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II.8.

Iso-enzyme analysis
Iso-enzyme analysis was done from the shoot and inflorescence apex at different life
phase from both the Normal and Mutant Beta palonga.
i.

Extraction of enzymes:

Enzyme was extracted following the method of Wetter and Dyck (1993). Two
grams of sample (shoot and inflorescence apex) were frozen first in liquid
nitrogen and then crushed with mortar and pestle and 5 ml ice cold (4C)
extraction buffer was added (Table: 3). The macerates were centrifuged at
12,000 rpm for 20 minutes at 4C to collect the supernatant containing the
extracted enzymes. Finally the sample was stored at -20C.

Table:3 Solution required for plant iso-enzyme extraction


Solution

Composition

Amount

Storing condition

Plant iso-enzyme
extraction buffer

Tris-base
Sucrose
2-marcaptoethanol
H2O upto

2.422 gm
34.230 gm
0.4 ml
100 ml

4 0C

pH 8.5
ii.

Extraction of enzymes:

Protein content was estimated by modified Lowrys method using bovine


serum albumin as a standard (Scope, 1982)
iii.

Casting of polyacrylamide gels


a. Separating gel: Two slab gel plates were assembled in a casting
mode using 1.50 mm thick spacers. Required amount of stock
solutions according to table-4 and table-5 were mixed thoroughly
excepting ammonium persulphate and TEMED. The mixture was
degassed in 250 ml slide arm vaccum flask followed by addition of
ammonium per sulphate and TEMED. The gel solution after proper
mixing was pipetted into glass sandwich to a level of 4 cm from the
top. A water layer of about 2 mm thick was applied over the solution
to make the gel surface uniform as evident from sharp water gel
interface visible after polymerization. After polymerization water
was poured off and 1 ml of running gel overlay solution was applied

on gel surface and then the comb was placed over the gel surface
and finally the gel was allowed to stand for 2-3 hours.
b. Stacking gel: Measured amount of stock solutions were mixed for
stacking gels as mentioned in table-4. The mixture was degassed
prior to the addition of ammonium per sulphate and TEMED. The
running gel was poured off and stacking gel solution was gently
poured into the sandwich over polymerized separating gel. A comb
was inserted into the sandwich to make wells in the stacking gel at
regular interval for application of sample proteins. Finally the
stacking gel was allowed to polymerize for half an hour.
iv.

Electrophoresis of the enzyme extracts

Polyacrylamide gel electrophoresis of the enzyme extracts was carried out


using electrophoretic apparatus, DESEAGA, DISAPHOR VA. The polymerized
gel was removed from the casting mode having two spacers on two edges and
lower free surface of the separating gel fitted with the electrophoretic
apparatus. Then the comb was removed straight up from the stacking gel.
Lower tank was filled with running buffer up to a specific mark in such a way
that the lower free surface of separating gel remained immersed in the
running buffer.
The enzyme extracts were under layered in the wells of the stacking gel.
Volume of each sample enzyme extract was adjusted in such a way that fixed
amount of enzyme extract used for electrophoresis for a particular enzyme
system contained equal amount of protein. The protein content was estimated
by modified Lowrys method using bovine serum albumin as a standard
(Scope, 1982). A constant current of 50 mA was applied across 1.5 mm thick
gel. Initially the current applied was 30 mA but increased to 50 mA when dye
front entered into the separating gel. The electrophoresis was performed in a
cold chamber having a constant temperature of 4 0C. With the completion of
electrophoresis the gel was stained as per staining protocol of different
enzyme system.

Table:4 Stock solutions required for PAGE analysis of iso-enzyme


Solution

Composition

Amount

Storing condition

Tris-HCl
H2O upto

18.17 gm
100 ml

4 0C

Tris-HCl
H2O upto

3.02 gm
50 ml

Acrylamide
monomer
Bis-Acrylamide
H2O upto

29.20 gm
0.80 gm
100 ml

Solution-A
1.5 M Tris HCl
buffer
(pH adjusted with conc. HCl)

pH 8.8
Solution-B
0.5 M Tris HCl
buffer
(pH adjusted with conc. HCl)

4 0C

pH 6.8

Solution-C
30% Monomer
Acrylamide solution

40C

Solution-D
10% Ammonium
per-sulphate
solution

Ammonium
per-sulphate
H2O upto

0.10 gm
1 ml

Freshly prepared

Tris HCl
Glycine
H2O upto

3.02 gm
14.41 gm
1 liter

Room temperature

Running buffer
0.025 M Tris
0.192 M Glycine

Table:5 Stock solutions required for PAGE analysis of iso-enzyme

v.

Solutions

Separating gel
(8.5%)

Stacking gel (4%)

Solution-A
Solution-B
Solution-C
Solution-D
TEMED
H2O

4.00 ml
5.66 ml
0.1 ml
10 l
10.04 ml

0.50 ml
0.675 ml
50 l
5 l
3.75 ml

Staining methods of different enzymes


a. Peroxidase (E.C. 1.11.1.7).
O-dianisidine (100 mg) was
dissolved in 1 ml Glacial acetic acid and made up the volume to 100
ml with ddH2O and filtered. The gel was placed under this solution
and then H2O2 (30% H2O2, 0.2 ml) was added quickly. Bands
became visible very rapidly and as soon as the back ground of the
gel started to took the color, solution was pored out and washed for
2-3 time with dd H2O (Brewbaker et al., 1968).
b. Esterase (E.C. 3.1.1.1): Fast Blue RR Salt (76 mg) was dissolved
in 100ml 0.2 M Phosphate buffer (Table: 6), then filtered and
stored. Prior to use the substrate 40mg -napthyl acetate was
dissolved in 1ml Acetone and 1ml distilled water were mixed with
the previously prepared buffer solution and the whole mixture was
pored over the gel. The gel was incubated at 37 0C until brownish
black bands appeared. Generally 30 to 40 minutes was required for
clear development of bands (Brewbaker et al., 1968).
c. Acid phosphatase (E.C. 3.1.3.2): The gel was first incubated in
Acetate B buffer (Table: 6) at 5 0C for 30 minutes (as pH of the
running buffer was greater than 7.0). After discarding the buffer the
gel was incubated with the staining solution (Table: 7) till the
optimum development of blackish brown bands (Pasteur et al.,
1988).

Table:6 Composition of buffers used for preparing staining

solutions of different iso-enzyme


Solution

pH

Compositions

Amoun
t

0.2 M Phosphate
buffer

6.0

NaH2PO4, 2H2O
Na2HPO4
ddH2O upto

2.08 gm
0.94 gm
100 ml

0.2 M Phosphate
buffer

7.5

NaH2PO4, 2H2O
Na2HPO4
ddH2O upto

2.83 gm
0.30 gm
100 ml

Acetate-B buffer

5.0

Glacial Acetic Acid


NaOH
ddH2O upto

2.06 ml
1.12 gm
200 ml

Tris-A buffer

8.0

Na-EDTA
Tris
ddH2O upto
pH adjust with conc.
HCl

80 mg
4.84 gm
200 ml

Tris
ddH2O upto
pH adjust with conc.
HCl

1.21 gm
100 ml

0.1 M Tris buffer

7.5

d. Glutamate oxoglutarate transaminase (E.C. 2.6.1.1):


Staining buffer of GOT (Table: 6) was prepared freshly and then
incubated the gel at 370C for 30 to 45 minutes until the bands
appeared. After staining, the stained gel was washed thoroughly
with ice cold ddH2O to stop the reaction and the prominent band
appeared (Das and Mukherjee, 1997).
e. Superoxide dismutase (E.C. 1.15.1.1): The gel was incubated in
SOD staining solution (Table: 7). Then the gel was incubated at
room temperature under a cool white fluorescent light. After one or
two hours SOD appeared as a light band over a blue background.
Bands became visible very slowly and as soon as the back ground of
the gel started to took the color, solution was pored out and washed
with ice cold ddH2O for 2-3 times (Beauchamp and Fridravich,
1971).
f. Phosphogluconate dehydrogenase (E.C. 1.1.1.49): The gel
was incubated over night in the PGD staining solution (Table: 7) in

dark at 300C. Next day the blue bands developed which indicated
the activity of this enzyme (Wendel and Weeden, 1989).

Table:7 Composition of staining solutions used for staining gels for


different iso-enzyme
Enzymes

Staining ingredients

Quantity

Acid Phosphatase

- napthyl acid phosphate


Fast Blue BB salt
Acetate-B buffer

0.1 gm
0.1 gm
100 ml

GOT

0.2 M Na-Phosphate Buffer (pH-7.5)


pyridoxal- 5- phosphate BSA
L-Aspartic acid
-keto-glutaric acid
PVP
Fast violet Bsalt
ddH2O upto

SOD

PGD

MgCl2

82 ml
1 mg
100 mg
400 mg
64 mg
2.24 mg
136 mg
100 ml

Tris-A buffer
MgCl2 (0.5 M)
NAD (1%)
Before use add
NBT (0.1%)
PMS (1%)

40.00 ml
0.20 ml
1.00 ml

0.1 M Tris buffer

100 ml
98 mg
20 mg
15 mg
20 mg
4 mg

Phosphogluconic acid
NADP
MTT
PMS

1.00 ml
0.50 ml
6-

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