Profiling of Catalase and Hydrogen Peroxide activity in Tryptophan administered final instar larvae of Bombyx mori L

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Sebastian, C.D. et al Int. J. Pure App. Biosci. 3 (3): 201-207 (2015)
ISSN: 2320 – 7051
Copyright © June, 2015; IJPAB 201
Profiling of Catalase and Hydrogen Peroxide Activity in Tryptophan
Administered final instar Larvae of Bombyx mori L.
Priya Bhaskaran, K.P., Bindu, P.U., Akhilesh, V.P., Rukhsana, K., Jisha Krishnan, E.K. and
Sebastian, C.D.*
Molecular Biology Laboratory, Department of Zoology, University of Calicut, Kerala, 673635 India
*Corresponding Author E-mail: drcdsebastian@gmail.com
INTRODUCTION
Ageing has been defined as the progressive loss of function accompanied by decreasing fertility and
increasing mortality and disability. Altered response to therapeutic interventions might be considered in
any future definitions of ageing
1
. Oxidative modification of DNA, proteins, lipids and small cellular
molecules by reactive oxygen species (ROS) along with impaired antioxidant mechanism play some role
in a wide range of common diseases and age-related degenerative conditions. To protect molecules
against toxic free radicals and other ROS, cells have developed antioxidant defence system that include
the enzymes super oxide dismutase (SOD), which dismutates toxic superoxide and glutathione
peroxidase. External sources of antioxidant vitamins like vitamin C, vitamin E and phytochemicals from
plant rich diets provide important protection against oxidant damage
2
. However, the natural antioxidant
mechanism can be inefficient and hence dietary intake of anti oxidant compounds become important. In
addition to this there is an inverse relationship between dietary intake of antioxidant rich foods and
incidence of a number of diseases. Therefore search into the determination of antioxidant capacity of
different compounds become important.
Ageing is the process pertains to individuals and hence elucidation of age related physiological activities
should ideally be carried on the same individual during its entire lifespan and further the cellular and
molecular level patterns can rarely be obtained without killing the organism.
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Int. J. Pure App. Biosci. 3 (3): 201-207 (2015)
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Research Article
ABSTRACT
Ageing is a universal, biological and natural phenomenon in all living organism. Oxidant damage
by reactive oxygen species along with impaired antioxidant mechanism play a vital role in common
and age related degenerative diseases in animals. To protect molecules against toxic free radicals
and other ROS, cells have developed antioxidant defence system that include the enzymes super
oxide dismutase (SOD), which dismutates toxic superoxide and glutathione peroxidase. Despite of
the absence of glutathione peroxidase (GPx) in insects, the catalase mediates the main mechanism
for hydrogen peroxide (H
2
O
2
) breakdown. The natural antioxidant mechanism can be inefficient and
hence dietary intake of anti oxidant compounds become important. In the present study, a reducing
amino acid tryptophan was administered through the feed material of final instar larvae of
silkworm, Bombyx mori, in order to study its antioxidant effects on the growth and longevity of the
larvae. The profiles of pro-oxidant H
2
O
2
and antioxidant enzyme catalase during the development of
the larvae were evaluated. H
2
O
2
levels in haemolymph and fat body of the normal larvae increased
gradually and recorded a peak value at 72h and decreased thereafter. Catalase activity haemolymph
of normal larvae significantly increased from the beginning of fifth instar larval period and recorded
a peak value at 72h followed by a sharp dip.
Key words: Bombyx mori, tryptophan, ageing, H
2
O
2
, catalase, ROS

Sebastian, C.D. et al Int. J. Pure App. Biosci. 3 (3): 201-207 (2015)
ISSN: 2320 – 7051
Copyright © June, 2015; IJPAB 202
As ageing process in multi-cellular animals is universal, such difficulties can be tide over by using insects
as experimental animals. Despite of the absence of glutathione peroxidase (GPx) in insects, the catalase
mediates the main mechanism for H
2
O
2
breakdown. So insects are most useful in analyzing the effect of
H
2
O
2
. It was found that H
2
O
2
alone and in combination with other substances cause deleterious damages
in the living tissue and the direct interaction of transition metal (copper) to DNA in the presence of H
2
O
2
caused destabilization and fragmentation of naked DNA
3
. In most eukaryotic cells super oxide dismutase
exists in two different forms, cytosolic type containing copper and zinc and a mitochondrial type
containing manganese
4
. The natural combination of dismutase and catalase contributes to remove H
2
O
2
and thus has a true cellular antioxidant activity. Catalase is highly effective in the breakdown of H
2
O
2
at
high concentrations and also has the unique characteristic that it can degrade H
2
O
2
by catalytic or by
peroxidatic mechanism
5
. Antioxidants may offer resistance against oxidative stress by scavenging free
radials, inhibiting lipid peroxidation and or some other mechanism
6
. All organisms possess antioxidant
defence system, which protect against oxidative damage and numerous damage removal and repair
enzymes, which remove and repair damaged molecules. However, the natural antioxidant mechanism can
be inefficient, dietary intake of antioxidant compounds become important
7
. Superoxide dismutases (SOD)
are metallic enzymes that are essential for dismutation of O
2
to H
2
O
2
and O
2
. SODs are important initial
components in the cellular defence against oxygen toxicity. In most eukaryotic cells SOD exists in two
forms, a cytosolic type containing copper and zinc and a mitochondrial type containing manganese
4, 8
.
Glutathion peroxidase is believed to play an important role in the breakdown of H
2
O
2
, because it is much
more efficient than catalase at low concentrations of the substrate
9
.
Catalase is primarily localized with in peroxisomes and to a lesser extent in mitochondria which is the
main site of H
2
O
2
production from O
2
dismutation. Catalase is highly effective in the breakdown of H
2
O
2
at high concentrations and also has a unique characteristic that it can degrade H
2
O
2
by catalytic
mechanism
5
. In a recent study on protective effects of catalase over expression on UVB - induced
apoptosis in normal human keratinocytes, pointed out that catalase over expression has a protective role
against UVB irradiation by preventing DNA damage mediated by the late ROS increase
10
. Catalase
activity revealed differences in time related changes across a 24 h period that are more obvious in peak
levels between the brain, kidney and liver tissues of mouse
11
. Their results also showed significant organ
differences with the highest catalase activity in liver compared with kidney and brain. This might be
related to several factors such as their respective physiological function, the risk of exposure to oxidative
damage and the balance between synthesis and degradation of proteins during normal metabolism.
In the present study, a reducing amino acid tryptophan was administered through the feed material of final
instar larvae of silkworm, Bombyx mori, in order to study its antioxidant effects on the growth and
longevity of the larvae. The specific objectives of the study are to evaluate the physiological and
biochemical changes during the development of the final instar larvae on the administration of antioxidant
amino acid, tryptophan. The profiles of pro-oxidant H
2
O
2
and antioxidant enzyme catalase during the
development of the larvae were evaluated.
MATERIALS AND METHODS
The experimental animal
The silkworm Bombyx mori L. belongs to the Phylum Arthropoda, Class Insecta and Order Lepidoptera.
The bivoltine silkworm hybrid, Elite- CSR 2x4 was used for the study. The silkworm rearing was
undertaken by procuring newly hatched larvae immediately after their brushing from Serifed, Palakkad,
Kerala. The rearing house and all the appliances were disinfected in advance with chlorine
dioxide/bleaching powder to free the rearing environment and the surroundings from pathogens. The
rearing bed was made up by chopped leaves having the size of half to one cm. squares and covered with
blue polythene sheet. Clean wet sand in trays was placed around the rearing bed to ensure 80-90%

Sebastian, C.D. et al Int. J. Pure App. Biosci. 3 (3): 201-207 (2015)
ISSN: 2320 – 7051
Copyright © June, 2015; IJPAB 203
humidity and the rearing room temperature was maintained at 27°C. Tender mulberry leaves with more
moisture content, protein and carbohydrates were used for feeding the worms in the initial stages of
rearing and were fed three times a day.
The duration of 5th instar was normally about 6-7 days and the larvae started to spin cocoon by the end of
this stage. During 5th instar the larvae were fed with fully matured mulberry leaves and for the last two
days it was coarse leaves. The present work was done on the fifth instar larvae, beginning from the newly
moulted stage and continued till the last day of the instar, just before spinning began. The larval period
was divided into seven chronologically identified stages: i.e., 0h, 24 h, 48h, 72h, 96h, 120h and 144h.
After the fourth moult the larvae were segregated into two sets. One set was fed with mulberry leaves
dipped in 5mM tryptophan solution and drained in air for half an hour. The other set of larvae were fed
with leaves dipped in distilled water and drained in air for half an hour.
Biochemical analysis
For various biochemical estimations pooled haemolymph samples were extracted from appropriate
number of both normal and treated larvae separately. For the estimations of fat body samples the tissue
was homogenized and diluted to appropriate volume with water for all assays except enzymes. The
analysis was carried out at 24 hour intervals in on the basis of unit volume and total volume in the case of
haemolymph and on the basis of unit weight of fresh tissue and total tissue in the case of fat body. The
pooled haemolymph and fat body samples were isolated from the larvae of each set. The tissues were
stored at
-
20° C until the estimations were carried out.
Estimation of Hydrogen peroxide level and Catalase activity
Both estimations were done based on the formation of soluble coloured peroxotitanium complex in a
reaction of H
2
O
2
with potassium titanium oxalate and the measurement of the orange/ yellow colour
developed, at 410 nm
12
. For estimation of H
2
O
2
, the homogenized tissue was deproteinised with 20%
trichloroacetic acid (TCA) and centrifuged at 3000 rpm for 10 min. 0.5 ml of the protein- free solution
was mixed with 1.5 ml of acidic potassium titanium oxalate reagent and the bright yellow colour formed
was measured at 410 nm in a spectrophotometer. The determination of catalase activity was based on
estimating the amount of residual H
2
O
2
in the assay mixture after incubation of a known amount of H
2
O
2
with the enzyme extract for a fixed time interval. The data were subjected to statistical analysis to
evaluate whether the variations are significant between normal and treated insects using student’s t test
method.
RESULTS
The H
2
O
2
levels estimated per unit fresh tissue in the haemolymph and fat body of both normal and
treated larvae are given in Figure 1 and Figure 2. H
2
O
2
levels per unit volume haemolymph of the normal
larvae increased gradually and recorded a peak value at 72h and decreased thereafter. H
2
O
2
level at the
final stage of the larvae was almost one half of that recorded in the previous stage. A different pattern of
change was found in treated larvae where the values reached a maximum at 48h and then dropped down
sharply. The pattern of change in H
2
O
2
levels of the normal and treated larval fat body was more or less
similar to that observed per unit volume of haemolymph.
The activities of the antioxidant haemoprotein catalase per unit fresh tissue was estimated in the
haemolymph and fat body of both normal and treated larvae and are presented in Figure 3 and 4
respectively. Catalase activity of per unit volume haemolymph of normal larvae significantly increased
from the beginning of fifth instar larval period and recorded a peak value at 72h followed by a sharp dip.
There was a significant reduction in catalase activity in the treated larvae when compared with normal.
The activity of catalase in fat body of normal larvae remained more or less the same throughout its
development except that it was low in its initial stage. The activity of catalase in the treated larvae was
high in the active feeding stages of the larvae but declined sharply towards the end of larval development.

Sebastian, C.D. et al Int. J. Pure App. Biosci. 3 (3): 201-207 (2015)
ISSN: 2320 – 7051
Copyright © June, 2015; IJPAB 204
Fig. 1: Changes in the hydrogen peroxide levels in haemolymph during final larval instar of B. mori on
administration of tryptophan
Fig. 2: Changes in the hydrogen peroxide levels in fat body during final larval instar of B. mori on
administration of tryptophan
**
**
**
** ** **
0
1
2
3
4
5
6
7
8
9
0h
24h
48h
72h
96h
120h
144h
mg/ml
Larval age
Changes in the hydrogen peroxide levels in
haemolymph
mg/ml Normal
mg/ml Treated
*
** *
*
*
0
1
2
3
4
5
6
7
8
9
10
0h
24h
48h
72h
96h
120h
144h
mg/mg fresh tissue
Larval age
Changes in the hydrogen peroxide levels in the fat
body
mg/mg fresh tissue Normal
(x10-3)
mg/mg fresh tissue Treated
(x10–3)

Sebastian, C.D. et al Int. J. Pure App. Biosci. 3 (3): 201-207 (2015)
ISSN: 2320 – 7051
Copyright © June, 2015; IJPAB 205
Fig. 3: Changes in the catalase activity levels in haemolymph during final larval instar of B. mori on
administration of tryptophan
Fig. 4: Changes in the catalase activity levels in fat body during final larval instar of B. mori on
administration of tryptophan
DISCUSSION
Accumulation of hydrogen peroxide and a decline in catalase activity is associated with ageing process of
organisms. Accumulation of tryptophan residues in the transmembrane domains of integral membrane
proteins performs vital antioxidant functions
13
. The accumulations of histological biomarkers of ageing
were also delayed after tryptophan restriction in some organs. The tryptophan hydroxylase activity is
affected by ageing process, which can drastically affect the levels of serotonin in brain
14
. The dietary
effects of non-enzymatic antioxidant such as carotenoides, selenium and beta carotenoids have been
demonstrated in organisms
15,16
.
*** **
**
0
20
40
60
80
100
120
140
160
180
0h
24h
48h
72h
96h
120h
144h
mg/min/ml
Larval age
Changes in the catalase activity levels in haemolymph
mg/min/ml Normal
mg/min/ml Treated
*
*
**
0
0.5
1
1.5
2
2.5
3
3.5
4
0h 24h 48h 72h 96h 120h 144h
mg/min/mg fresh tissue
Larval age
Changes in the catalase activity levels in fat
body
mg/min/mg fresh tissue
Normal
mg/min/mg fresh tissue
Treated

Sebastian, C.D. et al Int. J. Pure App. Biosci. 3 (3): 201-207 (2015)
ISSN: 2320 – 7051
Copyright © June, 2015; IJPAB 206
Cytosolic enzyme catalase is a component of antioxidant defense system that reduces hydrogen peroxide
to water and protect the cell from oxidative damage
17
. Generation of H
2
O
2
associated with life expectancy
is proved by different workers
9,18
. H
2
O
2
is normally eliminated from the cells by the activity of catalase
and peroxidases. Catalase is primarily localized within peroxisomes, and to a lesser extent in
mitochondria, which is the main site of H
2
O
2
production
5
. In vitro studies on the role of reactive oxygen
species on fibroblasts from patients with systemic sclerosis revealed that H
2
O
2
act as a mediator of a
positive autocrine feed back mechanism of ROS generation. The effects of H
2
O
2
and H
2
O
2
with the
antioxidant agent N-acetyl-1-cystein or catalase on mean arterial pressure and heart rate of rats and found
that H
2
O
2
produced as a result of oxidative reaction causes misfunction of organs
19
. They also concluded
that even 1.0 to 1.5 µmol doses of H
2
O
2
can produce long lasting bradycardia.
The development of the final instar larvae of B. mori is marked by distinctive feeding and non- feeding
stages. At the end of the non- feeding stage, the larva is transformed into a pupa with entirely different
morphological features. The larvae of B. mori maintain a high level of H
2
O
2
in its feeding stages and
showed a sharp dip in the non- feeding stage. H
2
O
2
levels in the tryptophan treated larvae showed a
conspicuous reduction apparently indicating its scavenging action on the free radicals. Similar reduction
in peroxide level on the administration of mixed antioxidant supplemented diet in adults of Zaprionus
paravittiger, after the administration of mixed antioxidant supplemented diet
20
. Studies have been
conducted on Bombyx mori larvae and proved that the administration of ascorbic acid and tyrosine caused
significant changes in the organism’s body by scavenging the free radicals
21
.
Although steady state level of oxidative stress depends on both pro-oxidant generation and
antioxidant defenses, most of the studies pertaining to ageing have focused on antioxidant defences.
Catalase activity has been studied in a variety of insects
22,23
. Catalase activity increased with age and
decreased during the latter part of life. Studies on catalase activity and lifespan of flies have shown that
complete lack or reduced level of catalase activity does not affect their lifespan. An over expression of
catalase (about 50%) had no effect on the lifespan of flies, nor did it improve their viability to an
experimentally enhanced level of oxidative stress induced by parquet intake or hyperoxia
24
. The increased
levels of catalase activity in the haemolymph of normal and treated larvae in the active feeding stages and
its decline in the non-feeding stages observed in the present study can be explained in the above context.
The treated larvae exhibited a significant reduction in haemolymph catalase activity which is in tune with
the reduced H
2
O
2
levels in the tissue. The results of the present study also demonstrate that there is a
corresponding increase in the antioxidant activity with an increase in pro-oxidant generation. Studies on
the variation of catalase activity in brain, kidney and liver of adult male mice showed that the activity was
highest in the liver compared with kidney and brain
11
. The difference in the levels of enzymes in different
tissues have been attributed several factors such as their respective physiological functions, the risk of
exposure to oxidative damage and the balance between synthesis and degradation of proteins during
normal metabolism. The reduced levels of catalase activity in the treated larvae compared to the normal
also indicate a reduced pro-oxidant generation in the latter.
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