Materials and methods

Glassware: pipette, Beaker, glass rods, Test tube, Test tube stand, Eppendorf tubes,
centrifuge tube, conical flasks, muslin clothes, Dialysis bag, etc.

Instruments: centrifuge machine, Incubator, magnetic stirrer, pH meter, weighing machine,

colorimeter, Grinder, mixer etc.

Chemicals: sodium hydroxide(NaOH), sodium chloride(NaCl), Trichloro acetic acid (TCA),

L-Tyrosine, Tris-HCl, Sodium dodecyl sulphate(SDS), Polyacrylamide, (BSA)bovine serum
albumin, Folin Ciocalteau (FC )reagent, Ammonium sulphate, Di potassium hydrogen
phosphate(K2HPo4), potassium dihydrogen phosphate(KH2Po4), Cupric Sulphate(CuSo4),
Sodium potassium tartarate, Sodium carbonate(Na2Co3) ,Haemoglobin, ammonium per
sulphate, Glycine , Sodium acetate, Methanol, Acetic acid(Ch3COOH), Coomassie brilliant
blue.

Buffers and other reagents

 Phosphate buffer - Solution-A (K2HPo4) Dissolve 174.18g of K2HPo4 in 30ml of
distilled water. Solution-B (KH2Po4) Dissolve 136.09g of KH2Po4 in 50ml of distilled
water.
Mix both the solutions A and B before use.
 Standard BSA -Stock solution (1mg/ml) - 10mg of BSA was dissolved and made upto
10ml using distilled water. Working solution (200ug/ml) – 1ml of the above stock was
made up to 5ml using distilled water.
 Lowry reagent– Copper A – dissolve 2g of anhydrous sodium carbonate and 0.4g of
sodium hydroxide in 100 ml distilled water. Copper B – dissolve 0.025g of copper
sulphate and 0.05g of sodium potassium tartarate in 5ml distilled water. Mix solutions
A and B in the ratio 50:1 just before use.
 Folin Ciocalteau reagent (FC)-Dilute commercially available FC reagent two-fold
before use.
 Acetate Buffer(0.1m) (pH5 &pH6): 0.1M of Sodium acetate i.e. 0.164g of Sodium
acetate mixed in 20ml of autoclave water. 2 sets of this solution one for pH5 and other
for pH6.
 Glycine Buffer(0.1m) (pH9 &pH10):Weigh 0.105g of glycine and dissolve in 20ml of
autoclave water. 2 sets of this solution one for pH9 and other for pH10

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3.1. ISOLATION OF ANTICOAGULANT ENZYME FROM DIFFERENT

PLANT SOURCES

17 different plant sources were taken for the preliminary examination to the detect the
presence of enzyme; 1)lemongrass, 2)soybean seed, 3)pepper, 4)fennel,5) amla, 6)garlic,
7)cabbage, 8)turmeric, 9)fenugreek, 10)flax seeds, 11)ginger, 12)Radish , 13)pomegranates,
14)pineapple,15)green chillies,16)cinnamon spice, 17)orange

1) SCREENING:

Milk coagulation and blood coagulation method:

A drop of different plant samples was added to a thin layer of milk taken in a petri dish.
The formation of clear zone by the breakage of milk protein by the latex showed the presence
of anticoagulant enzymes.

The selected samples were ,cinnamon, orange, amla radish, garlic, pineapple, soybean, lemon
grass, green chilli, cabbage which were tested with 100µl of blood. The blood clotting time
was noted for all samples by using 50μL of whole sample in 100μL of blood to deter-mine the
anticoagulant property, while for control 50 μL of phosphate buffer was used in blood. The
samples were selected depending on the longest coagulation time. The selected samples were
subjected to enzyme assay.

ENZYME ASSAY(heme assay)

Heme assay is based on a aqueous alkaline solution method, in which the heme is
converted into a uniform coloured form, producing a colorimetric (660 nm) result, directly
proportional to the heme concentration in the sample. The optimized formulation reduces
interference and exhibits high sensitivity. Proteolytic activity was determined by using 0.1%
haemoglobin as substrate. The protease activity was expressed as amount of haemoglobin
hydrolysed to produce colour equivalent to µg of tyrosine/min/mg of protein at 37°. The colour
intensity is estimated by detecting the optical density at 660 nm. Into a series of tubes, 5 ml of
haemoglobin (prepared in phosphate buffer) was added as substrate except blank where only
phosphate buffer was added in same volume as substrate. The enzyme (plant extracts) 0.5ml
were added to each tube and kept for incubation at 37°for 10.min. Immediately TCA (0.5 ml)
was added to all the tubes to stop the enzymatic reaction, and kept for incubation for 30min
@37°. Filter the solutions and add 2ml of filtrates to other set of fresh tubes, then Na2Co3

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(5ml) and FC reagent(0.5ml) was added and kept in incubation for 30min in dark for colour
development. The absorbance was read in spectrophotometer at 660nm.

CONSTRUCTION OF STANDARD GRAPH.

Tyrosine standard curve was performed to determine the enzyme activity. Different
concentration of tyrosine (1mg/ml) was added to 5 tubes (0.2 to 1 ml aliquots) and made up to
2 ml with distilled water. 0.5M of Na2Co3(5 ml) and FC reagent(0.5ml)t was added and kept in
dark for 30min incubation. Absorbance was read at 660 nm in UV spectrophotometer.

CALCULATON:

Enzyme activity= µmol of L-tyrosine liberated ×total volume of assay ×dilution factor
________________________________________________________
volume of enzyme ×incubation time ×volume of assay
Enzyme unit : Amount of enzyme that catalyze the conversion of one micromole of
substrate per minute under the specialized condition

PROTEIN CONTENT BY LOWRYS METHOD

To determine the amount of protein, Lowry’s estimation was performed. The method
was first proposed by Lowry in 1951. The Lowry protein assay uses copper, which bonds with
the peptide’s bonds in proteins under alkaline conditions. This forms a monovalent copper ion
which can then react with the Folin reagent, which in turn can be reduced into blue coloured
substance. This blue colour can be measured using spectrophotometer to determine the
concentration of blue sample. Thus, the concentration of protein can be determined by using
Bovine Serum Albumin (BSA) as the standards. Since the protein content of the BSA is known
it can be used to determine the protein percentage for the absorbance obtained from the sample.
Standard curve was prepared by taking 0,0.2,0.4,0.6,0.8, and 1ml of the working standard BSA
solution(200µg/ml) and the volume was made up to 1ml by adding distilled water. 5ml of
alkaline copper reagent was added and incubated in room temperature for ten minutes. Later,
0.5ml of Folin- Ciocalteau reagent was added and incubated in dark condition for 30 minutes.
The absorbance was recorded at a wavelength of 660 nm. The samples were also processed in
the same manner by taking 100µL of the sample(plant extracts ) making up the volume to 1ml
with distilled water. The standard graph was plotted with Concentration of BSA versus
absorbance at 660nm and the amount of proteins in each sample was calculated.

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 Through the above analysis it was found that amla(Phyllanthus emblica) was having the
highest enzyme activity, compared to others as well as it took longer coagulating time than
compared to other samples in screening hence, amla was selected for extraction of the
required anticoagulant enzyme and was purified.

3.2 EXTRACTION OF ANTICOAGULANT FROM PLANT SOURCE

(Phyllanthus emblica).

COLLECTION OF SAMPLES

Whole amla (Phyllanthus emblica), was collected from the local market of HSR layout,
Bangalore. Seeds were removed and washed two to three times with distilled water to remove
surface dust and other foreign particles, and then stored in clean dry container.

EXTRACTION OF CRUDE EXTRACT

The stored Phyllanthus emblica was taken out and 350 g of sample was weighed,
washed under tap water then rinsed with distilled water. Sample are ground in a chilled mortar
and pestle with 250 ml of 0.1M phosphate buffer of pH7.5 at 4°C. The homogenate is passed
through cheese cloth, and the crude was collected in a clean 250 ml conical flask.

DETERMINATION OF ENZYME ACTIVITY(AMLA CRUDE )

Proteolytic activity was determined by using 0.1% haemoglobin as substrate. The

protease activity was expressed as amount of haemoglobin hydrolysed to produce colour
equivalent to µg of tyrosine/min/mg of protein at 37°. The colour intensity is estimated by
detecting the optical density at 660 nm. Into a series of tubes, 5 ml of haemoglobin (prepared
in phosphate buffer) was added as substrate except blank where only phosphate buffer was
added in same volume as substrate. The enzyme (amla extract) was added to each tube and kept
for incubation at 37°for 10.min. Immediately TCA (0.5 ml) was added to all the tubes to stop
the enzymatic reaction, and kept for incubation for 30min @37°. Filter the solutions and add
2ml of filtrates to other set of fresh tubes, then Na2Co3 (5ml) and FC reagent(0.5ml) were
added and kept in incubation for 30min in dark for colour development. The absorbance was
read in spectrophotometer at 660nm.

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DETERMINATION OF PROTEIN CONTENT BY FC METHOD

Standard curve was prepared by taking 0,0.2,0.4,0.6,0.8, and 1ml of the working
standard BSA solution(200µg/ml) and the volume was made upto1ml by adding distilled
water. 5ml of alkaline copper reagent (Reagent A: 2% sodium carbonate in 0.1N NaOH;
Reagent B: 0.5% cupric sulphate in 1% potassium sodium tartarate. Mix reagent A and
reagent B in the ratio of 50:1 prior to use) was added and incubated in room temperature for
ten minutes. Later, 0.5ml of Folin- Ciocalteau reagent was added and incubated in dark
condition for 30 minutes. The absorbance was recorded at a wavelength of 660 nm. The
samples were also processed in the same manner by taking 100µL of the sample making up
the volume to 1ml with distilled water. The standard graph was plotted with Concentration
of BSA versus absorbance at 660nm and the amount of proteins in each sample was
calculated.

3.3 PURIFICATION OF ENZYME

Protein purification is a series of processes intended to isolate one or a

few proteins from a complex mixture, usually cells, tissues or whole organisms. Protein
purification is vital for the characterization of the function, structure and interactions of the
protein of interest. The purification process may separate the protein and non-protein parts of
the mixture, and finally separate the desired protein from all other proteins. Separation of one
protein from all others is typically the most laborious aspect of protein purification. Separation
steps usually exploit differences in protein size, physio-chemical properties, binding affinity
and biological activity. The pure result may be termed protein isolate.

AMMONIUM SULPHATE PRECIPITATION:

The mechanism of salting-out is based on preferential solvation due to exclusion of the

cosolvent (salt) from the layer of water closely associated with the surface of the protein
(hydration layer). The hydration layer, typically 0.3 to 0.4 g water per gram protein (Rupley et
al., 1983), plays a critical role in maintaining solubility and the correctly folded native
conformation. When salt is added to the solution, the surface tension of the water increases,
resulting in increased hydrophobic interaction between protein and water. The protein responds
to this situation by decreasing its surface area in an attempt to minimize contact with the
solvent—as manifested by folding (the folded conformation is more compact than the unfolded
one) and then self-association leading to precipitation. Both folding and precipitation free up

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bound water, increasing the entropy of the system and making these processes energetically
favourable. (Timasheff and Arakawa, 1997).

The crude enzyme of Phyllanthus emblica was precipitated by addition of 44.2g of

ammonium sulphate to 70% saturation. The enzyme was incubated overnight at 4°C. The
enzyme was then centrifuged at 10000 rpm for 15 minutes and the supernatant was discarded.
The pellet was dissolved in 10ml of 10mM Tris HCl. 1ml of it is separated and stored in
eppendorf, remaining was subjected to dialysis .

DIALYSIS:

Dialysis works by diffusion, a process that results from the thermal, random movement
of molecules in solution and leads to the net movement from areas of higher to lower
concentration (until an equilibrium is reached). In dialysis, unwanted molecules inside a
sample-chamber diffuse through a semi-permeable membrane into a second chamber of liquid
or dialysate. Because large molecules cannot pass through the pores of the membrane, they will
remain in the sample chamber. By contrast, the small molecules will freely diffuse across the
membrane and obtain equilibrium across the entire solution volume, effectively reducing the
concentration of those small molecules within the sample.

Cellulose acetate membrane with a molecular weight cut off 8kDa was used for the
dialysis. The membrane was activated by adding 100ml of boiling water taken in a beaker to
which 2% Sodium Bicarbonate was added and boiled for 20 minutes. The membrane was then
transferred to a beaker of 100ml of boiling water and boiled for 20 minutes. The membrane
was cooled and one end of the membrane was tied making sure there is no leakage. The
precipitated sample was added of the membrane and the open end was tied. The membrane
bags are suspended in a beaker containing water and incubated overnight at 4°C. The water in
the beaker was changed and stirred using magnetic stirrer for 30 minutes. The water was again
changed and stirred three times.

ION-EXCHANGE CHROMATOGRAPHY-:

Ion-exchange chromatography is a process that allows the separation of ions and polar
molecules based on their affinity to the ion exchanger. It can be used for almost any kind of
charged molecule including large proteins, small nucleotides and amino acids. Ion-exchange
column was cleaned with distilled water and methanol and sonicated with distilled water for
15 minutes, to check the flow rate of the column and water was removed and DEAE anion

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exchanger (diethyl amino ether) resin was added to the column and allowed to settle, then 7
elution buffers were prepared using Tris HCL(1M) , NaCl(1M) and water. Once the resin
settles down elution buffer 1 was added and completely eluted to equilibrate the column.
Ammonium sulphate precipitated sample was added into the column and the upper layer was
marked with the marker. Different elution buffers were now added and were collected into
different test tubes naming each tube as (E1, E2 etc). Enzyme activity and protein content was
estimated for all the tubes. The tube having high enzyme activity was further purified by gel
filtration.

Procedure:

Preparation of reagent: Take 1.576g of Tris HCl dissolved in a 10ml of water. 1M Tris Hcl

Preparation of 1M NaCl: Take 0.5864g of Nacl dissolved in a 10ml of water.

After elution, samples were subjected for estimation of protein estimation and enzyme activity.
highest enzyme activity elute was chosen and was further purified through gel filtration.

GEL FILTRATION CHROMATOGRAPHY.

Gel filtration chromatography is a separation based on size. It is also called molecular

exclusion or gel permeation chromatography. In gel filtration chromatography, the stationary
phase consists of G75 Sephadex beads with a well-defined range of pore sizes (3000-80000).
The stationary phase for gel filtration is said to have a fractionation range, meaning that
molecules within that molecular weight range can be separated. Then the chromatography
column was cleaned with distilled water and methanol and sonicated along with water for 5
minutes to check the flow rate of the column. Then the column was filled with Sephadex G-75
gel and allowed to settle down. And highest, elution sample from ion exchange was added and
marked the upper layer with a marker, then 20 ml of phosphate buffer of pH 7 was added to
the column above the elution sample and collected in 32 eppendorf tubes as 1ml fractions. Then
Protein estimation was performed for all the eppendorf tubes by 280 nm method

PROTEIN ESTIMATION BY 280nm

PROCEDURE:

Phosphate buffer of 0.1 ml and autoclave water 2.9 ml was added to the cuvette and
was used to set the blank. Further from each of the 32 eppendorfs 0.1 ml was taken and 2.9ml
of autoclave water was added in cuvette and the absorbance was read at 280nm in UV
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spectrophotometer. Few samples with the highest protein contents were mixed . Enzyme assay
was performed for each mixture and activity was determined.

3.4 CHARACTERIZATION OF ENZYME

Enzyme characterisation simply refers to the determination of the various chemical and
physical properties (characteristics) of an enzyme. An enzyme attracts substrates to its active
site, catalyses the chemical reaction by which products are formed, and then allows the
products to dissociate. Enzyme Kinetics which is done by Effect of pH, Effect of temperature,
Effect of incubation Concentration ,and Effect of Substrate Concentration.

3.4.1 EFFECT OF pH

The optimum pH determination of the enzyme was carried out in different buffers with varying
pH range (5-10). For pH 5-6 Sodium acetate buffer was used, for pH 7-8 phosphate buffer was
used and pH 9-10 glycine buffer was used. Blank consists of buffers according to the range of
pH values chosen but without enzyme. 100µl of enzyme and 5000µl of substrate were assayed
in solutions containing buffers ranging from 5-10. The samples were assayed using FC reagent
and absorbance was read at 660nm against their respective blanks. The buffer solution in which
the absorbance was highest was considered as the optimum pH for that enzyme.

3.4.2 EFFECT OF TEMPERATURE

Effects of temperature on the enzymatic activity were determined by the reaction

between 100µl of enzyme and 5000µl of substrate at various temperature ranging from 25ºC
to 45ºC. Standard enzyme assay protocol was carried out, ea.ch was arrested by addition of
trichloro acetic acid. The samples were assayed using FC Reagent and the absorbance was read
at 660nm against their respective blanks. The temperature at which OD was highest was
considered as the optimum temperature for the enzyme.

3.4.3 EFFECT OF SUBSTRATE CONCENTRATION

The optimum Substrate concentration of the enzyme was carried out in different
concentration range 0.05% to 0.3% of haemoglobin. Effect of substrate concentration on the
enzyme activity were determined by the reaction between 0.1ml of enzyme and 5 ml of
haemoglobin substrate. Standard enzyme assay protocol is carried out. Each reaction was
arrested by addition of trichloro acetic acid after completion of incubation period. The samples

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were assayed using FC reagent and absorbance was read at 660nm against the blank. The
substrate concentration at which the OD was highest was considered as the optimum substrate
concentration for the enzyme.

3.4.4 EFFECT OF INCUBATION TIME.

The optimum incubation time of the enzyme was carried out in different time range (3,
6,9,12,15,18mins). Effect of incubation time on the enzyme activity were determined by the
reaction between 0.1ml of enzyme and 5 ml of haemoglobin substrate. Standard enzyme assay
protocol is carried out. Each reaction was arrested by addition of trichloro acetic acid after
completion of incubation period. The samples were assayed using FC reagent and absorbance
was read at 660nm against the blank. The substrate concentration at which the OD was highest
was considered as the optimum substrate concentration for the enzyme.

3.5 MOLECULAR WEIGHT DETERMINATION BY SDS-PAGE

SDS-PAGE

A very common method for separating proteins is by electrophoresis uses a

discontinuous polyacrylamide gel as a support medium and sodium dodecyl sulphate (SDS) to
denature proteins. The method is called sodium dodecyl sulphate polyacrylamide gel
electrophoresis (SDS-PAGE). The technique applies a negative charge so proteins move
towards a positive charge. Clean and dry the glass plates and spacers and assemble them
properly. Hold the assembly together with the clips. Clamp in an upright position with
petroleum jelly or 2% agar which is applied around the edges of the spacers to hold them to
place and seal the chamber between the plates. The separating gel is prepared around the edges
of spacers to hold them in place and seal the chamber between the plates. The separating gel is
prepared by adding 2.ml stock solution, 1ml separating buffer, 2ml of distilled water.
Ultrasonicate for 10 minutes and add 0.1 ml of ammonium per sulphate (APS), 0.1 ml of SDS
and 5µl of TEMED. Mix gently and carefully pour the gel solution in a chamber between the
glass plates and leave it for 30-50 minutes. Ultrasonicate for 5 minutes and then add 100µl of
SDS, 50µl of APS and 6µl of TEMED. Pour the stacking gel mixture, place the comb in the
stacking gel and allow the gel to set. After the stacking gel has polymerized, remove the comb
without distorting the shape of the gel. Carefully install the gel after removing the clips, in the
electrophoresis apparatus. Fill it with electrode buffer and remove any trapped air bubble at the
bottom of the gel. Connect the cathode at the top and turn on the DC power briefly to check

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the electric circuit. The electrode buffer and the plates can be kept cooled using suitable facility
so that the heat generated during their run is dissipated and does not affect the gel and
resolution. The sample solution was mixed with the dye and was heated in boiling water bath
10 minutes to ensure complete interaction in protein. Cool the sample solution and take up the
required volume(50µl) in a micro syringe and carefully inject into a well . Turn on the current
to 10-15µvolts for initial 10-15 minutes until the samples travel through the stacking gel. Then
continue to run at 100volts until the samples travels through the stacking gel. Power supply
was switched off. After electrophoresis, the gel was transferred into a clean plastic container
and the gel was soaked in the staining solution for overnight, later staining solution was
removed and the gel subjected to destaining. Then destaining solution was discarded and the
gel was rinsed with plenty of water and the previous step is repeated for every 2-3 hours. When
sufficient intensity of bands has developed the process was stopped. The protein banding
pattern was recorded by photography.

3.6 APTT TEST :

The APTT in contrast to the Prothrombin Time [PT], measures the activity of the
intrinsic and common pathways of coagulation. The division of the clotting cascade into the
intrinsic, extrinsic and common pathways has little in vivo validity but remains a useful
concept for interpreting the results of laboratory investigations.

PROCEDURE:2ml of blood was collected in a clean Eppendorf tube and immediately

centrifuged at 2500 rpm for 6 min. The upper layer was the plasma.

3 fresh vials were taken

CONTROL:100µl plasma+ 100µl CaCl2 + 100µl thromboplastin was added and used as
control

TEST 1: 100µl plasma+ 100µlCaCl2 + 100µlthromboplastin +100µl crude sample was added

TEST 2: 100µl plasma+100µlCaCl2 +100µlthromboplastin+100µl purified sample was added

All the three vials were continuously shaken and coagulation check was done.

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