MATERIALS
AND
METHODS
3.0 MATERIALS AND METHODS
3.1 Materials
Plant sources were procured from Green Earth products Pvt. Ltd., New Delhi. Few plants were
obtained from farm house of Amity University, Noida.
3.1.1 Chemicals
All chemicals were of reagent grade and obtained from standard commercial firms.
3.2 Methods
3.2.1 Screening of various plants for Superoxide Dismutase (SOD) enzyme
Screening of seventy two plants was done for SOD activity using standard protocols mentioned
below.
3.2.2 Extraction of SOD enzyme
The pre-weighed samples were crushed in Phosphate buffer (pH 7.0, 0.05 M). The crushed
material was filtered through whatman filter paper and centrifuged for 20 minutes at 10,000 rpm
at 0-4°C. The pellet was discarded and the supernatant was treated as crude extract.
3.2.3 SOD Assay
Superoxide dismutase (SOD) activity was determined using NBT method (Kakkar et al., 1984).
Principle:
NADH + 2O2 + PMS → NAD+ +2O2- + H+
....…
(1)
O2-+ NBT (oxidized) → O2 + NBT (reduced)
……
(2)
The assay involves the production of superoxide from O2 (using reduced β-nicotinamide adenine
dinucleotide (NADH) as a reductant, and phenazine methosulphate (PMS) as a catalyst in the
presence of an indicator, nitro blue tetrazolium (NBT), which turns blue when reduced by
superoxide. The color change during the reaction was monitored spectrophotometrically in the
visible range at 560 nm. When SOD enzyme is added to the reaction, (superoxide scavengers i.e.,
antioxidants) compete with NBT to react with superoxide. The percent inhibition of NBT
reduction was used to quantify superoxide-scavenging.
One unit of SOD: One unit of activity was taken as the enzyme reaction which gave 50%
inhibition of NBT reduction in one minute.
Assay buffer: 100 mM phosphate buffer, pH-8.3
Reagent stock solutions in assay buffer:
1. 12.72 mM NADH
2. 0.18 mM NBT
3. 1.28 mM PMS
Each of these reagents were freshly prepared and stored in refrigerator.
Determination of specific activity:
Specific activity of SOD was determined by using the following relationship:
Specific activity= Total enzyme Units /Total protein (mg)
3.2.4 Protein Estimation
The protein was estimated by Lowry (1951) method using BSA as a standard.
3.2.5 Purification of SOD enzyme from the screened spice
3.2.5.1 Ammonium sulphate fractionation
The crude extract of the screened spice was subjected to precipitation using salting out process.
Ammonium sulphate fractionation (0-30%) was done by adding salt in the extract according to
the required saturation level, slowly while keeping on ice. The ice-cold saturated solution of the
protein was stirred continuously and kept at 0-4°C for at least one hour followed by
centrifugation at 10,000 rpm for 10-15 min. Pellets were collected and were dissolved in minimal
amount of phosphate buffer (100 mM, pH 7.0) and used as 0-30% fraction, the supernatant being
subjected to next fractionation steps and further two fractions (30-60% and 60-90-%) were
achieved in the similar manner.
3.2.5.2 Dialysis
The sample was poured into the dialysis tubing and kept overnight at 0-4°C in Tris-HCl buffer
(0.01 M, pH 7.5).
Figure 3.1 Experimental setup of Dialysis
3.2.5.3 Anion-Exchange Chromatography using DEAE-Cellulose as matrix
Procedure:
I. Column packing
 DEAE-cellulose was poured carefully into a column chromatogram of size 500×40 mm
and flow rate of 2ml/min preventing air bubble entrapment.
II. Activation of resin:
 Resin was charged by passing 50 ml of 2 M NaCl.
 Column was then washed with about 150 ml of distilled water.
III. Equilibration
 The column was equilibrated with 30-50 ml of Equilibration Buffer (0.01M Tris-HCl
buffer pH 7.5).
IV. Sample Loading
 Sample was loaded into the column and one hundred ninety six fractions (2 ml each) were
collected in tubes kept over ice.
V. Elution

Sample was eluted by step gradient ascending method. Fractions (2 ml each) were
collected by eluting the column with 10 mM, 50 mM, 100 mM, 200 mM 300 mM, 400
mM and 500 mM NaCl in Tris-HCl buffer (0.01M, pH 7.5). The alternate fractions were
tested for protein amount and SOD enzyme activity, the fractions corresponding to the
peaks in the chromatogram being pooled. The specific activity of the pools obtained was
determined and the one having maximum value was further dialysed and taken over to the
next chromatographic separation.
3.2.5.4 Gel Permeation Chromatography (GPC) using Sephadex G-150 as matrix
Figure 3.2 Sehadex column setup
Procedure:
Sephadex G-150 was allowed to swell by suspending overnight in distilled water. It was then
packed into chromatographic column (200 mm × 10 mm) and washed with equilibration buffer
(0.01 M Tris-HCl buffer pH 7.5). The pool obtained from DEAE-cellulose column was loaded
onto the column into a column chromatogram of size 200 mm × 10 mm and flow rate of 2ml/min
and eluted with Tris-HCl buffer (0.01 M, pH 7.5). Fractions of 2 ml each were collected and the
alternate samples were tested for protein amount and SOD enzyme activity. The fractions
corresponding to the peaks obtained in the chromatogram were pooled and assayed for their
specific activity. Further the samples of all stages of purification i.e. crude, ammonium sulphate
fraction (dialyzed), pools of DEAE cellulose and Sephadex G-150 columns were run on SDSPAGE to study their band pattern.
3.2.5.5 Sodium Dodecyl Sulphate-Polyacrylamide Gel Electrophoresis (SDS-PAGE)

Materials-
1. Vertical slab gel electrophoresis unit which include:
a. Two glass plates.
b. Gel casting assembly.
c. Comb with teeth and spacers.
d. Electrophoretic tank with upper and lower buffer reservoirs.
e. Electrophoretic heads.
2. Power pack

Reagents -

Resolving Gel buffer:
Gel concentration of 12% in 1.5 M Tris-HCl pH 8.3
Reagents
Volume (ml)
30% Acrylamide stock*
4.0
H2O
3.3
1.5 M Tris-HCl pH 8.8
2.5
10% SDS
0.1
10% Ammonium persulphate
0.1
TEMED (added last)
0.004
* 29:1 – Acrylamide: N, N’-methylene bis-acrylamide

Stacking Gel buffer:
Gel concentration of 5% in 1.0 M Tris-HCl pH 6.8
Reagents
Volume (ml)
30% Acrylamide stock*
0.5
H2O
2.1
1 M Tris-HCl pH 6.8
0.38
10% SDS
0.03
10% Ammonium persulphate
0.03
TEMED (added last)
0.003
5x Sample Buffer:
Reagents
Concentration
SDS
2 % (w/v)
Dithiothreitol, or beta-mercapto-ethanol
100 mM
Glycerol
10%
Tris-HCl, pH 6.8
50 mM
Bromophenol blue
0.1%
1x Running Buffer:
Reagents
Concentration
Tris-HCl
25 mM
Glycine
200 mM
SDS
0.1 % w/v
Staining Solution:
Reagents
Concentration
Coomassie Brilliant Blue (CBB)
0.025 g/100 ml
Acetic acid
10 ml
Methanol
50 ml
Distilled water
40 ml
Reagents
Concentration
Acetic acid
10 ml
Methanol
50 ml
Distilled water
40 ml
Destaining Solution:
Procedure
Glass plates were assembled and sealed properly. Resolving gel was then poured and allowed to
polymerize. Stacking gel was poured over polymerized resolving gel and comb was inserted
straight down. Gel casted plates were set in electrophoresis apparatus and Tris-glycine buffer
was poured into the upper and lower chambers of apparatus. Gel was then loaded with sample
prepared in 1X loading buffer. Voltage (16 V) was applied across the gel. After the dye front has
moved into resolving gel, voltage was increased to 30V. Gel was stained with Coomassie
Brilliant Blue (CBB) and then destained to visualize protein bands and photographed.
3.3 Biochemical characterization of partially purified SOD enzyme
The SOD enzyme was characterized as follows:-
3.3.1 pH optima
To determine the pH optima, suitable buffers of different pH values ranging from 3.0 to 9.0 were
used. The reaction mixture was incubated for optimum time period and activity was determined
using standard assay.
3.3.2 pH stability
The enzyme alone was incubated for 2 hours with suitable buffers of pH ranging from 3.0 to 10.0
and further assayed using optimum assay conditions.
3.3.3 Temperature Optima
Temperature ranging from 10 ºC to 90 ºC was used to incubate the standard reaction mixture to
determine the optimum temperature for SOD activity.
3.3.4 Thermal stability
The SOD enzyme alone was incubated at different temperatures viz. 10 ºC, 20 ºC, 30 ºC, 40 ºC,
50 ºC, 60 ºC, 70 ºC, 80 ºC, 90 ºC for 72 hours followed by assay under pre-optimized conditions.
3.3.5 Determination of Kinetic parameters
The kinetic parameters (Km and Vmax) of the SOD enzyme from selected plant sources with high
specific activity were determined from the Lineweaver-Burk (1934) plot by using different
concentrations (from 0.5 to 5 mM) of the substrate.
3.3.6 Temperature coefficient
The temperature coefficient (Q10) is calculated by the following formula:
where,
1.
2.
3.
4.
R1 is the intial rate
R2 is the final rate
T1 is the intial temperature in (°C)
T2 is the final temperature in (°C)
3.3.7 Energy of activation
The energy of activation (Ea) is calculated by the following formula:
𝐾
𝐸
𝑇2 −𝑇1
𝑎
log 𝐾2 = - 2.303
(
𝑅
1
𝑇1 𝑇2
)
where,
1.
2.
3.
4.
5.
6.
7.
K1 is the initial rate constant
K2 is the final rate constant
A is called the frequency factor
Ea is the activation energy
R is universal gas constant
T1 is initial temperature (°C)
T2 is the final temperature (°C)
3.3.8 Effect of detergents on SOD activity
Tween-20, Tween-80, Cween-20, Cween-60, Triton X-100 and Sodium Lauryl Sulphate (SLS) at
the concentration level of 0.1% (using detergent blanks as control) were added and their effect on
SOD activity from selected plant sources with high specific activity were studied.
3.3.9 Effect of metal ions on SOD activity
The effect of various metal ions viz. Ni2+, Fe3+, Co2+, Cu2+, Mg2+, Zn2+, Mn2+, Ca2+, K+, Ba2+ and
Na+ in the form of their respective salts i.e.; NiCl2, FeCl3, CoCl2, CuSO4, MgSO4, ZnSO4, MnCl2,
CaCl2, KCl, BaCl2 and NaCl were studied on SOD enzyme from selected plant sources with high
specific activity at 0.25 mM concentration by comparing with additive blanks.
3.3.10 Determination of Inhibition constant (Ki) of detergents as inhibitors: For the potential
detergents as inhibitors of SOD enzyme from selected plant sources with high specific activity;
inhibition constants (Ki) were determined by varying their concentration (I0) and plotting 1/V0
versus I0.
3.3.11 Determination of Inhibition constant (Ki) of Metal ions as inhibitors: Inhibition
constants (Ki) were determined by varying the concentration of Metal ions (I0) as inhibitors of
SOD enzyme from selected plant sources with high specific activity; by plotting 1/V0 versus I0.
3.3.12 Determination of type of inhibition To determine the type of inhibition, the
concentration of substrate (from 0.1 to 0.5 mM) was varied with fixed concentration of inhibitors
(I0) and compared with negative control i.e. reaction mixture without inhibitor. LineweaverBurk plot was prepared and the type of inhibition was determined.
3.3.13 Studies on effect of SOD on cancer cell lines: MCF-7 and HepG-2
The cytotoxic effect on plant source with highest SOD activity were tested at different stages of
purification using (3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide) MTT-assay.
Cells of human cancer cell lines were obtained in T-flasks. Adhered cells were scraped off and
centrifuged with DMEM medium, Pellet containing cell was used for MTT assay. Equal volumes
of cells were obtained in 96 well plate, first well “Blank” with just medium, second well control
and the remaining with plant extract were studied with different dilution concentrations till 72
hrs of incubation. Selected plant source with highest SOD activity was tested at different stages
of purification against MCF-7 Breast cancer cell line and HepG-2 Liver cancer cell line in
concentrations of 0.65, 1.25, 2.5 and 5 % (v/v) upto 96 hrs of incubation and cell counted by
standard method. The final study was done at Dabur research foundation, Ghaziabad, Uttar
Pradesh.