Wednesday, May 31, 2006

PROTOCOLS IN STRESS PHYSIOLOGY AND BIOCHEMISTRY

Estimation of magnitude of oxidative stress or lipid peroxidation or MDA, proline and assays of plant non-enzymatic and enzymatic antioxidants

Estimation of thiobarbituric acid reactive substances

Thiobarbituric acid reactive substances (TBARS), considered as oxidative damage products, may be determined in leaf samples by the method of (Heath and Packer 1968) modified slightly by our research group. Homogenize one gram of fresh tissue in 10 cm3 (ml) 0.1% TCA (trichloroacetic acid) and centrifuged at 7826 g 5 min. Add one cm3 of supernatant with 4.0 cm3 of 0.5% thiobarbituric acid (TBA), which was prepared in 20% TCA. The mixture was heated at 95 0C for 30 min, cooled and centrifuged at 1957 g for 5 min. Read the absorbance of the supernatant at 532 nm and corrected for unspecific turbidity after subtraction from the value obtained at 600 nm. Express TBARS in nmol g–1 fw, quantified using an extinction coefficient of 155/mM-1 cm-1.

Estimation of proline content

Proline content in leaf samples may be estimated by the method of Bates et al. (1973). Homogenize half gram of fresh leaf in 10 cm3 of 3% sulphosalicylic acid and centrifuged at 7826 g for 10 min. Add two cm3 of supernatant with 2 cm3 of acid ninhydrin reagent (1.25% ninhydrin, 30% glacial acetic acid and 20% 6 N o-phosphoric acid) and 2 cm3 of glacial acetic acid. Boil the resultant mixture at 100 0C in a water bath for 30 min, stop reaction in an ice bath and add 4 cm3 of toluene to each sample followed by vigorous cyclomixing for 10-15 s. Lift the toluene (upper) layer to read at 520 nm using UV-Vis Spectrophotometer. The corresponding concentration of proline should be determined against the standard curve of L-proline, express as μ g-1 fw.

Esimation of Asc and Dasc

Ascorbate (Asc), dehydroascorbate (DAsc) and total ascorbate (Asc + DAsc) should be estimated by a modified method of Law et al. (1983). Grind half gram of fresh leaf tissue in two cm3 of Na-phosphate buffer (0.1 M, pH 7, 1 mM EDTA) and centrifuge at 7826 g for 10 min. Distribute the supernatant in two separate sets of microcentrifuge tubes (400 μl in each) for the assay of total ascorbate (Asc + DAsc) and ascorbate (Asc) in first and second set, respectively. To each sample, add 200 μl of 10% TCA. After 5 min, add 10 μl of NaOH (5 M) solution, mix and centrifuge for 2 min in a microfuge. To 200 μl of the supernatant, add 200 μl of Na-phosphate buffer (150 mM, pH 7.4) and 200 μl of DDW. To another 200 μl of supernatant add 200 μl of Na-phosphate buffer (150 mM, pH 7.4) and 100 μl of 10 mM dithiothritol (DTT) and, after a thorough mixing, was leave at room temperature for 15 min. Add 100 μl of 0.5 % N-ethylmaleimide to each of the tubes and incubate at 24 0C for >30 s. Further, add 400 μl of 10% TCA, 400 μl of 44% H3PO4, 400 μl of 4% bipyridyl and 200 μl of 3% FeCl3. After being vortex-mixed, incubated the samples at 37 0C for 60 min record absorbance at 525 nm on a UV-Vis Spectrophotometer. A standard curve in the range of 0-100 nmol of Asc should be used for calibration. Compute dehydroascorbate (DAsc) concentrations from the difference (total ascorbate – ascorbate). Estimated the amounts in nmol g–1 fw.

Estimation of GSH and GSSG

Reduced (GSH), oxidised (GSSG) and total glutathione (GSH + GSSG) may be determined by the glutathione recycling method of Anderson (1985). Homogenize half gram of fresh leaf in 2 cm3 of 5% sulphosalicylic acid at 4 0C. Centrifuge the homogenate at 7826 g for 10 min. To a 0.5 cm3 of supernatant, add 600 µl of reaction buffer (0.1 M Na-phosphate, pH 7, 1 mM EDTA) and 40 μl of 0.15% 5,5-Dithiolbis 2-nitrobenzoic acid (DTNB), and read at 412 nm after 2 min. To the same, add 50 μl of 0.4% NADPH and 2 μl of GR (glutathione reductase; 0.5 enzyme unit) and let the reaction run for 30 min at 25 0C. Read the samples again at 412 nm to determine the total glutathione. Actual values of glutathione content may be determined against GSH standard curve (10-100 nmol). By subtracting GSH from total glutathione (GSH+GSSG), estimate GSSG. The amounts of GSH, GSSH and (GSH + GSSG) should be expressed in nmol g-1 fw.

Enzymatic assays:

Superoxide dismutase (SOD)

Follow the method of Dhindsa et al. (1981) with our slight modification for estimating SOD activity. Homogenized fresh leaf material (0.2 g) in 2.0 cm3 of extraction mixture containing 0.5 M Na-phosphate buffer, pH 7.3, 3 mM EDTA, 1% PVP, 1% Triton X 100 and centrifuge at 11269 g at 4 0C. Assay SOD activity in the supernatant by its ability to inhibit photochemical reduction of nitroblue tetrazolium (NBT). The assay mixture should consist of 1.5 cm3 reaction buffer (0.1 M Na-phosphate buffer, pH 7.5, 1% PVP), 0.2 cm3 of L-methionine (200 mM), 0.1 cm3 enzyme extract with equal amount of 1M NaHCO3, 2.25 mM NBT solution, 3 mM EDTA, 60 µM riboflavin and 1.0 cm3 of DDW in test tubes to incubated under illumination of 15W inflorescent-light lamp for 10 min at 28 0C. Blank A, containing all substances of the reaction mixture, along with enzyme extract, must be placed in the dark. Blank B, containing all substances of reaction mixture without enzyme must be placed in light along with the sample. Terminate the reaction after 10 min by switching off light and cover the tubes with the black cloth. The non-irradiated reaction mixture containing the enzyme extract will not develop bluish-gray color. Read absorbance of samples along with blank B at 560 nm against blank A. The difference of % reduction in colour between blank B and the sample should be calculated. 50% reduction in colour is considered as one unit of enzyme activity expressed in enzyme unit (EU) mg-1 protein h-1.

SOD activity (EU mg-1 protein h-1) = % Reduction in color between blank and sample x Dilution factor x 60 / 50 x Incubation time x mg protein in sample*

*For the estimation of protein content in the sample, use Bradford reagent and quantify the amount of protein against the standard curve of BSA (Bovine Serum Albumin)

Ascorbate Peroxidase (APX)

Ascorbate peroxidase (APX) activity may be estimated by the method of Nakano and Asada (1981). Grind one gram of the fresh leaf material in 5 cm3 of extraction buffer (0.1 M K-phosphate, pH 7, 3 mM EDTA, 1% PVP, 1% Triton X 100), centrifuge at 7826 g for 10 min at 4 0C. Determine APX activity in supernatant by the decrease in absorbance of ascorbate at 290 nm, due to its enzymatic breakdown. One cm3 of reaction buffer should contain 0.5 mM ascorbate, 0.1 mM H2O2, 0.1 mM EDTA and 50 µl extract containing enzyme. Let the reaction run for 5 min at 25 0C. APX activity may be calculated by using extinction coefficient 2.8 mM–1 cm–1 and expressed in enzyme units (EU) mg-1 protein. One unit of enzyme determines the amount necessary to decompose 1 μmol of ascorbate consumed per min at 25 0C.

Glutathione reductase (GR)

Glutathione reductase (GR) activity may be determined by the method of Foyer and Halliwell (1976) modified by Rao (1992). Grind half gram fresh leaf material in 2 cm3 of extraction buffer (0.1 M Na-phosphate, pH 7.0, 3 mM EDTA, 1% PVP, 1% Triton X 100) and centrifuge at 7826 g for 10 min. The supernatant must be immediately assayed for GR activity through glutathione-dependent oxidation of NADPH at 340 nm. One cm3 reaction mixture containing 0.2 mM NADPH, 0.5 mM GSSG and 50 μl of enzyme extract is kept for 5 min at 25 0C. Corrections should be made for any GSSG oxidation in the absence of NADPH. The activity may be calculated by using extinction coefficient 6.2 mM–1 cm–1 and expressed in enzyme units (EU) mg-1 protein. One unit of enzyme determines its amount necessary to decompose 1 μmol of NADPH per min at 25 0C.

Catalase (CAT)

Catalase (CAT) activity may be determined by the method of Aebi (1984). Half gram of the fresh leaf material, ground in 5 cm3 of extraction buffer (0.5 M Na-phosphate, pH 7.3, 3 mM EDTA, 1% PVP, 1% Triton X 100) is centrifuged at 7826 g for 20 min at 4 0 C. Assay the catalase activity in supernatant by monitoring the disappearance of H2O2, measuring a decrease in absorbance at 240 nm. Reaction should be run in a final volume of 2 cm3 of reaction buffer (0.5 M Na-phosphate, pH 7.3) containing 0.1 cm3 3 mM EDTA, 0.1 cm3 of enzyme extract and 0.1 cm3 of 3 mM H2O2 for 5 min. CAT activity should be calculated using an extinction coefficient (ζ = 0.036 mM –1 cm-1) and is expressed in enzyme units (EU) mg-1 protein. One unit of enzyme determines the amount necessary to decompose 1 μmol of H2O2 per min at 25 0C.

3 Comments:

At 7:13 AM, Anonymous Anonymous said...

Dear Dr Irfan,
I am very happy to note that you are making us feel proud ....remember me??????
Amjad. I like the way you have created this website. WISH YOU ALL THE BEST IN YOUR LIFE.

 
At 6:47 AM, Blogger abhirichster said...

thanks..

 
At 9:35 PM, Anonymous Anonymous said...

dr irfan
i am a student doing mphil. i want to ask how we can calculate the volume of chemicals(if their molarity is given) 2 be added in a 3 ml assay mixture (containing .2 ml enzyme)? eg 1.5 cm3 reaction buffer (0.1 M Na-phosphate buffer, pH 7.5, 1% PVP), 0.2 cm3 of L-methionine (200 mM), 0.1 cm3 enzyme extract with equal amount of 1M NaHCO3, 2.25 mM NBT solution, 3 mM EDTA, 60 µM riboflavin

 

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