Article ID: WMC004270
ISSN 2046-1690
A Comparative Study of Catalase Activities in
Different Vertebrates
Corresponding Author:
Dr. Dipak K Sahoo,
Post Doctoral Researcher, KTRDC, University of Kentucky, Cooper & University Drives , 40546-0236 - United
States of America
Submitting Author:
Dr. Dipak K Sahoo,
Post Doctoral Researcher, KTRDC, University of Kentucky, Cooper & University Drives , 40546-0236 - United
States of America
Other Authors:
Mr. Sripad C Patnaik,
Lecturer, Department of Biotechnology, College of Engineering and Technology (BPUT), Techno Campus,
Ghatikia Bhubaneswar, Orissa, INDIA, 751003 - India
Dr. Gagan B Chainy,
Professor, Departments of Zoology and Biotechnology, Utkal University, Bhubaneswar-751004, Odisha - India
Article ID: WMC004270
Article Type: Research articles
Submitted on:05-Jun-2013, 02:00:36 AM GMT
Published on: 05-Jun-2013, 07:00:19 AM GMT
Article URL: http://www.webmedcentral.com/article_view/4270
Subject Categories:ZOOLOGY
Keywords:Fish, Amphibia, Reptiles, Birds, Mammals, Catalase, Catalase activity staining
How to cite the article:Patnaik SC, Sahoo DK, Chainy GB. A Comparative Study of Catalase Activities in
Different Vertebrates. WebmedCentral ZOOLOGY 2013;4(6):WMC004270
Copyright: This is an open-access article distributed under the terms of the Creative Commons Attribution
License(CC-BY), which permits unrestricted use, distribution, and reproduction in any medium, provided the
original author and source are credited.
Source(s) of Funding:
None
Competing Interests:
None
WebmedCentral > Research articles
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A Comparative Study of Catalase Activities in
Different Vertebrates
Author(s): Patnaik SC, Sahoo DK, Chainy GB
Abstract
Catalase is one of the most active antioxidant defence
enzymes known for being highly cooperative with SOD
and other H2O2 producers at high flux of hydrogen
peroxide. In the present study, catalase activity was
analyzed in liver samples of different animals both by
gel activity staining method as well as
spectrophotometric biochemical method. Five different
animals like fish (Cirrhinus mrigala), amphibia (Bufo
melanistictus.), reptile (Calotes versicolor.), bird
(Gallus domesticus.), and mammal (Rattus rattus)
from five different classes of vertebrates were taken
for the study. Catalases from these animals differ in
activities as shown by biochemical assay method and
activity staining method.
Introduction
Virtually all organisms that survive in oxic
environments, whetherthey are capable of aerobic or
anaerobic lifestyles or both,contain enzymes known as
antioxidant defence enzymes that convert reactive
oxygen intermediates (ROI)to undisruptive compounds.
If not dismutated, ROI such as superoxide,hydrogen
peroxide, and hydroxyl radical will interact with
macromolecules,their derivatives and cellular
structures thereby leading toconformational changes
and loss of integrity. Catalase is one of the most active
antioxidant defence enzymes known for being highly
cooperative with SOD and other H2O2 producers at
high flux of hydrogen peroxide. Catalase (EC1.11.1.6;
hydrogen peroxide: hydrogen peroxide oxidoreductase;
CAT) is an iron porphyrin enzyme that catalyzes the
breaking down of H2O2 to water and oxygen molecule.
Catalase along with glutathione peroxidase and
superoxide dismutase serves as an efficient
scavenger of reactive oxygen species (ROS)
preventing cellular damage. Catalase of vertebrate
source has a relative molecular mass of 225,000 to
250,000 and composed of four identical protein
subunits, each of which contains a ferric heam group
bound to its active site (Reid et al., 1981). Each
subunit usually has one molecule of NADPH bound to
it. This NADP may serve to protect the enzyme from
oxidation by its H 2 O 2 substrate. Catalase activity
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decreases due to dissociation of its subunits on long
storage; freeze-drying or exposure to acid or alkali and
also its activity is inhibited by azide, cyanide and
HOCL (Das and Chainy, 2001).
Affinity of catalase towards H2O2 is very low and the
enzyme has a very high Km value (25 mM) for its
substrate H 2 O 2 (Lehninger et al., 1993). The
physiological functions of catalase as a H 2 O 2
scavenging enzyme would be limited to situations in
which mean or local cellular concentration of the
inorganic hydroperoxide reaches high value (Chance
et al., 1979).Rate wise catalase is considered a
relatively slow enzyme. One catalase enzyme
molecule, for example, will completely break down 5.6
million hydrogen peroxide molecules per minute. One
of the fastest enzymes, carbonic anhydrase, will break
down 36 million carbonic acid molecules per min.
According to structural and functional similarities
catalases can be divided in three subgroups (Zamocky
and Koller, 1999). These enzymes are homotetramers,
200-340 kDa in size with four prosthetic haem groups
(Guan and Scandalios, 2000). The native quaternary
structure of typical catalases is strictly required for
maintaining their catalytic function. The crystal
structures of several catalases of bacterial, fungal, or
mammalian origin have been resolved and reveal an
extremely well conserved “catalase fold”.
Catalase protects aerobic organisms against the toxic
effects of hydrogen peroxide, which they cleave into
water and molecular oxygen. Typical catalases,
forming the largest of three subgroups, are found in
almost all aerobically respiring organisms, both
prokaryotes and eukaryotes. Peroxisomal catalase
plays an important nonspecific role in peroxisomal
metabolism (Horiguchi et al. 2001). Catalase is able to
protect human fibroblasts against free radicals
generated by acetaldehyde-xanthine oxidase system.
Catalase has predominant role to combat oxidative
stress than glutathione peroxidase particularly in
hypothyroid rat testis as marked by significant
increase in CAT activities and a simultaneous
decrease in total GPx (Sahoo et al., 2008a, Sahoo and
Roy, 2012).
Catalase also inhibits nitrofurantoin-induced toxicity on
lung parenchymal cells (Michiels et al. 1994). The
activities of the pulmonary antioxidant enzymes
(AOE), superoxidedismutase (SOD), glutathione
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peroxidase (GPx) and catalase,increase in the final
10–20 % of gestation in themammalian lung, to protect
the lung from attack by increasinglevels of reactive
oxygen species at birth (Starrs et al. 2001). It is also
believed that cell signaling molecules regulate
catalase to control cell mitogenesis (Yanos, 2002).
The gene encoding human Catalase is located on
chromosome 11, mutation of which results in
catalasaemia with an observable clinical problem of
increase in mouth ulceration. Aniridia is a disease
associated with mental retardation and cancer known
as Wilm’s tumor resulting from deletion in
chromosome11 (Das and Chainy, 2001).
Comparative studies of Catalase among different
species and organs can help understanding
physiological significance of free radicals and the
evolutionary trend. In the present study, we measured
catalase activity biochemically both by
spectrophotometric and native-gel electrophoretic
staining methods from five different animals from five
different classes of vertebrates.
Materials and methods
1. Animals
Five different animals from five different classes of
vertebrates were taken. They are fish (Cirrhinus
mrigala), amphibia (Bufo melanistictus.), reptile
(Calotes versicolor.), bird (Gallus domesticus.),
mammal (Rattus rattus). The fish and fowl were
procured from Bhubaneswar, Orissa, India. The toads
and garden lizards were obtained from Bhubaneswar
habitat, Orissa, India. They were acclimatized in the
standard conditions. Wistar strain rats were obtained
from National Institute of Nutrition, Hyderabad, India.
Adult male rats of age 90-120 days weighing about
250-350 g were used for present study. Rats were
maintained in the standard conditions (Sahoo et al.,
2008a). Healthy and active animals were taken for
study. Animal care, maintenance, and experiments
were conducted under the supervision of the
Institutional Animal Ethics Committee (IAEC) regulated
by the Committee for the Purpose of Control and
Supervision of Experiments on Animals (CPCSEA),
Government of India.
2. Chemicals
Catalase, cumenehydroperoxide, bovine serum
albumin, TEMED, DEAE cellulose was obtained from
Sigma Chemical Company, USA. N’ N’ –bis acrylamid
were obtained from Merck-Schudant, GERMANY.
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Horse radish peroxidase (HRP), Ammonium
persulphate and Sodium dodecylsulphate (SDS),
diaminobenzidine tetrachloride (DAB), acrylamide
were purchased from SISCO research laboratory,
India. Freund’s complete and incomplete adjuvant,
goat-antirabbit-IgG
biotinylated,
streptavidin-peroxidase
conjugate
and
goat-antirabbit-IgG-peroxidase conjugate were
obtained from Bangalore Genie, Bangalore, India.
Nitrocellulose membrane was purchased from
Scheinrer and Shull, Switzerland. All other chemicals
and buffers used were of highest purified grade
commercially available.
3. Tissue processing
A. Homogenization
Animals were sacrificed and livers were dissected out
quickly, cleaned in ice-cold normal saline (0.9%, w/v),
pat dried in filter paper. The tissue were weighed and
kept in ice for further processing. A 20% (w/v)
homogenate of the tissue was prepared in 0.25 M
ice-cold sucrose prepared in phosphate buffer (50 mM
pH 7.4). The homogenate was done with the help of
Potter - Elvehjem type motor driven glass Teflon
homogenizer.
B. Isolation of sub cellular organelles
The crude liver homogenate was filtered through
four-layered sterilized cheesecloth and the filtrate was
centrifuged at 600 ´ g for 10 minutes at 4°C to
precipitate nuclei and cellular debris. The supernatant
was further centrifuged at 10,000 ´ g for 20 minutes at
4°C to separate mitochondrial pellet and the
post-mitochondrial fraction (PMF) as the supernatant
(Sahoo, 2013). The PMF was processed immediately
for various biochemical analyses. All the centrifugation
steps were performed in Sigma laboratory centrifuge
model 3K30.
4. Protein Estimation
Protein contents of liver PMF samples were estimated
by the method of Lowry et al. (1951). Biuret reagent
was prepared freshly by mixing solutions A, B and C in
a proportion of 100:2:2. Solution A was 2% (w/v)
sodium carbonate (Na2CO3) in 0.1 N sodium
hydroxide (NaOH) solution; solution B contained 0.5%
(w/v) copper sulphate (CuSO4) in distilled water and
solution C contained 1% potassium sodium tartarate
(KNaC4H4O6) in distilled water. Commercially
available Folin & Ciocalteu’s phenol reagent was used
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after diluting it with distilled water in a proportion of 1:2
(v/v). Samples were diluted suitably and 0.1ml of the
sample was taken and the volume was made up to 0.5
ml with distilled water. Then 5 ml of biuret reagent was
added to the tubes, vortexed and allowed to stand for
10 minutes at room temperature. To this 0.5 ml of
Folin-Ciocalteu’s phenol reagent was added, vortexed
and then incubated at room temperature for 30
minutes. The color of the reaction was read against a
blank at 700 nm.
Bovine serum albumin (BSA) solution (1 mg/ml of
distilled water) was used as working standard. It gave
a linear curve in the concentration range of 25-125 mg.
Protein content was expressed as mg/g wet weight of
tissue (Sahoo, 2013).
5. Estimation of catalase activity
Catalase activity was measured in the PMF by the
method of Aebi (1974). To 0.5 ml of sample 5 µl of
absolute ethanol was added and incubated for 30
minutes in ice bath. To 0.45 ml of this aliquot 50 µl of
10 % triton x-100 (1% final concentration) was added
(Cohen et al., 1970). Catalase forms an inactive
complex with H2O2, which is called Complex II.
Ethanol reverses the inactivation, which occurs during
the time interval from the preparation of tissue
homogenate to the subsequent assay of catalase
activity. Triton x-100 increases observable catalase
levels, as the activation is complete. The enzyme
reaction was started by adding 0.1 ml of sample
(0.4-0.5 mg protein) to 2.9 ml of 50 mM phosphate
buffer, pH 7.0 containing 12 mM H2O2. The
absorbance was recorded at 240 nm immediately at
15 seconds interval till 2 minutes. A blank was taken
without sample. Absorbance was read at 240 nm and
?A/ min was calculated. Calculation was done by
taking the extinction coefficient of H2O2 i.e. 43.6 M-1
cm-1 (Sahoo et al., 2005; Sahoo, 2013). Activity was
expressed as n Kat per mg protein. (1 katal (Kat) = 1
mol sec-1)
6. Isoenzyme pattern study through native
poly-acrylamide gel electrophoresis and catalase
activity staining
The PMF samples were separated using native
poly-acrylamide gel electrophoresis. The activity of
Catalase in PMF was visualized on native
polyacrylamide gel by the method of Woodbury et al.
(1971) with slight modification. The principle involves
the reaction of hydrogen peroxide with potassium
ferricyanide (III) by reducing it to ferrocyanide (II). The
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peroxide is oxidized to molecular oxygen. Ferric
chloride reacts with ferrocyanide (II) to form stable
insoluble Prussian blue pigment. Catalase signaled its
location by scavenging H2O2 causing transparent
bands on the blue gel. The protein run gel was
washed two times with distilled water. Then it was
soaked in H2O2 solution (0.01% v/v) for 5 minutes
followed by washing two times for removing excess
H2O2 contents. Then it was stained in 0.5% solution
of ferric chloride and potassium ferricyanide (1:1
proportion) for 4-5 minutes. When transparent bands
were observed, immediately 1% HCl was added to
stop the further reaction. Then the gel was washed
with distilled water and photographed.
7. Western blotting analysis
Protein contents in plant extracts after determined
(Lowry et al., 1951) were separated by
SDS–polyacrylamide gel electrophoresis as described
by Laemmli (1970) and transferred onto a
nitrocellulose membrane (Bio-Rad) and subjected to
Western blot analysis as described earlier (Sahoo et
al., 2013). For catalase detection, the membrane was
incubated with primary catalase polyclonal antibody (1:
5000), then with biotinylated goat-antirabbit-IgG (1:
5000) and streptavidin-peroxidase conjugate and
finally developed by using diaminobenzene (DAB) and
hydrogen peroxide following manufacturer’s
instructions (Thermo Scientific, Rockford, IL, USA).
8. Statistical analyses
All data were subjected to one way analysis of
variance (ANOVA) to find out significance among
mean values. A difference was considered significant
at P<0.05 levels.
Results and Discussion
Catalase is present almost in every aerobic organism,
but the type of Catalase found differs from organism to
organism. Enzymes that reduce hydrogen peroxide or
use it as a reductantare generally termed
hydroperoxidases (HP) (Klotz and Loewen, 2003).
Catalatic hydroperoxidases(CHPs) primarily dismutate
hydrogen peroxide to water and dioxygenby
two-electron transfer redox reactions, and there is
considerablediversity among the enzymes that exhibit
catalatic activity. CHPs have a variety of subunitsizes,
a number of quaternary structures, at least two
differentheme prosthetic groups, and the reductant for
bifunctional catalase-peroxidases (CPX)can vary.
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Generally, CHPs can be placed into four main groups:
(1) the heme-containing monofunctional catalases
(hydrogen peroxide oxidoreductase E.C. 1.11.1.6) for
which hydrogen peroxide are both electron donor and
acceptor. It is again divided into three groups group I,
group II, group III (2) the heme-containing bifunctional
CPXs in which the catalatic activity is much higher
than
the
peroxidatic
activity,
(3)
thenonheme-containing catalases, and (4) a
miscellaneous groupcontaining proteins with minor
catalatic but no peroxidaticactivities (Jones and Wilson,
1978). Bacteria contain any of the enzymes or
combination of them. Among eukaryotes, fungi have
mainly group II, group III catalases but lack group I
and nonheme catalases. Similarly protists and higher
animals lack group I and nonheme catalases.
Chordates mainly contain group III catalase and thiol
peroxidase (TPX). Plant kingdom have only group I
type of catalases.
Most of the catalase is contained within organelles
inside cells that contain a large collection of different
oxygen-reacting enzymes like peroxisomes. In
vertebrates, catalse concentration is more in liver
(Chattopadhyay et al., 2007), as it is the site where
most metabolic and anabolic processes occur for the
entire body, and due to which larger amount of
peroxides are generated in liver. Hence, liver needs
more catalase to nullify the produced peroxides. In the
liver, like all cells, catalase is primarily contained
inside the peroxisomes. Besides liver, catalase is also
concentrated in erythrocytes (Das and Chainy, 2001).
In contrast to liver and erythrocytes, brain, heart and
skeletal muscle contain less amount of catalase
(Chattopadhyay et al., 2003; Sahoo and Chainy,
2007). The catalase concentration varies between
muscles and even different regions of same type of
muscle. Catalase is largely or completely located in
subcellular organelle peroxisomes (Chance et al.,
1979). In mammals presence of catalase is reported in
the mitochondria of heart (Redi et al., 1991) and also
in ER. Mouse liver catalase has a capacity to bind to a
variety of subcellular membranes in vivo and this
interaction may be consistent with a general protective
role for the enzyme, as well as being compatible with a
model of peroxisomal biogenesis which involves the
interaction of catalase with microsomal membranes
(Pegg et al., 1989).
The present experiment was carried out in the liver
samples of five different animals of five different
classes of vertebrates. The spectrophotometric
catalase activity assay shows a marked difference in
the activity of catalase among the animals (Figure 1).
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While the reptile (Calotes) showed the maximum
catalase activity, bird (hen) showed the minimum
activity. It is contrary to the fact that the birds may
have more aerobic metabolic conditions than any
other animal. However, the environmental conditions
play a major role in the physiology as well as free
radical generation (Halliwell and Gutteridge, 2001). As
hens were kept in less stress environment, it may
cause less catalase activity in their liver. Similarly the
reptile and mammal showed a high degree of catalase
activity. Fish showed a higher range of catalase
activity, as it lives in the most oxygen stress condition
among the taken animals.
The fact that experimental conditions may act
differently on the expression of enzymes of different
animals resulting in unequal expressions might have
contributed to the huge marked difference in the value
of catalase activity. First, the catalase activity staining
was performed with taking standard catalase, with
different concentrations of catalase (Figure 2), with
different substrates (Figure 3), with different substrate
concentrations (Figure 4) and with different substrate
incubation time (Figure 5). The activity staining of
catalase in these animals also showed (Figure 6 and
Figure 7) different pattern of activity among them. The
Calotes catalase showed the maximum activity with
other animals having activity pattern same as the
spectrophotometric assay. The aerobic capacity of
liver strongly influences the activities of antioxidant
enzymes in the tissues across vertebrates (Campo et
al. 1993). The warm-blooded animals might have more
catalase since their metabolisms would tend to
produce more reactive oxygen species and hydrogen
peroxide requiring catalase to dismentle them. Our
finding also corroborates the earlier study in which
lesser amount of catalase in hen than rat was reported
(Campo et al. 1993).
The activity staining with same protein loading showed
different Rf values (Table 1). The fish showed the
highest Rf value while others have almost similar
values. It denotes the fish catalase enzyme may have
less molecular mass and higher acidic amino acid
content. The similarity among reptile, bird and
mammal may be due to evolution of these animals
from the common ancestor. The Rf values are more or
less in a decreasing pattern. This may validate the
evolution propagation from fish to mammal. It may so
happen that the fish had a light and simple catalase
enzyme, which got evolved to a bulky and
complex/compound one during the process of
evolution.
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The striking feature of the activity staining of catalase
with same activity loading is the almost similar
expression of activity among reptile, bird and
mammalian catalases (Figure 7). The similar
magnitude of activity expression also certifies the
correctness of the result of spectrophotometric assay.
In western blotting analysis, all the animal liver
samples except bird expressed two broad bands
(Figure 8). The bird sample showed only one thick
band. The reptile sample expressed an additional light
band.
The availability of oxygen differs between aquatic and
terrestrial environments (Nayak et al., 1999). The
requirement and consumption of oxygen also differs
between aquatic and terrestrial vertebrates. Similarly
poikilothermic vertebrates have lower rates of oxygen
consumption (metabolic rate) than homeotherms. A
strong positive relationship exists between the rate of
oxygen consumption of aerobic animals and rate of
generation of oxygen radicals in their tissues (Davies
et al., 1982; Sohal et al., 1989). Different organs have
different age related patterns in antioxidant enzymes.
Catalase activity increases with age in liver and brain
of garden lizard (Jena et al., 1998).
In addition to developmental regulation, CAT
expression is also influenced by many environmental
factors such as light, hormone, ozone, temperature,
xenobiotics and hydrogenperoxide (Guan and
Scandalios, 2000). The research in fish revealed that
environmental factors like water temperature, salinity,
season and feeding habits, exerted changes in
peroxisomal enzyme activities that, additionally, vary
greatly among species. It was also discovered that
season, age and gender affect the morphology of fish
liver peroxisomes.
As Catalase is a conserved protein the study of
structural and functional variations among organisms
can shed light on how the evolution line has
propagated among these species. The three
dimensional structure of proteins is often more
conserved than their amino acid sequences.
Comparison of three-dimensional structures can
reveal common origins and functions of evolutionarily
distant proteins and can provide information on
functionally important, conserved structural features.
Residues of Penicillium vitale catalase (PVC) have
been built into a 2 Å resolution electron density map
and the backbone of this structure is compared to that
of beef liver catalase (BLC). The two proteins have
many structural similarities including sharing the same
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catalytic function, binding heme groups in analogous
binding pockets at similar positions. Both have a
tyrosine as a proximal iron ligand, and a distal region
containing a histidine and an asparagine necessary for
activity. However, there are differences in the two
structures. PVC has an additional flavodoxin-like
domain at its carboxy terminus. BLC contains a bound
NADP molecule plus an extra 13 residues at the
amino-terminus that are absent in PVC. The NADP
molecule in BLC is bound in the region occupied by
the extra flavodoxin-like domain in PVC. The presence
of the flavodoxin-like domain in PVC may indicate the
binding of a nucleotide. The above comparison shows
that neither the flavodoxin-like domain of PVC nor the
NADP of BLC are absolutely required for catalase
function, but that the presence of catalase-bound
nucleotides is important, presumably to protect the
enzyme from oxidative damage. The structural
similarities point to strongly conserved mechanisms for
peroxide detoxification, since mammalian and fungal
catalases diverged from a common ancestor at least
as early as the first eukaryotes (Boon et al. 2001).
Wdzieczak et al., (1982) compared levels of SOD,
Catalase, peroxidase in erythrocytes and livers of
seven freshwater and three marine fish species.
Results of the studies clearly indicate marked inter
specific differences in the distribution of antioxidant
enzymes and a regulatory effect of seasons on these
enzymes. No CAT activity was observed in
erythrocytes of some primitive species like Polypterrus
senegalus (Rabie et al., 1972) and in milkfish Chanos
chanos (Smith, 1976). Marcon and Wilhelm (1999)
also observed acatalasemia in fresh water migratory
teleost Colossoma macropomum from Amazon. Filho
(1996) made a comparison of various marine species
from southwestern Brazilian coast and few fresh water
species from central Amazon basin. He opined that
more active marine species exhibit high CAT and SOD
activity in liver and blood than sedentary species. In
frog the principal decomposer of H2O2 is catalase. It
is possible that birds, which have very high rates of
oxidative metabolism produce more catalase than
mammals, but virtually all catalases, even from
horseradishes are simiar and it would be difficult to
determine differences between them. Cow catalase is
almost identical to the catalse found in Bakers Yeast,
and human catalse is almost exactly the same as that
found in dogs, mice, worms, frogs, zebrafish, lime
molds and wheat. If we look at the sequence of the
DNA in plants versus fungus versus humans for
catalase, they are actually quite similar as well,
implying that catalase(s) are all decended from a
common ancestral protein and the structural
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similarities point to strongly conserved mechanisms for
peroxide detoxification.
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Illustrations
Illustration 1
Figure 1. Catalase activities in liver postmitochondrial fraction of different animals. Data are
expressed as mean ± S.D. of eight observations. Data differ significantly (P < 0.05) from each
other.
Illustration 2
Figure 2. Native-PAGE and negative staining of catalase by ferric chloride and potassium
ferricyanide method. Different concentrations of standard catalase were taken for the study.
Lane 1: 0.004 µg/25 µl; Lane 2: 0.01 µg/25 µl; Lane 3: 0.025 µg/25 µl; Lane 4: 0.06 µg/25µl;
Lane 5: 0.16µg/25µl; Lane 6: 0.4 µg/25µl.
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Illustration 3
Figure 3. Native-PAGE and negative staining of catalase by ferric chloride and potassium
ferricyanide method. Different substrates were taken for the study. 1: Hyderogen peroxide
(H2O2); 2: Cumene hydroperoxide.
Illustration 4
Figure 4. Native-PAGE and negative staining of catalase by ferric chloride and potassium
ferricyanide method. Different concentrations of substrate (H2O2) were taken for the study. 1:
0.66% (v/v) H2O2; 2: 0.033% (v/v) H2O2; 3: 0.0165% (v/v) H2O2.
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Illustration 5
Figure 5. Native-PAGE and negative staining of catalase by ferric chloride and potassium
ferricyanide method. Different incubation time in substrate (H2O2) were studied. 1: 12 min of
incubation; 2: 6 min of incubation; 3: 3 min of incubation.
Illustration 6
Figure 6. Native-PAGE and negative staining of catalase by ferric chloride and potassium
ferricyanide method in post mitochondrial fraction of different vertebrates with constant protein
loading.
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Illustration 7
Figure 7. Native-PAGE and negative staining of catalase by ferric chloride and potassium
ferricyanide method in post mitochondrial fraction of different vertebrates with constant activity
loading.
Illustration 8
Figure 8. Western blot analysis of catalase from different vertebrates using
biotin-streptavidin-HRP method. Lane1: Fish (Cirrhinus mrigala); Lane 2: Amphibia (Bufo
melanistictus); Lane 3: Reptile (Calotes versicolor); Lane 4: Bird (Gallus domesticus); Lane 5:
Mammal (Rattus rattus).
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Illustration 9
Table 1. Rf value of catalase measured in Native-PAGE and negative staining method.
Animals
Rf value
Fish (Cirrhinus mrigala)
3.125
Amphibia (Bufo melanistictus)
2.275
Reptile (Calotes versicolor.)
2.575
Bird (Gallus domesticus)
2.250
Mammal (Rattus rattus)
2.475
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