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Assay of Enzyme Superoxide Dismutase (SOD)
by Hiroyuki
UKEDA
Summary
The enzyme superoxide
dismutase (SOD) catalyses the breakdown of superoxide anion
(O2-) and provides the first line of defense against
oxygen toxicity. The activity and the assay techniques are associated with
diverse fields such as medicine, biochemistry, plant physiology and food
chemistry. During the past few decades, various SOD assay methods have been
developed. However, those methods have some drawbacks in the selectivity,
rapidity, cost and convenience. Recently, we found that novel water-soluble
tetrazolium salts such as XTT, WST-1 and WST-8 are suitable for the detection of
O2- and are applicable to the SOD assay. Of these
tetrazolium salts, WST-1 appears to be the most promising for the SOD assay due
to its sensitivity, its low absorbance of the oxidized form and its water
solubility. The SOD assay method based on the use of WST-1 can be applied to
practical biochemical samples such as erythrocytes, liver and heart from rats. A
novel flow injection assay system for SOD was also developed using WST-1. In
this system, a rapid assay (sampling frequency=30 samples/hour) was achieved.
Key Words: Superoxide anion; tetrazolium salt; XTT;
WST-1
1. Introduction
SOD is an
enzyme that catalyzes dismutation of two superoxide anion
(O2-) into hydrogen peroxide and molecular oxygen (Scheme
1).
2O2- + 2H+ ->
H2O2 + O2 (Scheme 1)
SOD is one of the
most important enzymes in the front line of defense against oxidative stress.
According to Dr. Cutler, it is also a factor that controls organisms'
life-spans.1) Therefore, research on SOD activity will be important
for the understanding of various mechanisms of life. Since the discovery of SOD
by Dr. McCord and Dr. Fridovich in 1969, various SOD detection methods have been
developed. However, there is no satisfactory method available from the point of
selectivity, rapidity, simplicity and application range. In this article, I
describe a new WST-1-based SOD assay developed in order to resolve the
above-mentioned problems of conventional methods.
2.
Reactive Oxygen Species and SOD
The determination of SOD activity
is necessary for all research fields related with organism. First, I will
describe involvement of reactive oxygen species (ROS) and SOD in research fields
concerning human, plants and food, then consider the roles of SOD assay
techniques performed in these research fields.
2.1 Humans
A
given amount of the oxygen taken in by the body is always converted to
O2-, H2O2, hydroxy radical
(OH.) and other molecules by various enzymatic metabolism systems.
Among these molecular species, the life spans of OH. and
O2-, which has an unpaired electron, are the shortest.
OH. has the highest reactivity and it reacts with various molecules
with diffusion controlled rate. Although it is believed that
O2- may not directly react with lipids, proteins, sugars
or nucleotides, it is transformed into OH. when it interacts with
metal ions (Fenton reaction) and it reacts with nitric oxide (NO) to quench the
physiological activities such as vascular relaxation. At the same time,
O2- generates peroxinitrite (ONOO.), which
causes oxidative damage. In order to protect the body from highly toxic ROS, the
body has acquired anti-oxidative stress mechanisms including SOD. These
anti-oxidative stress mechanisms are localized in tissues and inside the cells
where ROS are generated. If the amount of ROS exceeds the limit of the defense
mechanism of the body for any reason, serious disease may be induced. The
typical example is cancer and life-style related diseases such as
arteriosclerosis are also included. Researchers are currently exploring the role
of oxidatively aggregated deposition of Beta-amyloid to senile plaques in
causing Alzheimer disease and other vascular-damage-related brain diseases.
Furthermore, cell damage by ROS is considered to be one of the main causes of
various aging-related diseases. The use of radical trapping agents as a possible
cure for these diseases is also being studied.
It is speculated that the
level of O2- scavenging activity by SOD is related to the
appearance of these diseases. So far, there are several examples that clearly
indicate the correlation between SOD activity and human diseases such as Werner
syndrome, amyotrophic lateral sclerosis (ALS, decrease in SOD activity), and
Down syndrome (increase in SOD activity). Furthermore, since SOD activity is
dramatically decreased by in vivo Maillard reaction, the SOD activity
level of diabetes patients tends to be low. This decrease in SOD activity also
decreases the diabetes patient's defense capability against oxidative stress,
and various diseases may result as diabetes complications. It is expected that
the correlation between these various diseases and SOD activity will continue to
be revealed. Additionally, SOD activity determination will be utilized not only
for the research of the mechanisms that cause diseases but also for the
diagnosis and indication of health conditions.
2.2
Plants
Since plants cannot move around freely, they have highly
sophisticated defense mechanisms toward environmental alterations. For example,
the SOD activity of a plant is increased by the use of herbicides such as
paraquat, by an increase in SO2 concentration in the atmosphere, by drought, or
by exposure to high concentration of zinc and magnesium. These phenomena cause
ROS to be generated in the plant. Findings also suggest that the reduction in
the toxicity of O2- is a very important defense system
against oxidative stress. Therefore, the role of SOD assay techniques is very
important in the study of plant physiology.
2.3 Food
It is
thought that the risk of ROS-related diseases is decreased by reinforcement of
the defense mechanism against oxidative stress. Many people are interested in
the anti-oxidation qualities of plants and their products such as red wine, tea
and so on. There is an element in these plants that induces
O2- quenching activities (also called SOSA (superoxide
anion scavenging activity)), which are similar to those of SOD. Currently, the
O2- scavenging activities of animal proteins are also
being studied. It is expected that the development of food materials that have
high SOD activity will continue to advance.
3. Conventional
SOD Assays
Since SOD activity detection methods are used in many
research fields, samples to be analyzed vary widely. Therefore, the most
important technical aspect of SOD assay is the applicability of the assay to
different samples. In other words, high selectivity with little interference
from other components in a sample solution is desired. For the production of
O2-, which is a substrate of SOD, a xanthine-xanthine
oxidase reaction is utilized. A probe for the detection of
O2- is included in the reaction solution. The change of
the probe without any sample is indicated as a blank control, and the
suppression ratio of the change of the probe by the sample solution is indicated
as the inhibition ratio.
Generally, 50% inhibition by the sample solution is
used for the activity determination (IC50). On the other hand,
O2- generated by the xanthine-xanthine oxidase reaction is
spontaneously transformed to oxygen and hydrogen peroxide. This spontaneous
dismutation reaction occurs rapidly in acidic conditions, and at the rate of
8.5x105 - 8.5x104 M-1s-1 at
physiological pH (pH 7 - 8). Therefore, the second-order rate constant of the
reaction between O2- and the probe should exceed the rate
constant of the dismutation reaction. In the case where the rate constants are
almost the same, the concentration of the probe should be increased. There are
several different types of probes: those which change their color (colorimetric
probes) when they react with O2-, those which emit light
(chemiluminescence probes) when they react with O2-, and
those which produce specific radicals (spin trap agents) when they react with
O2-.
3.1 Spectrophotometric
Detection
The most typical SOD detection method is the one based on
spectrophotometric detection. This method uses either cytochrome C or nitroblue
tetrazolium (NBT) (Fig. 1). The detection of O2- by the
cytochrome C reducing method is based on the color change to generate purple
color dye from reduced cytochrome C (scheme 2). This is the most commonly
utilized method since the discovery of SOD.
Cyt(FeIII) +
O2- -> Cyt(FeII) + O2 (Scheme
2)
Since cytochrome C is easily reduced by reductases such as NADPH
reductase and other reducing agents, it is necessary to consider contaminants in
the samples. Moreover, this method requires continuous monitoring in 1.5-minute
intervals, so it is not suitable for high-throughput detection. The NBT method
is based on the generation of water-insoluble blue formazan dye (lmax: 560 nm) by a reaction with O2-.
Because the dye is not water-soluble, a non-homogeneous suspension is created
during long-term analysis that causes problems in the reproducibility of the
data. In order to solubilize formazan, alternative methods such as addition of
BSA have been developed. However, the addition of unnecessary proteins may make
the data analyses much more complicated. Moreover, NBT is reduced by various
reducing agents. This characteristic of NBT is utilized for the detection of
keto-amine, which is a marker of diabetes and is the intermediate in the
Maillard reaction. The most significant disadvantage of the NBT method is that
100% inhibition cannot be achieved even with the addition of excessive amount of
SOD into assay solution. The direct interaction between NBT and xanthine oxidase
is speculated to be the cause.
3.2 Chemiluminescence
Method
The chemiluminescence probe used for O2-
detection can also be applied for SOD assay detection. There are two types of
these probes. One is a lucigenin, and the other is a luciferin derivative
(MCLA). These chemiluminescence reactions are highly pH dependent. For example,
lucigenin shows extremely intense chemiluminescence at pH 9.0 and
higher.15) Therefore, SOD detection by chemiluminescence under
physiological pH conditions is not feasible. On the other hand, MCLA emits
strong luminescence under physiological conditions, and is therefore used for
the detection of Cu, Zn-SOD activity in the human brain. However, MCLA is not a
suitable SOD assay probe since it reacts not only with O2- but also
with singlet oxygen. Moreover, MCLA reacts with dissolved oxygen to emit
background luminescence, and the transitional metal ions accelerate the
oxidation reaction.
3.3 Electron Spin Resonance Spectroscopy (ESR)
Method
At room temperature, the ESR signal of
O2- in solution cannot be detected directly, but can be
indirectly detected by a spin trap method. The most common trapping agent is
5,5-dimethyl-1-pyrroline N-oxide (DMPO). Since O2-
trapping DMPO indicates a particular ESR spectrum pattern, ESR detection is the
most specific method for O2- detection. However, the
second-order rate constant between DMPO and O2- is
relatively lower than the reaction constant of the spontaneous reaction of
O2-. Therefore, a large amount of DMPO should be added to
the solution (e.g., using a final concentration of 0.45 M). Unfortunately, this
large volume of DMPO increases the cost per assay. Another problem with this
method is the requirement of a relatively expensive ESR instrument.
4. New SOD Assay by Use of Water-soluble Tetrazolium
Salts
In order to overcome above-mentioned problems, the following
aspects need to be considered for the development of SOD assay: an economical
method using a simple instrument, a less pH-sensitive method, and a highly
O2--specific method. If a general spectrophotometer is
applied as a simple instrument, colorimetric probes similar to cytochrome C and
NBT are preferable. The most important point for the assay is to be able to
determine 100% inhibition by SOD without interference from other components. We
examined new tetrazolium salts that generate water-soluble formazan by reduction
in order to establish a new SOD assay.
4.1 XTT
XTT is a
water-soluble tetrazolium salt first reported in 1988. Since then, it has been
utilized as a substrate for the electron transfer system of bacteria cells or
mammalian cells. Its structure is indicated in Fig.1. While NBT has a
bis-tetrazolium structure, the XTT structure is monotetrazolium and it has two
sulfonic acid groups.
Fig. 1: Structures of
tetrazolium salts used in SOD assay.
We used XTT for the SOD assay.
As indicated in Fig. 2, 100% inhibition was observed in proportion to the
increase in the SOD concentration. This 100% inhibition could not be observed by
NBT method before. At an optimized condition, 100% inhibition was still observed
at various pH. The observation of 100% inhibition by XTT means that XTT is
specifically reduced by O2-, and XTT overcomes the problem
associated with NBT.
Fig. 2: SOD inhibition
curves using NBT (black circle) and XTT (white circle).
The reaction
mixture contained 2.5 ml of 50 mM carbonate buffer (B, pH 9.4; C, 10.2) or 50 mM
phosphate buffer (A, pH 8.0) and 0.1 ml each of 3 mM EDTA, 3 mM xanthine, 56.1
mU/ml xanthine oxidase, 0.75 mM XTT or NBT, and sample solution containing SOD
at the concentration shown at abscissa. In the NBT method, 0.1 ml of BSA was
also added.
It is also reported that NBT directly interacts with glucose
oxidase except for xanthine oxidase.20) We added NBT or XTT in a
solution of glucose oxidation reaction with glucose oxidase, and measured the
production of its formazan (Fig. 3). Even in the glucose-glucose oxidase
reaction, which does not generate O2-, NBT was reduced to
generate its formazan in a time dependent manner, but no O.D. increase was
observed in the case of XTT. Initial O.D. increased with an increase in the XTT
concentration. This is due to the background O.D. from XTT tetrazolium. From
these results, XTT does not seem to have any direct interaction with the reduced
form of some enzymes that are generated during the oxidase reaction process, and
XTT was shown to be a suitable probe for SOD assay.
Fig. 3: Time course of
the reduction of NBT and XTT during the oxidation of glucose by glucose
oxidase.
The assay mixture (2.8 ml) contained the following components as
the final concentration: 50 mM glycine-NaOH (pH 9.5), 0.1 mM glucose, and 0.1 mM
NBT (black circle), 0.1 mM XTT (triangle) or 0.2 mM XTT (white circle). The
reaction was initiated by the addition of glucose oxidase solution. The
absorbance at 470 (XTT) or 560 nm (NBT) was monitored at
25oC.
In order to test the applicability of XTT for practical
samples, we determined SOD activity of rabbit erythrocytes and compared with the
data obtained by NBT method (Fig. 4: the assay was carried out at pH 10.2).
Fig. 4: Relationship
between SOD activity in rabbit erythrocytes obtained by XTT and NBT
methods.
A crude extract of SOD from blood erythrocytes was separated
according to the conventional method.21) The XTT method data showed a
high correlation with the NBT method data with a correlation coefficient of
0.954. The value obtained by XTT method is almost double the NBT value,
reflecting the difference in the sensitivity of these assays. Next, we applied
this method to SOSA detection of food samples.22) As we know, food
samples are very complicated multi-component mixtures. Samples such as red wine,
green tea, coffee and cocoa were chosen to determine SOSA by XTT because these
samples are already known to have SOSA. As expected, the color from food samples
with no dilution interfered with the assay, and the materials in the samples
reduced XTT directly. To achieve 10% or less absorbance change by these
materials, tea, red wine, instant coffee or cocoa need to be diluted 100 times,
10 times, 50 times or 10 times, respectively at pH 8. Each diluted sample
solution had over 50% inhibition activity at each dilution rate, thus it was
shown that XTT assay was useful for the determination of SOSA in food samples.
These results were consistent with the data obtained by ESR method. From these
results, it was shown that XTT method is applicable to the use with biological
samples and food samples.
XTT overcomes the problems associated with NBT
and it seems that XTT is an ideal reagent for SOD assay. However, we encountered
several new problems during the experiment using XTT. One of the problems is the
pH dependent sensitivity change (Fig. 2). The data obtained by NBT was stable in
the range between pH 8 and 10.2, however the sensitivity in the case of XTT
decreased as the pH was lowered. Therefore, the sensitivity of XTT at pH 8.0 was
lower than that of NBT. Another problem is the water-solubility of XTT. The
water-solubility of XTT is around 2 mM and a heating process was necessary to
prepare an optimal XTT solution (0.75 mM). Moreover, the high background O.D
indicated in Fig. 3 is another concerning issue. Thus, we tried to further
improve the assay by using different types of tetrazolium salts.
4.2
WST-1 and WST-8
From 1993 to 1998, Dr. Ishiyama and his group
developed several types of water-soluble tetrazolium salts. Since the
water-solubilities of these compounds are from several 10 mM to several 100 mM,
we applied these water-soluble tetrazolium salts for SOD assay in place of XTT.
For this assay, we used WST-1 and WST-8 (the chemical structures shown in Fig.
1). Both WST-1 and WST-8 are mono-tetrazolium salts that have sulfonate group(s)
in their structure. At first, we optimized assay conditions and prepared
inhibition curves by using standard SOD at pH 8.0, 9.4 and 10.2 (Fig.
5).
Fig. 5: SOD inhibition
curves using the WST-1 (black circle) and WST-8 (white circle)
systems.
The reaction mixture contained 2.5 ml of a phosphate buffer (A,
pH 8.0) or 50 mM carbonate buffer (B, pH 9.4; C, pH 10.2) and 0.1 ml of 3 mM
EDTA, 3 mM xanthine, 58 mU/ml of xanthine oxidase, 0.75 mM WST and the sample
solution containing SOD at the concentration shown on the abscissa.
Both
WST-1 and WST-8 showed 100% inhibition as seen in XTT assay at high
concentrations of SOD. The most remarkable point was to be able to obtain the
almost same IC50 in different pH solutions. This result indicated
that WSTs can overcome several shortcomings associated with XTT and NBT, making
them ideal reagents for SOD assay. Since 100% inhibition was observed, we used
WST-1 and WST-8 to determine whether the formazan is produced by the
glucose-glucose oxidase reaction as in the XTT experiment (Fig. 6).
Fig. 6: Time course of
the reduction of NBT and WST during the oxidation of glucose by glucose
oxidase.
The assay conditions were same as those in Fig. 3. The assay
mixture contained 0.1 mM NBT (black square), 0.2 mM WST-1 (black circle) or 0.2
mM WST-8 (white circle). The absorbance at 438 (WST-1), 460 (WST-8) or 560 nm
(NBT) was monitored at 25oC.
As expected, no formazan
production was observed by a glucose-glucose oxidase reaction. Moreover, no
background increase was observed with the increase in the concentration of WST.
Therefore, WSTs enable us to determine the SOD activity at lower background
conditions. In order to compare the sensitivity of WST, we compared
IC50 data obtained using NBT assay and XTT assay with that of WST
assay (Table 1).
|
Table 1: Comparison of IC50 obtained by varioustetrazolium salts at pH 10.2 |
|
|
|
|
|
tetrazolium |
IC50(ug/ml) |
|
|
|
|
WST-1 |
0.22 |
|
WST-8 |
0.75 |
|
XTT |
0.26 |
|
NBT |
0.55 |
These
IC50 values are determined at pH 10.2, and it was indicated that
WST-1 could determine SOD activity with the highest sensitivity among several
other tetrazolium salts. Since WST-1 seemed to show high sensitivity, we used it
to determine SOD activity of rat erythrocytes and compared the data with the
ones obtained by using XTT method. As a result, a good linear correlation
between WST-1 method and XTT method was observed with the correlation
coefficient of 0.968 (n=7).
Recently, Dr. Winterbourn and his group
recognized the usefulness of WST-1 in SOD activity detection and developed a
microplate assay for SOD activity detection. They used a microplate assay to
determine the SOD activity of human erythrocytes and rat liver and heart
homogenates, and reported that the result obtained by this assay was consistent
with the data reported previously. Thus, it seems that WST-1 has a wide range of
applicability for biological samples. Currently, we are investigating the
applications for other samples such as plant tissues and food samples.
In
order to establish an automatic analytical system based on WST-1 assay, flow
injection analysis (FIA) method was utilized. A diagram of our FIA system is
shown in Fig. 7.
Fig. 7: FIA manifold for
SOD assay.
P, pump; IV, injection valve; D, detector
In this
system, we used a xanthine oxidase-immobilized reactor to avoid the exogenous
addition of the enzyme in each experiment, making this analytical system a rapid
and economical method. After series of experiments, we immobilized catalase in
the reactor at the same time and used hypoxanthine as a substrate in order to
increase the stability of xanthine oxidase. Prior to the assay, we prepared
hypoxanthine and WST-1 mixed solution and combined it with the sample solution
at 1:9 volume ratio, then injected 20 ul of the mixture into the reactor. When
no SOD is contained in the sample solution, a maximum amount of WST-1 formazan
is produced and maximum peak height is observed. If the sample solution contains
SOD, the maximum peak is decreased in proportion to the SOD activity
(Fig.8).
Fig. 8: Typical response
curve of SOD preparation obtained under the optimum conditions.
A, 0; B,
1; C, 2.5; D, 5; E, 10; F, 25; G, 50; H, 100; I, 250; J, 500 ug/ml
The
SOD inhibition curve was prepared by the inhibition rate determined from the
decreasing ratio of the peaks. From the inhibition curve, IC50 was
determined to be 2.7 ug/ml and the absolute value was 50 ng. This value is
larger compared with the IC50 value of 20 ng obtained by a batch
method. However, this FIA method enables us to determine over 30 samples per
hour, thus the detection speed is extremely fast. We determined the SOD activity
of rat erythrocytes using this method, and compared it with the data obtained by
NBT method (Fig. 9).
Fig. 9: Relationship
between SOD activity in rabbit erythrocytes obtained by NBT method and WST-1-FIA
method.
There was a very good correlation in the data between the FIA
method and NBT method. Even though this assay is a rapid assay system, the
results obtained by this method were sufficiently consistent with the data
obtained by conventional methods.
5.
Conclusion
We described the importance of SOD assay and several
detection methods. Since the discovery of SOD, various assay systems have been
proposed every year. This fact suggests that currently there is no single
satisfactory method available. The WST-1 based SOD assay we developed seems to
be the most attractive method that overcomes many problems associated with
conventional methods. In order to render this method as a standard method for
SOD assay, we plan to investigate the specificity of WST-1 as an
O2- probe.
Author:
Hiroyuki UKEDA, Ph.D.
Department of
Agriculture
Khochi University
200 Mononobe Otsu
Nangoku-city, Khochi
783-8502
Japan
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