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4.1) Parameters and sampling frequency
At the height of two to three meters, fully expanded mature leaves were
collected from each plant in the polythene bags and transported to the
laboratory. The leaf samples were collected on seasonal basis and this
frequency was strictly maintained throughout the year (November 2009 to
October 2010).
The following investigations were carried out in all the five plants Ficus
religiosa, Ficus benghalensis, Ficus glomerata, Azadirachta indica and
Polyalthia longifolia.
Dust fall on leaves was studied seasonally (winter, summer and rainy).For
photosynthetic study the chlorophyll pigments (Chlorophyll a, chlorophyll b,
and total chlorophyll) were studied and biochemical changes in leaves (Starch,
phenols, and sugars- total, reducing and non-reducing) were studied on seasonal
basis. Air pollution tolerance index and enzymatic activities i.e protease,
catalase, peroxidase and invertase were studied in winter season.
Experiment
Physiological Metabolism
Dustfall Chlorophyll APTI Biochemical Enzymatic
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Biochemical Enzymatic
Sugars Phenol Starch Protease Oxidase Invertase
Total Reducing Non-reducing Catalase Peroxidase
4.2) Method of measuring dust fall, chlorophyll pigments (total, a,
and b) and air pollution tolerance index (APTI).
A: Measurement of dust falls on the leaves
From each plant, ten matured leaves were collected in the separate polythene
bags during winter, summer and rainy from November 2009 to October 2010.
Leaves were collected at the height of three to four meters from all the sites.
For dust fall measurement, the method of Dry technique described by Das and
Pattanayak (1997) was followed. In this technique first the intact leaf was
weighted (in mg) then dust particulates from leaf surfaces were gently collected
with the help of camel hair brushes and the weight of leaf was measured again.
The amount of dust – deposition in mg/cm2 was calculaed as:-
Weight of intact leaf- initial weight of leaf
Dust content (mg/cm2) = -----------------------------------------------------
Total surface area of leaf (cm2)
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B: Measurement of chlorophyll pigments
The chlorophyll pigments in the leaves were estimated following the method of
Arnon (1949).
The fully expanded leaves from all the sites were collected in the poly-thene
bags and transported to the laboratory. The leaves were washed out thoroughly
with distilled water. Three replicates were used for each plant.
Weighted fresh leaf material was homogenized and extracted thrice in chilled
80% acetone (v/v). The volume of the acetone extract was made up to a known
one and the optical density was read at 645nm and 663nm wavelengths on a
spectrophotometer. The concentration of the chlorophyll pigments was
calculated using the following formula and the results are expressed in mg/g
fresh weight.
Chlorophyll a = [(12.7 X OD at 663) – (2.69 X OD at 645)] X dilution factor
Chlorophyll b = [(22.9 X OD at 645) – (4.68 X OD at 663)] X dilution factor
Total chlorophyll = [(20.2 X OD at 645) – (8.02 X OD at 663)] X dilution
factor.
C: Measurement of Air Pollution Tolerance Index
Air pollution tolerance index (APTI) was determined by the method given by
Singh and Rao, 1983. The samples were estimated for Leaf-extract pH (Singh
and Rao, 1983), relative moisture content (Wealtherly, 1965), total chlorophyll
(Arnon, 1949) and ascorbic acid (Abida begum et al., 2010). The fully
expanded leaves from all the sites were collected in the poly-thene bags and
transported to the laboratory. The leaves were washed out thoroughly with
distilled water. Three replicates were used for each plant.
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Estimation of Leaf-extract pH (Singh and Rao, 1983): 0.5 g of leaf material was
ground to paste and dissolved in 50 ml of distilled water and Leaf-extract pH
was measured by using calibrated digital pH meter.
Relative moisture content (Wealtherly, 1965): Estimation of relative moisture
content: Fresh leaf samples collected from the study area and were brought
immediately to the laboratory and washed thoroughly. The excess water was
removed with the help of filter paper. The initial weight of samples were taken
(W1 g) and kept in oven at 600 oC until constant weight was obtained and the
final weight was taken (W2 g).
Total Chlorophyll content was measured by the method of Arnon (1949) as
mentioned above.
Ascorbic acid content (AA) (mg/g) was measured using spectrophotemetric
method. 1 g of the fresh foliage was put in a test-tube, 4 mL oxalic acid - EDTA
extracting solution was added, then 1 mL of orthophosphoric acid and then 1
mL 5% tetraoxosulphate(VI) acid added to this mixture, 2 mL of ammonium
molybdate was added and then 3 mL of water. The solution was then allowed to
stand for 15 minutes. After which the absorbance at 760 nm was measured with
a spectrophotometer (Abida Begum and Krishna, 2010).
APTI given as:
APTI = [AA (T + P) + R]
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(Where AA is the ascorbic acid in mg/g, T is the total chlorophyll in mg/g, P is
pH of leaf sample and R is relative water content in mg/g).
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4.3) Method of measuring sugar (total, reducing and non-
reducing), phenol and starch.
A: Measurement of Sugars (Nelson, 1944)
Principle: Monosaccharide readily reduces oxidizing agents such as Ferric
cyanide, hydrogen peroxide or cupric ions (Cu++
). In such reactions the sugar is
oxidized at carbonyl group and the oxidizing agent becomes reduced glucose or
other sugars capable of reducing oxidizing agents are called reducing sugars.
Thus by measuring the amount of oxidizing agent that is reduced by a sugar
solution, it is possible to estimate the sugar concentration. The method involves
the reduction of cupric ions (Cu++
) to cuprous ions (Cu+) which in alkaline
solution and forms yellow cuprous hydroxide, which in turn is converted by
heat of the reaction to insoluble red cuprous oxide (Cu2O).
The amount of Cu2O formed can be increased by adding arsenomolybdic acid
which in turn is reduced to lower oxides of molybdenum by Cu2O. The
coloured complex produced is known as molybdenum blue. The intensity of the
colour is related to the concentration of the reducing sugars in the sample.
Procedure: 100mg plant material was weighed and homogenate with 10ml
80% ethanol. It was centrifuged for 10rpm for 10minutes. Supernatant 1 was
collected; while 10 ml 80% ethanol was added again to the residue, centrifuge it
and supernatant 2 was mixed with supernatant 1. Residue was discarded.
Total sugars: To 1ml alcoholic aliquot, 1ml 1N H2SO4 was added and heated
at 490 C in water bath for 30 minutes for hydrolysis of the mixture. 1-2 drop of
methyl red indicator was added. 1N NaOH was added drop wise for the
neutralization (colour was to yellow from pink). 1ml Nelson Somogyi’s reagent
was added to it and the tube was kept in boiling water bath for 20 minutes.
After cooling of the test tube, 1ml arsenomolybdate was added and final
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volume was made up to 20ml with DW. O.D. was noted at 540nm. Blank was
prepared in the same manner.
Reducing sugars: To 1ml alcoholic aliquot, Nelson Somogyi’s reagent was
added and kept in boiling waterbath for 20min. After cooling of the test tube,
1ml arsenomolybdate was added and final volume was made upto 20ml with
DW. O.D. was noted at 540nm. Blank was prepared in the same manner.
Non-reducing sugar= Total sugar – Reducing sugar.
The result was expressed as mg/gm plant material.
Preparation of reagents:
1.80%Ethanol: 80ml Ethanol was diluted up to 100ml DW.
2.1N Sulphuric acid (H2SO4): 2.77ml conc. H2SO4 (95-98%) was diluted up to
100 ml with DW.
3. 0.1N Sodium hydroxide (NaOH): 4gm NaOH was dissolved up to 100ml
with DW.
4. Methyl red indicator (1N): 0.1gm Methyl red powder was dissolved in 5ml
0.02M NaOH and final volume was made up to 250ml with DW.
5. Nelson Somogyi’s reagent:
Nelson A: 12.5gm Na2CO3, 12.5gm Na-K-tartrate, 10gm NaHCO3, 100gm
Na2SO4 were dissolved one by one and final volume made up to 50ml with
DW.
Nelson B: 15gm CuSO4.7H2O dissolved up to 100ml with DW.
Nelson Somogyi’s reagent: 50ml Nelson A and 1ml Nelson B were mixed.
6. Arsenomolybdate reagent: 25gm Ammonium molybdate was dissolved in
450ml DW, 21ml conc. H2SO4 was added to it. 3gm Sodium arsenate was
dissolved in 25ml DW and both solutions were mixed. It was incubated at 350C
for overnight before use of it.
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CHART- 4.3(a): FLOW CHART FOR TOTAL SUGARS AND
REDUCING SUGARS
100 mg plant material was weighed
Homogenized with 10 ml 80% ethanol
Centrifuged at 5,000- 10,000rpm for 10 minutes
Supernatant I Residue +10ml 80% ethanol
Centrifuged at 5000-10000g for 10 minutes
Supernatant II Residue
Supernatant I+II
Total sugar Reducing sugar
1 ml aliquot + 1ml aliquot +
1 ml 1 N H2SO4 1ml Nelson Somogyi’s reagent
Incubated in water bath Incubated in boiling
at 490 C for 30 min. water bath for 20min
Add 1-2 drop of methyl red indicator Add 1ml Arsenomolybdate
Add 1N NaOH drop wise Final vol. made upto 20ml
for neutralization with D.W
(Colour change Pink to Yellow) OD was noted at 540 nm
1ml Nelson Somogyi’s reagent
Incubate for 20mins in boiling water bath
Add 1ml Arsenomolybdate
Final volume was made up to 20ml with D.W.
O.D. at 540nm.
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The readings were compared with a standard which was prepared by using
different concentration of glucose. The results were expressed as mg/g plant
material.
B: Measurement of Total phenols (Bray et al., 1954)
Principle: Estimation of phenols using Folin- Ciocalteu’s reagent is based on
the reaction between phenols and an oxidizing agent phosphomolybdate which
results in the formation of a blue complex (Bray et al., 1954).
Procedure: 100mg plant material was weighed and homogenate with 10ml
80% ethanol. It was centrifuged at 5000-10,000rpm for 10minutes. Supernatant
1 was collected; while 10 ml 80% ethanol was added again to the residue,
centrifuge it and supernatant 2 was mixed with supernatant 1 and used for
estimation. Residue was discarded.1ml alcoholic aliquot was mixed with 1ml
20% Na2CO3 and 0.5ml Folin-Ciocalteau’s reagent. It was boiled for 10minutes
at 1000C in water bath. Final volume was made up to 20ml with DW and O.D.
was noted at 660nm. PPT were filtered or centrifuged before reading. Blank
was prepared in the same manner.
The result was expressed as mg/gm plant material.
Preparation of reagents:
1. 80%Ethanol: 80ml Ethanol was diluted up to 100ml DW.
1. 20% Sodium carbonate (Na2CO3):20gm Na2CO3 was dissolved into 100ml
DW.
2. Folin-ciocalteau’s reagent (1N): Commercially available reagent (2N) was
diluted with an equal volume of DW.
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CHART4.3 (b): FLOW CHART FOR TOTAL PHENOLS
100mg plant material crushed in 10ml 80% ethanol
Centrifuge at 5000-10,000 g for 10mins
Supernatant 1 residue + 10ml 80% ethanol
Centrifuge
Supernatant 2 + Supernatant 1 residue discarded
1ml aliquot + 1ml 20% Na2CO3
0.5 ml Folin-Ciocalteau’s reagent
Boil in water bath for 10mins
Final volume made up to 20ml with DW
Ppt. filtered
O.D. at 660nm
The readings were compared with a standard which was prepared by using
tannic acid. The results were expressed as mg/g plant material.
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C: Measurement of Starch (Chinoy, 1939)
Principle: The plant material is treated with aqueous sodium hydroxide in the
cold to dissolve the starch. This dissolved starch reacts with I2KI and gives a
coloured product and the starch content is determined by calculated with the
standard curve.
Procedure: 100mg plant material was weighed and homogenate with 10ml
80% ethanol. It was centrifuged for 10minutes. Supernatant 1 was collected;
while 10 ml 80% ethanol was added again to the residue centrifuge it and
supernatant 2 was mixed with supernatant 1 and removed. Residue was used for
starch estimation. The residue was dissolved in 20ml 0.7% KOH and boiled for
gelatinization for 40 minutes. It was centrifuged after cooling and 1ml aliquot
(Supernatant), 0.5ml 20% acetic acid; 1ml citrate buffer (0.05M, pH 5.0) and
1ml I2KI were added and incubated at room temperature for 10minutes. O.D.
was taken at 600nm. Blank was prepared in the same manner.
The result was expressed as mg/gm plant material.
Preparation of reagents:
1.80%Ethanol: 80ml Ethanol was diluted up to 100ml DW.
2. 0.7% Potassium hydroxide (KOH): 700 mg KOH was dissolved into 100ml
DW.
3. 20%acetic acid: 20ml glacial acetic acid was diluted up to 100ml with DW.
4. I2KI Solution: 200mg iodine crystal and 2gm KI were dissolved up to 100ml
with DW.
5. Citrate buffer: (0.05M, pH 5.0)
Citrate X: 0.1M Citric acid (2.19gm Citric acid was dissolved into 100ml DW.)
Citrate Y: 0.1M Sodium Citrate (2.94gm Sodium Citrate was dissolved into
100ml DW.)
Citrate buffer: 20.5ml Citrate X and 29.5ml Citrate Y were dissolved up to
100ml with DW.
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CHART4.3(c): FLOW CHART FOR STARCH
100mg plant material crushed in 10ml 80% ethanol
Centrifuge at 5000-10,000 g for 10mins
Supernatant 1 residue + 10ml 80% ethanol
Centrifuge at 5,000-10,000g for 10min
Supernatant 2 + Supernatant 1 residue
Discarded
Dissolved in 20ml 0.7% KOH and
boiled for gelatinization for 40mins.
Cooled and centrifuged
1ml aliquot
0.5ml 20% acetic acid + 1ml citrate buffer + 1ml I2KI
Incubate at room temperature for 10mins
O.D. at 600nm
The readings were compared with a standard which was prepared by using
starch. The results were expressed as mg/g plant material.
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4.4) Method of measuring enzyme activity (Protease, Catalase,
Peroxide, Invertase).
Method for enzyme extraction: Grind 1gm plant material in 10ml phosphate
buffer. Centrifuge the extract at 10,000 rpm for 15minutes at 40C refrigerated
centrifuge.
Preparation of reagents:
1. Phosphate buffer (0.1M, pH=7):
Phosphate A: 0.2 M dibasic sodium phosphate (35.61g Na2HPO4.7H2O was
dissolved up to 1,000ml with DW.
Phosphate B: 0.2 M monobasic sodium phosphate (31.21g NaH2PO4.2H2O was
dissolved up to 1,000ml with DW.)
Phosphate buffer: 61ml Phosphate A and 39ml Phosphate B were diluted up to
100ml DW.
CHART: FLOW CHART FOR ENZYME EXTRACTION
Take 1gm plant material
Grind it in 10ml phosphate buffer
Centrifuge at 10,000 rpm for 15mins at 40C
Use supernatant for enzyme activity.
A: Measurement of Protease activity (Cruz et al., 1970)
Principle: Protease hydrolyses protein into its constituent amino-acids. By
estimating the amount of protein hydrolyzed in a solution, the activity of the
protease enzyme can be known.
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The Folin- Lowry assay used for protein estimation was used here for
determining the activity of the protease enzyme. Protein reacts with Folin
reagent to give a coloured complex. The intensity of the colour depends upon
the amount and type of aromatic amino-acid produced due to hydrolyses by the
enzyme. The type of amino-acid varies for different proteins.
Procedure: 3 test tube, 1ml enzyme aliquot, 1ml phosphate buffer (0.2 M,
pH=7.0 and 1ml 1% casein solution were mixed. It was incubated at room
temperature for 60 min. 1ml 20% Trichloro acetic acid (TCA) was added to it.
Standard tube had 1ml DW, 1ml Phosphate buffer, 1ml Trichloro acetic acid
(TCA) and 1ml casein. Blank has 2ml DW, 1ml phosphate buffer and 1ml
Trichloro acetic acid (TCA). All test tubes were incubated at room temperature
for 60 minutes. All the 3 test tubes were centrifuged and residue was discarded.
1ml aliquot (supernatant) was mixed with 5ml Lowry ‘C’ and incubated at
room temperature for 10 min. 0.5 ml Folin - Ciocalteau’s reagent was added in
each and again incubated for 10 minutes at room temperature. O.D of blank was
related with zero setting, standard gave the reading of casein, while test gave
reading of reduced amount of protein at 600nm. Subtraction of control and test
gave protease activity.
Result was expressed as mg/protein reduced/gm plant material.
Preparation of reagent:
1. Phosphate buffer (0.2M, pH=7):
Phosphate A: 0.2M Monobasic sodium phosphate.
Phosphate B: 0.2M Dibasic sodium phosphate.
Phosphate buffer: 39ml phosphate A was mixed with 61ml phosphate B.
2. 1% casein solution: 1gm casein was dissolved in 5ml 1N NaOH and final
volume was made up to 100ml with DW.
3. 20% Trichloro acetic acid (TCA): 20gm TCA was dissolved in 100ml DW.
4. Lowry’s reagent:
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Lowry A: 2% Na2CO3 in 0.1N NaOH.
Lowry B: 0.5% CuSO4 in 1% of Na-K-tartrate.
Lowry C: 50ml Lowry A was mixed with 1ml Lowry B.
5. Folin-Ciocalteaus reagent (1N):
Commercially available reagent (2N) was diluted with an equal volume of DW.
CHART4.4 (a): FLOW CHART FOR PROTEASE
Test Standard Blank
1ml enzyme extract 1ml DW 2ml DW
1ml 20% TCA (Trichloro Acetic Acid)
Incubate for 1hr.
Supernatant Residue
(Discarded)
1ml Supernatant
5ml Lowry Reagent
1ml Phosphate Buffer
1ml 1% Casein
Incubated for 1hr
Centrifuge
d
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Incubated for 10minutes
Add 0.5ml Folin Reagent
Incubated for 10min
O.D. at 600
The readings were compared with a standard which was prepared by using
different concentration of tyrosine. The results were expressed in mg tyrosine
liberated/hr/mg protein.
B: Measurement of Catalase activity (Chance and Maehly, 1955)
Principle: The enzyme catalase is an endogenous antioxidant present in all
aerobic cells helping to facilitate the removal of hydrogen peroxide. The
enzyme consists of 4 subunits of the same size, each of which contains a heme
active site to accelerate the decomposition of H2O2 to water and oxygen.
Procedure: This activity was done by titration method. Reaction, mixture was
prepared by mixing 3ml of phosphate buffer (0.1M, pH= 6.8), 1ml 0.1M H2O2
and 1ml enzyme aliquot. It was incubated at room temperature for 1min. Futher
reaction was stopped by addition of 10ml 20% H2SO4. This mixture was titrated
against 0.01N KMnO4 to estimate the residual H2O2 until a faint pink colour
persisted for at least 15secs. This enzyme activity was expressed as amount of
enzyme break down by H2O2/min/gm plant material.
Preparation of reagent:-
1) Phosphate buffer (0.1M, pH=6.8):
Phosphate A: 0.2M Monobasic sodium phosphate.
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Phosphate B: 0.2M Dibasic sodium phosphate.
Phosphate buffer: 51ml phosphate A and 49ml phosphate B were diluted
upto 200ml DW.
2) 2% Sulphuric acid (H2SO4): 2ml conc. H2SO4 was diluted upto 100ml
DW.
3) 0.01N Potassium permanganate (KMnO4): 158.04mg KMnO4 was
dissolved upto 100ml DW.
4) 0.1M Hydrogen peroxide (H2O2): 3.041ml H2O2 (100v/v, 30%) was
diluted upto 1000ml DW.
CHART4.4(b): FLOW CHART FOR CATALASE
1ml Enzyme aliquot
+ 3ml phosphate buffer (0.1M, pH=6.8)
+ 1ml 0.1MH2O2
Incubated at room temperature for 1min
Added 10ml 2% H2SO4
Titrated against 0.01N KMnO4 to estimate the residual H2O2
until a faint pink colour persisted for at least 15sec
Expressed enzyme activity as amount of ml enzyme broke
Down by H2O2 /minute/gm plant material.
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C: Measurement of Peroxidase activity (George, 1955)
Procedure: 1ml enzyme aliquot was mixed with 1ml phosphate buffer (0.1M,
pH= 6.4) and 1ml 20mM guaiacol. Optical density was noted at 420nm. 0.5ml
H2O2 was added and reading was noted after 2 mins. Calculation was done
expressed as O.D difference/minute/gm plant material. Blank was prepared in
the same manner.
Preparation of the reagent:
1) Phosphate buffer (0.1 M, 6.4= pH):
Phosphate A: 0.2 M monobasic sodium phosphate (27.8g
NaH2PO4.2H2O was dissolved upto 1,000ml with DW.
Phosphate B: 0.2 M dibasic sodium phosphate (53.65g Na2HPO4.7H2O
was dissolved upto 1,000 with DW.
Phosphate buffer: 73.5ml Phosphate A and 23.5ml Phosphate B were
diluted upto 200ml DW.
2) 20mM Guaiacol: 0.22ml Guaiacol was upto 100ml with DW.
CHART4.4(c): FLOW CHART FOR PEROXIDASE
Test Blank
1ml Enzyme + 1ml DW +
1ml Phosphate buffer + 1ml Phosphate buffer +
1ml Guaiacol (20mM) 1ml Guaiacol (20 mM)
0.5ml H2O2
Incubate for 2min
O.D. read at 420nm
O.D. at 420 nm
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The results of enzyme activity were expressed as O.D. difference/min/mg
protein.
D: Measurement of Invertase (Hatch and Glasziou, 1963)
Principle:Invertase is an enzyme which hydrolyses disaccharides into
monosaccharide. Sucrose is the substance that gets reduced to monosaccharide
that is reducing sugar which can be produces by invertase and can also be
measured.
Monosaccharide acts as an oxidizing agent and oxidizes Cu++
. The sugar that
reduces the oxidizing agent is known as reducing sugar. The method involves
reduction of cupric ions (Cu++
) into cuprous ion (Cu+) which is alkaline in
nature and forms yellow cuprous hydroxide, which in turn is converted by heat
of the reaction to insoluble red cuprous oxide (Cu2O). The amount of Cu2O is
dissolved in arsenomolybdate and forms a coloured complex. The developed
colour is related to the concentration of the reducing sugars and is measured
using the spectrometer.
Procedure: 3 test tubes were taken for this activity (1) test (2) control (3)
blank.1ml enzyme aliquot, 1ml 0.1M sucrose solution in citrate buffer (0.1M,
pH=5.4) and 1ml citrate buffer were added to the test tube.1ml DW, 1ml
sucrose solution and 1ml citrate buffer were added to the control tube.2ml DW
and 1ml citrate buffer were added to the blank tube. All test tubes were
incubated at room temperature for 60minutes. After the incubation, 2ml
absolute alcohol and 2ml 5% Sodium sulphate (Na2SO4) were added in all 3t.t.
All test tubes were again incubated in boiling water bath for removal or
evaporation of alcohol (10-20 minutes). Impurities were filtered or centrifuged
and each test tube had left 5ml solution. 1ml solution aliquot and 1ml Nelson
somogyi’s reagent were mixed and kept in boiling water bath for 20 minutes.
1ml arsenomolybdate was added to each after cooling. Final volume was made
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up to 20ml with DW. O.D. was noted at 540nm.Blank was related with zero
setting, control gave the reading of sucrose, while test gave reading of reduced
sucrose. Subtraction of control and test gave Invertase activity.
Result was expressed as glucose reduced/gm plant material.
Preparation of the reagent:
1. Citrate buffer (0.1 M, pH=5.4):
Citrate A: 0.1M citric acid (21.01gm citric acid was dissolved up to 1000ml
with DW.)
Citrate B: 0.1M Sodium citrate (29.41gm Sodium citrate was dissolved up to
1000ml with DW.)
Citrate buffer: 16ml citrate A and 34ml citrate B were mixed.
2.0.1M sucrose in citrate buffer: 3.42gm of sucrose was dissolved up to 1000ml
with citrate buffer (0.1 M, PH-5.4)
3. Sodium sulphate (Na2SO4) (5%):
5g Na2SO4 was dissolved in 100ml D.W.
4. Nelson somogyi’s reagent:
Nelson ‘A’: 12.5 gm Na2CO3, 12.5 gm Na-k tartrate, 10g NaHCO3, 100g
Na2SO4 were dissolved one by one and final volume was made up to 500 ml
DW.
Nelson ‘B’:15g CuSO4.7H2O was dissolved up to 100ml with DW.
Nelson Somogyi’s reagent: 50 ml Nelson ‘A’ and 2ml Nelson ‘B’ was mixed.
5. Arsenomolybdate reagent:-
25gm ammonium molybdate was dissolved in 450ml DW, 21 ml concentrated
H2SO4 was added to it. 3g sodium arsenate was dissolved in 25 ml DW and both
solutions were mixed. It was incubated at 370c over night before use of it.
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CHART4.4 (d): FLOW CHART FOR INVERTASE
Test Standard Blank
1ml enzyme extract 1ml DW 2ml DW
Incubate for 1 hr
2ml alcohol + 2ml 5% Sodium Sulphate (Na2SO4)
Incubate in boiling water bath for evaporation of alcohol (10-20 minutes)
Impurities were filtered or centrifuged
1ml solution aliquot + 1ml Nelson Somogyi’s reagent
Incubation in boiling water bath for 20 minutes
Add 1ml AMR (Arsenomolybdate reagent)
Prepare final volume 20ml with D.W.
O.D. at 540nm
1ml Sucrose
1ml citrate Buffer (0.5 M, pH 5.4)
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The O.D was compared with a standard which was prepared using different
concentration of glucose. The results of enzyme activity were expressed as mg
glucose released/hr/mg protein.
4.5 STATISTICAL RESULT
It is essential to understand the relationship between different parameter
when the study is completed.
4.5.1 Relationship between the parameters (Correlation)
Any relationship between the two variable is known as correlation. If
one variable increases or decreases with a corresponding increase or decrease of
the other variable, a direct positive correlation exists between the two variables.
If one variable decrease with an increase in the other variable, then there is a
negative or inverse correlation. There are two different methods to study
correlation
Graphic method
It is the simplest method of showing the relationship between two
variable. In this one variable is represented on X-axis and other variable on Y-
axis on graph paper. Data corresponding to X and Y axis were plotted in form
of dots. And then estimated lines joining first and last points was drawn on the
graph paper to find out correlation.
Correlation coefficient
The graphic method indicates the existence of a correlation. But it is not
possible to calculate the extent or degree of relationship using these graph.
So,this was calculated by using following formula
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∑ (dx . dy)
r =
√∑(dx)2 . ∑(dy)
2
Where,
r is the correlation coefficient,
x and y are the two variable
dx is the deviation from the x-mean of the x variable,
dy is the deviation from the y mean of the y variable,
∑ (dx . dy) is the sum of the products of the deviations,
∑ (dx)2 is the sum of the squares of the deviations of the x variable,
∑ (dy)2 is the sum of the squares of the deviations of the y variable,