One milliliter of reactions mixture contained d-fructose (20 g/l) in sodium phosphate buffer (50 mM, pH 8.0) and 200 μl crude enzyme.
Three milliliters of reaction mixture contained 50 mM phosphate buffer (pH7.8), 0.1 mM EDTA, 13 mM methionine, 75 μM NBT, 16.7 μM riboflavin, and 300 μL of enzyme extract.
One milliliter of this reaction mixture, diluted with 3.0 mL of double-distilled water, has instantaneously been taken for the UV-visible spectroscopic investigations.
For ascorbate peroxidase (APX, EC 1.11.1.11) activity, three milliliter of the reaction mixture contained 50 mM potassium phosphate buffer (pH 7.0), 0.1 mM EDTA, 0.5 mM ascorbate, 0.1 mM H2O2, and 0.1 mL enzyme extract.
One milliliter of the reaction mixture contained 100 μl of 28 mM 2-deoxy-2-ribose (dissolved in phosphate buffer, pH 7.4), 500 μl solution of various concentrations of solvent extracts of test plants (200 1000 μg/ml), 200 μl of 200 μM FeCl3 and 1.04 mM EDTA (1 1 v/v), 100 μl H2O2 (1 mM) and 100 μl ascorbic acid (1 mM).
One unit of endoxylanase and one unit of endoglucanase were defined as the amount of enzyme that liberated 1 μmole of xylose and 1 μmole of glucose respectively from their respective substrates per milliliter per minute of reaction.
Two milliliters of the reaction mixture were withdrawn at regular time intervals, and extracted twice with 2.0 ml of water saturated n-butanol.
One milliliter of supernatant from the reaction mixture was added to 1.0 ml of 0.5 N HCl along with 1.0 ml of nitrite molybdate reagent (10% w/v sodium nitrite + 10% w/v sodium molybdate) (a yellow coloration appeared) followed by the addition of 1.0 ml of 1.0 N NaOH (a red coloration appeared).
One milliliter of DNS reagent added, and the reaction was heated in boiling water for 5 min and rapidly cooled.
After mixing, 10 μL of 1 M sodium cyanoborohydride stock (in 10 mM NaOH) was added to each milliliter of the conjugation mixtures and the reaction mixtures were incubated at room temperature for 24 hours with gentle mixing on a rotating platform.
