2. LITERATURE REVIEW
3.4. Antioxidant enzyme and biochemical changes in T. pallida due to chromium
59 order to eliminate the interference of Cu-grid, Cu information was deducted from the spectra.
3.4. Antioxidant Enzyme and Biochemical Changes in T. pallida due to
60 reaction mixture (2.8 mL) contained 1.5-mL 200-mM sodium phosphate buffer (pH 7.8), 1.0-mL deionised water and 0.3-mL 0.1-M H2O2 prepared afresh prior to its use. The reaction mixture was then added with 0.5 mL enzyme extract, and the enzyme activity was measured by monitoring the decrease in absorbance at 240 nm due to H2O2 consumption. One unit of catalase activity was defined as change in absorbance of the mixture at 240 nm/min/g of fresh weight.
3.4.3. Estimation of Peroxidase Activity
The peroxidase activity was determined based on the oxidation of guaiacol, which was measured by an increase in the absorbance at 470 nm (Ambreen et al., 2000). The reaction mixture (total volume, 1 mL) contained 25 mM phosphate buffer (pH 7.0), 1.0 mM H2O2 (30%), 0.05% guaiacol and 0.1 mM EDTA and enzyme extract. The reaction was initiated by adding H2O2, and the increase in the absorbance was monitored at 470 nm (ε=26.6 mM/cm) at 1-min intervals up to 4 min. One unit of peroxidase activity was defined as the change in absorbance at 470 nm/min/mgfresh weight (fw) of leaf.
3.4.4. Estimation of Ascorbate Peroxidase Activity
Ascorbate peroxidase (APX) activity was measured according to a method described by Leonardis et al. (2000). Two-millilitre reaction mixture for this assay contained 50 mM phosphate buffer (pH 7.8), 0.1 mM EDTA, 0.3 mM ascorbate, 0.1 mM H2O2 and 100 μL enzyme extract. The reaction was initiated by the addition of H2O2, and oxidation of ascorbic acid was estimated by following the decrease in absorbance at 290 nm. APX activity was calculated using the molar extinction coefficient (ε =2.8 mM/cm) and was expressed as units per milligram of protein.
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61 3.4.5. Extraction and Estimation of Proline Content
Proline was extracted and estimated by following the method of Bates et al. (1973). Fresh plant tissue weighing 0.5 g was homogenized in a mortar and pestle with 10 ml of 3%
aqueous sulfosalicyclic acid. The homogenate obtained was then filtered through Whatmann No.1 filter paper. The residue was re-extracted and pooled, and thevolume was made upto 20 ml with aqueous sulfosalicylic acid before estimation of proline. To 2 ml of proline extract, 2 ml of acid ninhydrin and 2 ml of glacial acetic acid were added.
The mixture was incubated for an hour at 100°C in a boiling water bath and then transferred to an ice bath to terminate the reaction. 4 ml of toluene was added to the mixture and mixed vigorously using a test tube stirrer for 20 seconds, the toluene phase containing the chromophore was separated from the aqueous phase with the help of a separating funnel and the absorbance was measured at 520 nm in a spectrophotometer using a reagent blank. The proline content was determined from a standard curve obtained using pure proline, as shown in Fig. 3.3, and the results were expressed in milligram per gram fresh weight (Fig. 3.3).
Figure 3.3: Standard curve of proline concentration vs. absorbance at 520 nm used in the estimation of proline content.
y = 0.013x + 0.05 R² = 0.991
0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5
0 100 200 300 400
Absorbance (O.D. 520 nm)
Proline content (µm)
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62 Proline content on fresh-weight-basis is calculated using the equation 3.5:
µmoles per g tissue= (µ )/ ( )
. x (3.5) where, 115.5 is the molecular weight of proline
3.4.6. Estimation of Chloroplyll Content
Chloroplyll content was measured according to a method described by Arnon et al.
(1949). About 0.1 g fresh leaf sample was homogenised in 10 mL of cold 80% acetone using chilled mortar and pestle and the leaf homogenate was filtered through (Whatman No. 1) a the filter paper. The extract was then analysed using a UV-visible spectrophotometer (Cary 100, Varian, Australia) and to measure its absorbance at 663 nm and 645 nm for estimating the Chl concentration. Chlorophyll concentration was calculated using Arnon's equation (Eq. 3.6- 3.8) to convert the absorbance measurements to mg Chl/g leaf tissue.
Chl a (mg/g) = [(12.7 × A663) - (2.6 × A645)] × ml acetone / mg leaf tissue (3.6) Chl b (mg/g) = [(22.9 × A645) - (4.68 × A663)] × ml acetone / mg leaf tissue (3.7) Total Chl = Chl a + Chl b. (3.8)
3.4.7. Estimation of Total Carbohydrate Content
Total carbohydrate content was measured according to anthrone method (Raunkjer et al.
1994) using glucose as the standard and as detailed further. About 100 mg plant tissue sample was hydrolyzed for 3 hrs in water bath with 5 ml of 2.5N HCl. The reaction mixture was cooled to room temperature and neutralised with sodium carbonate powder till effervescence ceased. Final volume was made upto 100 ml with Milli Q water and centrifuged at 10000 ×g for 10 minutes. Supernatant was collected and 0.5 ml aliquot was used for analyses.
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63 For Anthrone method, 2.5 ml of anthrone reagent was mixed with 0.5 ml of a suitably diluted sample taken in a series of test tubes and heated in a boiling water bath for 15 min. A suitable blank prepared by mixing 2.5 ml of Anthrone reagent with 0.5 ml of Milli Q water and heated in a boiling water bath for 15 min.. The intensity of the colour developed was measured at 630 nm using a UV–visible spectrophotometer (Cary 100, Varian, Australia). A calibration curve prepared using known concentrations of glucose (0.02 to 0.2 g/l), as shown in Fig. 3.4, was used for estimating the total carbohydrate estimation in the samples.
Figure 3.4: Standard curve of glucose concentration vs. absorbance at 630 nm used in the estimation of total carbohydrate content.
3.4.8. Estimation of Total Protein Content
The soluble protein content in the samples was measured according to Lowry’s method (Lowry et al. 1951) using bovine serum albumin (BSA) as the standard protein. 2 ml of alkaline copper sulphate reagent was mixed with 0.5ml of a suitably diluted sample taken in a series of test tubes. The test tubes were mixed well and incubated at room
y = 7.696x + 0.026 R² = 0.980
0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8
0 0.05 0.1 0.15 0.2 0.25
Absorbance (O.D. 630nm)
Glucose concentration (mg/L)
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64 temperature for 10 minutes. Then 0.2 ml of Folin Ciocalteau solution (1N) was added to each test tube and incubated for 30 min. The intensity of the colour developed was measured at 660 nm using a UV–visible spectrophotometer (Cary 100, Varian, Australia).
A calibration curve prepared using known concentrations of BSA (0.05 to 1 mg/ ml), as shown in Fig. 3.5, was used for estimating the total protein concentration in the samples.
Figure 3.5: Standard curve of protein concentration vs. absorbance at 660 nm used in the estimation of total protein content.