2. Superoxide 1. Total SOD activity was assayed using a modified NBT
Dismutase (SOD) method (11) (see Note 7).
2. The 2 mL assay reaction mixture contained 50 mM phos-
phate buffer (pH 7.8) containing 2 mM EDTA, 9.9 mM
L-methionine, 55 μM NBT, and 0.025% Triton-X100.
3. Forty microliters of diluted (2×) sample and 20 μL of 1 mM
riboflavin were added and the reaction was initiated by illu-
minating the samples under a 15 W fluorescent tube (12).
4. During the 10-min exposure, the test tubes were placed in a
box lined with aluminum foil.
5. The box with the test tubes was placed on a slowly oscillating
platform at a distance of approximately 12 cm from the light
source.
6. Duplicate tubes with the same reaction mixture were kept in
the dark and used as blanks.
7. Absorbance of the samples was measured immediately after
the reaction was stopped at 560 nm (see Note 8)
8. The enzyme activity (grams per fresh weight) of a sample
was determined from a standard curve obtained by using
pure SOD.

3.3. Catalase (CAT) 1. CAT activity was determined according to Aebi and Lester
(13) (see Notes 1–4).
278 Elavarthi and Martin

2. The decomposition of H2 O2 was followed as a decrease in

absorbance at 240 nm in a UV/Vis spectrophotometer.
3. The 3 mL assay mixture contained 2 mL leaf extract (diluted
200 times in 50 mM potassium phosphate buffer, pH 7.0)
and 10 mM H2 O2 .
4. The extinction coefficient of H2 O2 (40 mM−1 cm−1 at
240 nm) was used to calculate the enzyme activity that was
expressed in terms of millimoles of H2 O2 per minute per
gram fresh weight).

3.4. Ascorbate 1. APX activity was assayed using a modified method of Nakano
Peroxidase (APX) and Asada (14) (see Notes 1–4).
2. APX activity was determined from the decrease in
absorbance at 290 nm due to oxidation of ascorbate in the
reaction.
3. The 1 mL assay mixture contained 50 mM potassium phos-
phate buffer (pH 7.0), 0.5 mM ascorbate, 0.5 mM H2 O2 ,
and 10 μL of crude leaf extract (see Note 9).
4. H2 O2 was added last to initiate the reaction, and the
decrease in absorbance was recorded for 3 min.
5. The extinction coefficient of 2.8 mM−1 cm−1 for reduced
ascorbate was used in calculating the enzyme activity that
was expressed in terms of millimole of ascorbate per minute
per gram fresh weight.

3.5. Glutathione 1. GR activity was assayed according to Smith et al. (15) (see
Reductase (GR) Notes 1–4).
2. The increase in absorbance at 412 nm was measured when
DTNB was reduced to TNB by GSH in the reaction.
3. Ten microliters of leaf extract was used in the assay along
with 0.75 mM DTNB, 0.1 mM NADPH, and 1 mM GSSG
in a total of 1 mL assay volume (see Notes 9 and 10).
4. GSSG was added last to initiate the reaction and the increase
in absorbance was recorded for 3 min.
5. The extinction coefficient of TNB (14.15 M−1 cm−1 ) was
used to calculate the activity of GR that was expressed in
terms of millimole TNB minute per gram fresh weight.

4. Notes

1. Unless otherwise stated, all buffers/solutions were pre-

pared using Millipore water and stored at 4◦ C until they
were used.
 Antioxidant Enzyme Assays 279

2. Store buffers at 4◦ C and bring them to room temperature

before the experiment except NADPH and GSSG, which
should be maintained at 4◦ C at all times prior to use.
3. Quartz cuvettes were used for assays using UV wavelengths
and clear plastic disposable cuvettes were used for assays in
the visible range.
4. For all assays, care must be taken to avoid air bubbles form-
ing in the cuvettes before making absorbance measure-
ments.
5. The crude leaf extract was prepared fresh daily from leaf
tissue and all assays were conducted within a few hours.
6. The crude leaf extract should at all times be kept on ice.
7. All solutions for SOD assay should be at room temperature
for optimal enzyme activity.
8. Absorbance readings for SOD samples should be taken as
soon as the assay is stopped, because the blue formazon will
quickly start precipitating out, resulting in errors.
9. Ascorbic acid in the APX assay and GSSG in the GR assay
should always be kept on ice.
10. The NADPH stock solution can be stored at −20◦ C and
reused for up to a week. Thawed stock solution should be
kept on ice until used.

Acknowledgments

The authors would like to thank Dr. Kalpalatha Melmaiee and

Shraddha Vadvalkar for valuable suggestions and help in the criti-
cal review of the chapter.

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