View
2
Download
0
Category
Preview:
Citation preview
MATERIALS AND METHODS
31
The present study was focused on the evaluation of the neuroprotective effect
of Silk Protein, Sericin on Morphometric- and Behavioural Aspects, Cholinergic
system, Antioxidant system, Histological Aspects and Bioinformatics aspects.
Selection of Experimental Model:
The present study was carried out on the male albino rat, Rattus norvegicus,
weighing 160 ± 20 gm. Rat was chosen as experimental model for the present study
because of the following reasons.
1. Rat is considered as an excellent animal model for experimental studies
(McEwen and Stephenson, 1979).
2. Albino rats are relatively small, can be handled easily and require less feed.
3. The rats are preferred because of the plethora of Alzheimer’s Disease data that
exists for rodents (Bruce, 1985).
4. Extrapolation of the rats to the human being (man) is usually done for risk
assessment (Moser, 1990) for a wide variety of compounds (Grotenet al.,
1997).
5. It is worth full to develop animal model to understand the pathogenesis of AD
and to explore existing strategies for prevention and treatment of this
neurodegenerative disorder (Albensi, 2001).
Procurement and Maintenance of Experimental Animals:
Healthy Wistar strain Albino rats, Rattus norvegicus of the same age group of
3 months, weighing 160 ± 20 grams, obtained from Sri Venkateswara enterprises,
Bangalore were used as the experimental model in the present investigation. Prior to
experimentation, the rats were acclimatized according to the instructions given by
Behringer (1973). They were housed in polypropylene cages under the controlled
conditions of 28 ± 2°C temperature with photoperiod of 12 hours light and 12 hours
dark and 75% relative humidity maintained in the animal house of the Department
Zoology, according to the ethical guidelines for animal protection and welfare bearing
the Resolution No. 04/(i)/a/CPCSEA/ IAEC/ SVU/ KY- KPR / Dt. 28-03-2011. The
rats were fed with standard pellet diet supplied by Sri Venkateswara Enterprises,
Bangalore and water ad libitum throughout the period of experimentation.
P
ob
(M
C
fo
P
F
Procuremen
All ch
btained from
Mumbai, In
Chemicals (B
ollowing equ
1. Barns
2. Hahnv
3. Kubot
4. Hitach
Physical Pro
D-Gal
Fang and Liu
Moleculand Stru
IUPAC n
Synonym
MoleculMelting Boiling pSolubilitDensity PubChemAppearaStorage
t of Chemic
hemicals use
m Sigma (S
ndia), Ranba
Bombay, Ind
uipments hav
stead Thermo
vapor Rotary
ta KR 2000T
hi UV-2800
operties of D
lactose (D-G
u., 2007).
lar formuucture
name
ms
lar weight point point ty in water
m ID ance
cals:
ed in the pre
St. Louis, M
axy (New D
dia). In the p
ve been used
oline water p
y Evaporator
T centrifuge
spectrophot
D-Galactose
Gal) was use
ula : C6H12O
: (3R,4S 2,3,4,5
: D(+)- d-gala
: 180.15: 165-16: 527.1 : 683.0 : 1.581 : 43935: White : Store i away f
32
esent study w
MO, USA), F
Delhi, India)
present inve
d.
purification p
r HS-2005V
for centrifug
tometerfor m
:
d to induce A
O6
S,5R,6R)-6-(5-tetrol
Galactose, Gactose, D-Ga
56 g mol−1 68°C °C at 760 mg/L g/cm3 7 powder
in cool and dfrom direct l
were of Ana
Fisher (Pitts
), Qualigens
stigation, fo
plant for Na
V for extract p
gation of tiss
measuring Op
AD in rat as
Picture
(Hydroxyme
Galactose, Balactopyrano
mmHg
dry area and ight.
alar Grade (A
sburg, PA,
s (Mumbai,
or biochemic
ano pure wat
preparation.
sue homogen
ptical Densit
per (Zhang
10: D- Gal
ethyl)oxane-
rain sugar, ose.
kept sealed
AR) and we
USA), Mer
India), Lob
cal assays, th
er.
nates.
ty.
g et al., 2006
Structure
-
ere
ck
ba
he
6;
33
Biological effect of D-Gal:
Old Age can be induced by intraperitonial (IP) injection of D-Galactose, a
reducing sugar, which reacts readily with the free amines of amino acids in proteins
and peptides both in vivo and in vitro to form Advanced Glycation End-products
(AGE) through non-enzymatic glycation (Song et al., 1999; Bassi et al., 2002). The
Advanced Glycation End-product activates its receptors, which are coupled to
biochemical pathways that stimulate free radical production (Yan et al., 1996). D-
Galactose is a physiological nutrient, but over supply of D-Galactose will result in
abnormality of metabolism. The oxidative metabolism of D-Galactose produces
Reactive Oxygen Species(ROS), which surpass the ability of the cells to eliminate
them, consequently causing impairment of cellular membrane, structure and gene
expression (Wang, 1999; Zhang et al., 2006). In addition, the non-enzymatic
glycation is another pathway that can enhance oxidative lesions in ageing and age-
associated disease such as Alzheimer’s Disease (Baynes, 2001). Thus, long-term
intraperitoneal injection of D-Galactose induces AD in normal rat (Zhang et al., 2006;
Fang and Liu, 2007).
Collection of silk cocoons and extraction of Sericin:
Extraction of Sericin:
Raw Silk cocoons, purchased from local market of Chittoor were boiled in
water for 1hour and the resulting solution was cooled and filtered. After repeating this
process for 3 times, the filtrate was concentrated by using Hahnvapor Rotary
Evaporator (HS-2005V). 95% ethanol was added to the extract to precipitate Sericin,
collected by filtration, dried at 40oC, powdered and finally preserved in clean
container for further use. 8% SDS PAGE was done to confirm the presence of Sericin,
based on the molecular weight.
34
Picture -11: Sericin extraction from Silk Cocoons Flow chart
Induction of Alzheimer’s Disease:
D- Gal infection has been generally accepted to establish an aging model for
brain aging or anti-aging pharmacological research (Cui et al., 2006; Ho et al., 2003).
In the present study Memory impairment was induced to rats by IP injection of
D-Galactose (120mg/kg body weight) by dissolving in distilled water for one month
(Holden et al., 2003; Chen et al., 2006; Cui et al., 2006).
35
Administration of tested substance:
Silk Protein, Sericin (SP-P) extract (200 mg/kg body weight) was dissolved in
distilled water and given to the rat. A gavage tube was used to deliver the substance by
oral route, which is clinically expected route for administration. The volume of Sericin
administered was kept at 1 ml to the animal.
Experimental Design:
• Animal model : Male Albino Rat • Age : Three months old • Weight of : 160±20 grams • Chemical agents used for Induction of AD in Rat
: D-Galactose (D-Gal)
• Route of Administration : Intraperitoneal injection (IP) • Test substance : Sericin • Route of Administration : Oral
• Tissue selected • Brain regions selected
: :
Brain Cerebral Cortex (CC) and Hippocampus (HC)
• Isolation of tissues for Biochemical estimation
: 60th day and 90th day
Picture-12: Schematic representation of experimental design:
36
Grouping of Animals:
After the rats were acclimated to the laboratory conditions for 10 days before
the experimentation, they were randomly divided into four groups. Each main group
was again divided in to 2 sub groups of six each and were housed in separate cages.
These different groups of rats except control were treated with selected doses of
Sericin and D-Gal as given below. Keeping in view the altered activity of rats during
the nights compared to day time, all doses were given once in the morning hours in
between 8 A.M. to 9 A.M.
Group-I
(Control)
Control Rat
Group-II
(SP-S)
Rat, orally administered with Sericin (200 mg/kg body weight) up to 60
days (31st day to 90th day) continuously once in a day. (Joung et al.,
2011)
Group-III
(AD)
Rat, Intraperitoneally (IP) administered with D-Gal (120 mg/kg body
weight) up to end of the experiment (1st day to 90th day). (Zhang et al.,
2006; Hua et al., 2007)
Group-IV
(AD+SP-S)
Rat, Intraperitoneally injected with D-Gal (120 mg/kg body weight)
once daily for first 30 days. From 31stday onwards rats were
administered with Sericin (orally; 200 mg/kg body weight) along with
D-Gal (IP) up to 90th day.
In the present study the experimental duration selected was 90days. D-Gal was
given for first 30 days period to observe AD symptoms with the assessment of
cognitive skills in rats (AD group). Further AD induced rats were again treated with
D-Gal as well as Sericin simultaneously. To observe the cognitive skills, the rats were
subjected to behavioral studies on selected days (as per the experimental design).
37
Table-5: STANDARD METHODS APPLIED FOR VARIOUS PARAMETERS:
S.NO NAME OF THE PARAMETER
METHODS EMPLOYED
I Behavioural Aspects Morris water maze experiment(1984)
II Cholinergic System
Acetylcholine (ACh) Metcalf method (1957) as given by
Augustinsson (1957)
Acetylcholinesterase (AChE) Ellmanet al., (1961)
III Antioxidant System
Superoxide dismutase (SOD) Misra and Fridovich, (1971)
Catalase (CAT) Modified version of Aebi (1984)
Glutathione Reductase(GR) Carlberg and Mannervik (1985)
Lipid peroxidation (LPO) Ohkawaet al., (1979)
IV Histological Aspects Light Microscopy
V Bioinformatics Aspects
Modeling of 3D Structure MODBASE server
(http://salilab.org/modbase)
3D Structure Validation Ramachandran Plot
Energy Minimization Argus Lab
Protein-Protein Docking (Sericin and AChE)
Hex-6.0
Molecular Visualization PyMol
38
1. Behavioural Aspects:
Behavioral experiments were performed by using the water maze (Morris,
1984) which was originally designed to test the learning and memory ability in
rodents. A great deal of knowledge has been obtained on the neurochemical,
neuroanatomical and neurophysiological basis for the behavior associated with this
paradigm. The apparatus consisted of a circular tank, 100 cm in diameter and 50 cm in
depth painted with non-toxic white paint. The tank was filled with water (21-26oC) up
to a height of 30cm and the transparent escape platform made of plexiglass measuring
10cm in diameter and 29cm in height was hidden 1.5 cm below the surface of water in
a fixed location. Water was made opaque by mixing with powdered non-fat milk. The
platform was not visible from just above the water level and transfer trials have
indicated that escape on to the platform was not achieved by visual or other proximal
cues (Morris, 1981). The time spent by the animal to reach the hidden platform was
called as the Escape Latency and used as the index of memory.
Isolation of tissues:
For all biochemical estimations, the above mentioned four groups of rat were
sacrificed on selected days i.e., on 60thdayand 90thdayby cervical dislocation. The
brain was isolated immediately and placed on a chilled glass plate. Cerebral Cortex
and Hippocampus were separated by following standard anatomical marks (Glowinski
and Iverson, 1966) and they were frozen in liquid nitrogen and stored at -80oC until
further use. At the time of biochemical analysis, the tissues were thawed and used. The
results obtained were analyzed statistically.
Biochemical Assays:
Estimation of total proteins:
The total protein content was estimated by the method of Lowry et al., (1951).
2% homogenates were prepared in 10 % TCA and centrifuged at 1000xg for 15
minutes. The supernatant was discarded and the residue was dissolved in a known
amount of 1N sodium hydroxide. From this, 0.2ml was taken and 4 ml of alkaline
39
copper reagent and 0.4ml of folin phenol reagent (1:1 folin phenol and distilled water)
was added. The contents were allowed to stand for 30 minutes at room temperature
and the developed color was read at 600 nm in a spectrophotometer against a reagent
blank. The amount of total proteins present in the sample was calculated by using
bovine albumin standard and the values were expressed as mg/g wet weight of tissue.
Total proteins was calculated with the following formula
2. Cholinergic system:
i) Acetylcholine (ACh):
Acetylcholine (ACh) content was estimated by the method of Metcalf (1951)
as given by Augustinsson (1957). The rat brain regions such as Cerebral Cortex (CC)
and Hippocampus (HC) were weighed accurately, transferred to test tubes and placed
in a boiling water bath for 5 minutes to terminate the Acetylcholinesterase enzyme
activity and also to release the bound ACh. Then the tissues were homogenized in 1ml
of distilled water. To the homogenate, 1ml of alkaline hydroxylamine hydrochloride
was added followed by 1ml of 50 % hydrochloric acid solution. The contents were
mixed thoroughly and centrifuged. To the supernatant, 0.5ml of 0.37M ferric chloride
solution was added and the brown colour developed was read at 540nm against a
reagent blank (1ml of alkaline hydroxylamine hydrochloride+1ml of 50%
hydrochloride + 1ml of distilled water + 0.5ml of 0.37M ferric chloride solution) in a
spectrophotometer. The Acetylcholine content was expressed as μ moles of ACh/gm
wet weight of tissue.
ACh content was calculated with the following formula
∆ .
40
ii) Acetylcholinesterase (AChE):(E.C.: 3.1.1.7; Acetylcholine acetyl hydroxylase):
Acetylcholinesterase activity was estimated by the method of Ellman et al.,
(1961). 10% homogenates of different regions of rat brain were prepared in 0.25M ice
cold sucrose solution. The reaction was started with the addition of 100 μ liters of
homogenate to the reaction mixture containing 3.0ml of phosphate buffer (PH 8.0) +
20 μ moles of substrate (0.075M) + 100 μ moles of Dithiobis Trinitrobenzene
(DTNB,0.01M). The contents were incubated at 37°C for 15 minutes. The developed
colour was read at 412 nm in a spectrophotometer against a reagent blank containing
3.0ml of phosphate buffer (pH8.0) + 20 μ moles of substrate (0.075M) + 100 μ moles
of Dithiobis Trinitrobenzene (DTNB,0.01M).The enzyme activity was expressed as μ
moles of ACh hydrolyzed/mg protein/hour.
AChE activity was calculated with the following formula:
. .
3. Antioxidant System:
i) Superoxide Dismutase :(SOD EC: 1.15.1.6):
Superoxide dismutase activity was determined according to the method of
Misra and Fridovich, (1971) at room temperature. Different areas of rat brain such as
Cerebral Cortex (CC), and Hippocampus (HC) were homogenized in ice cold 50 mM
phosphate buffer (pH 7.0) containing 0.1 mM EDTA to give 5% homogenate (w/v).
The homogenates were centrifuged at 10,000 rpm for 10 mm at 4°C in ice cold
centrifuge. The supernatant was separated and used for enzyme assay. 100 µl of tissue
extract was added to 880 µl (0.05 M, PH 10.2, containing 0.1 mM EDTA) carbonate
buffer and 20 µl of 30 mM epinephrine (in 0.05% acetic acid) was added to the
mixture and measured the optical density values at 480 nm for 4 min in UV
Spectrophotometer.SOD activity was expressed as the amount of enzyme that inhibits
the oxidation of epinephrine by 50%, which is equal to 1 unit activity.
41
SOD activity was calculated with the following formula
Where,
ii) Catalase (CATEC 1.11.1.6):
Catalase activity was measured by a slightly modified version of Aebi, (1984)
at room temperature. Rat brain regions such as Cerebral Cortex (CC) and
Hippocampus (HC)were homogenized in ice cold 50 mM phosphate buffer (pH 7.0)
containing 0.1 mM EDTA to give 5% homogenate (w/v). The homogenates were
centrifuged at 10,000 rpm for 10 min. at 4°C in ice cold centrifuge. The resulting
supernatant was used as enzyme source. 10 µl of 100% ethyl alcohol was added to 100
µl of tissue extract and then placed in an ice bath for 30 min. After 30 minutes the
tubes were kept at room temperature followed by the addition of 10 µl of Triton X-100
RS. In a cuvette containing 200 µl of phosphate buffer and 50 µl of tissue extract, 250
µl of 0.066 M H202 (in phosphate buffer) was added and optical density was measured
at 240 nm for 60 s in a UV Spectrophotometer. The molar extinction coefficient of
43.6 M cm-1 was used to determine CAT activity. One unit activity is equal to the
moles of H202 degraded / mg protein /min.
Catalase activity was calculated with the following formula
∆ .
Where,
∆OD = Maximum OD difference between the 0” Sec to 60”sec.
42
iii) Glutathione Reductase (GR-EC: 1.6.4.2):
Glutathione Reductase activity was determined by a slightly modified method
of Carlberg and Mannervik, (1985) at 37°C. Selected rat brain regions such as
Cerebral Cortex (CC), and Hippocampus (HC)were homogenized (5%-w/v) in 50 mM
phosphate buffer (pH 7.0) containing 0.1 mM EDTA. The homogenates were
centrifuged at 10,000 rpm for at 4°C in ice cold centrifuge. The separated supernatant
part was used as enzyme source. NADPH (50 µl, 2mM) in 10mMTris buffer (pH 7.0)
was added to the cuvette containing 50 µl of GSSG (20mM) in phosphate buffer (0.5
M, pH 7.0 containing 0.1 mM EDTA) and 800 µl of phosphate buffer. The tissue
extract (100 µl) was added to the NADPH-GSSG buffered solution and measured at
340 nm for 3 mm. The molar coefficient of 6.22 X 103 cm-1 was used to determine GR
activity. One unit of activity is equal to the mM of NADPH oxidized I mg protein /
mim. The enzyme activity was expressed in μ moles of NADPH oxidized / mg protein
/min.
Glutathione Reductase activity was calculated with the following formula
∆ .
Where ,
∆OD = Maximum OD difference between the 0’ min to 4’min.
iv) Lipid Peroxidation (LPO):
The MDA levels were measured as described by Ohkawa et al., (1979).
Different areas of rat brain such as Cerebral Cortex (CC) and Hippocampus
(HC)tissue were homogenized (5%- W/V) in 50 mM phosphate buffer (pH 7.0)
containing 0.1 nM EDTA. The homogenates were centrifuged at 10,000 rpm for 10n
min at 40C in cold centrifuge. The separated supernatant part was used for estimation.
200 μl of the tissue extract was added to 50 μl of 8.1% Sodium Dodecyle Sulphate
(SDS), vortexed and incubated for 10 min at room temperature. 375 μl of twenty
percent acetic acid (pH 3.5) and 375 μl of thiobarbituric acid (0.6%) were added and
placed in a boiling water bath for 60 min. The samples were allowed cool at room
temperature. A mixture of 1.25 ml of Butanol:Phyridine(15:1) was added vortexed
43
and centrifuged at 1000 rpm for 5min. The colored layer (500 μl) was measured at
532 nm using 1,1,3,3-tetraethoxypropane as a standard. The values were expressed in
μ moles of Malondialdehyde formed/gram wet weight of the tissue.
MDA levels was calculated with the following formula:
4. Histopathology:
Histological examinations of the tissues Viz. Cerebral Cortex and
Hippocampus were carried out according to Humason (1972).
Light Microscopy
On selected days of experimentation viz. 60th and 90th day, the rats were
sacrificed by cervical dislocation and the selected brain regions such as CC and HC
were isolated. Isolated tissue of control and experimental rats were gently rinsed with
physiological saline (0.9% NaCl) to remove blood and debris adhering to the tissues.
They were fixed in 5% formalin for 24 hrs. The fixative was removed by washing
through running tap water overnight. After dehydrating through a graded series of
alcohols, the tissues were cleared in methyl benzoate, embedded in paraffin wax.
Sections were cut at 6μ thickness and stained with hematoxylin (Harris, 1900) and
counter stained with eosin (dissolved in 95% alcohol). After dehydration and clearing,
sections were mounted with DPX. The stained sections were observed under
microscope and the histological changes were recorded with the help of a pathologist.
5. Bioinformatics Aspects:
In order to understand the mechanisms of binding and the interaction between
Sericin and the Acetylcholinesterase, a three-dimensional (3D) structure of the rat
Acetylcholinesterase is generated based Homology modeling methods by using
MODBASE server. The initial models were improved by energy minimization
methods, the final refined models are further assessed by Ramachandran Plot. With
44
these models, the protein-protein docking studies were performed to check their
binding affinities.
The Present work was mainly concentrated on molecular interactions between
AChE and Sericin and the bioinformatics study was executed in various steps as given
below.
Homology modeling:
This, also known as comparative protein modeling or knowledge-based
modelingis used when there exists a clear structural relationship or functionally related
proteins with similar sequences, as measured by sequence identity and have similar
structures, in which the unknown sequence, called the target, bears a sufficiently
strong sequence similarity with another sequence, called the template, for which the
structure is already known. Broadly the modelling procedure can be divided intofour
steps: selecting the template, alignment of target with template, building the model
and evaluating the model (Ginalski, 2006).
The most basic step in structure prediction for selecting suitable template(s)
(sequence identity ≥ 30%) related to the target sequences were selected from the
Protein Data Bank (PDB) (Berman et al., 2000). This can be accomplished using
several tools that have been developed to carry out this step among which, BLAST
(Altschul et al., 1990) and PSI-BLAST (Altschul et al., 1997), FASTA (Pearson,
1988) are the most accepted ones.
The Coordinates of the three-dimensional model are built based on the
alignment and template structures. This step can be divided into two main trends. One
is to model the structure by copying the coordinates of the template in the aligned core
regions. The variable regions are modelled by taking fragments with similar sequences
from a database of previously observed loops. Then, the mutated side chains are
replaced with protamers that satisfy the stereo chemical criteria, together with limited
energy optimization. The sequence was submitted to database of protein structures like
MODEBASE server, a well-known program to build the Structures (Fiser et al.,
2000). The model building process consists of three main steps; in first step, the back
bone of the structure was build, then side chains were placed in second step and in
45
finally step, the entire structure was optimized. These steps were repeated until
acceptable results were obtained. Finally, the modeled structures were evaluated. It is
necessary to ensure that the model satisfies physical constraints and principles, such as
non-interpenetration of the atoms. Next in degree of importance, comes the
stereochemistry, such as bond lengths, angles, etc. The Ramachandran plot is a very
suitable method of checking the geometry of the model (Ginalski, 2006).
1. Collection of protein sequence from UniProt:
The sequences and the required information for the Proteins viz. AChE (Rattus
norvegicus) and Sericin (Bombyx mori) were retrieved from protein database UniPort
by using URL: http://www. uniprot.org.UniProt is a comprehensive, high-quality and
freely accessible database of protein sequence and functional information where many
entries were derived from genome sequencing projects. It contains a large amount of
information about the biological function of proteins derived from the research
literature. The UniProt consortium comprises the European Bioinformatics Institute
(EBI), the Swiss Institute of Bioinformatics (SIB), and the Protein Information
Resource (PIR). EBI located at the welcome trust genome campus in Hinxton, UK,
hosts a large resource of bioinformatics databases and services. SIB, located in
Geneva, Switzerland, maintains the ExPASy (Expert Protein Analysis System) servers
that are central resource for proteomics tools and databases. PIR is a successor to the
oldest protein sequence database hosted by the National Biomedical Research
Foundation (NBRF) at the Georgetown University Medical Center in Washington,
DC, USA. Margaret Dayhoff's prepared the Atlas of Protein Sequence and Structure
and published it in 1965. Finally, EBI, SIB, and PIR joined forces as the UniProt
consortium (2002).
II. BLAST (Basic Local Alignment Search Tool)
BLAST available at http://www.ncbi.nlm.nih.gov/BLAST was developed by
Altschul et al., (1997) for a rapid sequence comparison. Basic Local Alignment
Search Tool (BLAST) uses a heuristic to execute an approximate version of the Smith-
Waterman algorithm, which seeks directly approximates alignments that optimize a
measure of local similarity and the maximal segment pair scorelocal asopposed to
46
global alignments and is therefore able to detect relationshipsamong sequences which
share only isolated regions of similarity (Altschul et al., 1990). An early version of
BLAST, released in 1990 provided significance estimates for ungapped pair wise
alignments. More recent versions have provided two improvements: pairwise
alignments that are gapped and iterative searches derived from multiple alignments
(Altschul et al., 1997).
Once BLAST has found a similar sequence to the query in the database, it is
helpful to have some idea of whether the alignment is “good” and whether it portrays a
possible biological relationship or whether the similarity observed is attributable to
chance alone. BLAST uses statistical theory http://www.ncbi.nlm.nih.gov/
BLAST/tutorial/ Altschul-1. html to produce a bit score and expect value (E-value) for
each alignment pair (query to hit). A key element in this calculation is the
“substitution matrix”, which assigns a score for aligning any possible pair of residues.
The BLOSUM62 matrix is the default for most BLAST programmes. The E-value
gives an indication of the statistical significance of a given pairwise alignment and
reflects the size of the database and the scoring system used. Lower the E-value, more
significant is the hit.
As per Altschul et al., (1997), the BLAST family of programmes include:
BLASTP, which compares a protein sequence with a protein database; BLASTN,
which compares a nucleotide sequence with a nucleotide sequence; BLASTX, which
compares a nucleotide sequence (in all reading frames) with a protein database;
TBLASTN, which compares a protein sequence with a nucleotide sequence (in all
reading frames); and TBLASTX, which compares a nucleotide sequence (in all
reading frames) with a nucleotide database (in all reading frames); PSI–BLAST
Position Specific Iterated)and PHI-BLAST (Pattern-Hit Initiated BLAST).
Pearson and Lipman (1988) developed the programme, FASTA, for
comparing protein and DNA sequences. This program was developed with optimized
searches for local alignments using substitution matrices, and it has high level of
sensitivity for similarity searching. It has several features, including the similarity
search with several sequence and structure databases, genomics, and proteomics. In
addition, it has several features to get the pairwise alignment details, sequences of
47
aligned proteins, visual representation, and so on. The executable files and databases
for the sequence alignment program, FASTA, can be obtained from ftp://
ftp.ebi.ac.uk/pub/software/unix/fasta.
AChE (Rattus norvegicus) and Sericin (Bombyx mori) protein FASTA
sequences were retrieved from UniProt and submitted to BLAST-P against PDB to
retrieve the similar homology structures for query sequences by using the URL.
http://www.ncbi.nlm.nih.gov/BLASTP.
III. Modeling of 3D Structure for selected Proteins:
MODBASE is a queryable database of annotated protein structure models. The
models are derived by ModPipe, an automated modeling pipeline relying on the
programs PSI-BLAST and MODELLER. The database also includes fold assignments
and alignments on which the models were based. MODBASE contains theoretically
calculated models, and store the quality of the models. Additionally to the protein
structure models, MODBASE contains information about putative ligand binding
sites, SNP annotation, and protein-protein interactions. AChE (Rattus norvegicus) and
Sericin (Bombyx mori) protein sequences were submitted to MODBASE server to
build the 3D structures (http://modbase.compbio.ucsf.edu/modbase-cgi/).
IV. Validation of 3D structure:
MODEBASE-generated 3D structures were submitted to Ramachandran Plot
by using the URL http://dicsoft1.physics.iisc.ernet.in/rp/select.html.
V. Energy Minimization by using Argus Lab:
Energy minimization is also called energy optimization or geometry
optimization. This method is used to compute the equilibrium configuration of
molecules and solids. Stable states of molecular systems correspond to global and
local minima on their potential energy surface. Energy minimization is an efficient
tool for molecular structure optimization. It is necessary to reduce the net forces (the
gradients of potential energy) on the atoms until they become negligible.
Aurgus Lab, http://www.arguslab.com/arguslab.com/ArgusLab.html, is a
molecular modeling, graphics, and drug design program for Windows Operating
48
Systems. To date, there are > 20,000 downloads. Argus Lab is freely licensed.
Anyone can use it as many copies are needed for teaching where many students might
benefit from using Argus Lab. It is not allowed to redistribute Argus Lab from other
websites or sources. Aurgus lab is used for energy minimization of the lead molecules
which are taken mol format as input after minimization saved in PDB format.
In Aurgus lab software, the procedure is : go to File and open PDB file and
then remove hetero atoms and water molecules and then add hydrogen’s and then go
to optimize geometry.
VI. Protein-Protein Docking Studies by using Hex-version 6:
In the field of molecular modeling, docking is a method which predicts the
preferred orientation of one molecule to a second when bound to each other to form a
stable complex. Docking is frequently used to predict the binding orientation of small
molecule drug candidates to their protein targets in order to predict the affinity and
activity of the small molecule. Hex is an interactive molecular graphics program for
calculating and displaying feasible docking modes of pairs of protein and DNA
molecules. Hex can also calculate protein-ligand docking, assuming the ligand is rigid,
and it can superpose pairs of molecules using only knowledge of their 3D shapes.
Hex is freely available online docking software using Spherical Polar Fourier
(SPF) which correlates and accelerates the docking calculations. Hex is one of the few
docking programs having built-in graphics to view the docking results. It is the first
protein docking program to be able to use modern Graphics Processor Units (GPUs) to
accelerate the calculations.
In Hex's docking calculations, each molecule is modeled using 3D expansions
of real orthogonal spherical polar basis functions to encode both surface shape and
electrostatic charge and potential distributions. Essentially, this allows each property
to be represented by a vector of coefficients (which are the components of the basic
function). Overall docking score will be obtained from interaction energy in a pair of
3D structures. This docking approach is similar to conventional Fast Fourier
Transform (FFT) docking methods, which use Cartesian grid representations of
protein shape and other properties. However, the Cartesian grid approach only
49
accelerates a docking search in three translational degrees of freedom. Whereas, the
SPF approach allows the effect of rotations and translations to be calculated directly
from the original expansion coefficients.
VII. Protein and Protien interaction and visualization by PyMol: PyMol is a
widely used open-source molecular visualization program maintained by DeLano
Scientific LLC as a user sponsored project. PyMol version 1.0 earned a reputation for
its ease of use and numerous features. A powerful set of scripts for calculating
physical properties of molecules is available among classical molecular models in
PyMol. The core of the program is a full featured Python interpreter, which is then
extended by an OpenGL 3D display, a Tcl/Tk based GUI and the PyMol API which
gives the possibility of creating plug-ins. Although, this later feature was mainly
intended for automatic coloring of atoms according to relevant properties, loading and
manipulating structures, it is also capable of inserting additional graphical primitives,
like spheres, cylinders, triangles and vertices called Compilsed Graphic Objects
(CGOs). PYMOL uses object-oriented code. In this software open pymol , goto file
and open PDB files of protein and ligand and go on interact to see the hydrogen bond
interactions, ionic interactions between the residues and atom.
Validity of experimental procedures:
General:
For all the enzyme studies in the present investigation, the assays were
standardized by conducting preliminary assay to determine the optimal PH,
temperature, enzyme and substrate concentration and these optimal conditions were
subsequently followed for each enzyme assay.
Aliquots for assay:
Aliquots were selected such that initial rates were approximated as nearly as
possible, yet proving sufficient product to fall in to a convenient range for
spectrophotometric measurement.
50
Enzyme units:
The soluble protein content of the tissue homogenate was used as the enzyme
source and protein was estimated using Folin phenol reagent (Lowry et al., 1951).
This was used for the expression of enzyme units, i.e., μ moles of product formed or
substrate cleaved/mg protein/hour or minute.
Substrate requirements:
All the enzyme assays were made at saturating substrate concentrations, i.e.,
following zero order kinetics.
Lambert-Beer law:
For colorimetric procedures this law is applicable, which states that the optical
density of the resulting colored complex is proportional to the concentration of the
reaction products.
Enzyme nomenclature:
The nomenclature of the enzymes used in this thesis is according to the report
of the commission of enzymes of the International Union of Biochemistry.
Statistical Analysis:
Values of the measured in different parameters were expressed as Mean ±
SEM. One way ANOVA was used to test the significance of difference among the
four different groups with Dunnett’s post-hoc test for multiple comparisons using
standard statistical software, SPSS (Version -16). The results were presented with the
F-value and p-value. In all cases, F-value was found to be significant with p-value
less than 0.01. This indicates that the effects of factors are statistically significant.
Recommended