Estimation of Biomolecules
The chlorophyll content in the fresh leaves was estimated by using the method of Arnon (1949). One gram of fresh leaf was weighed, homogenized and extracted in chilled 80% acetone (v/v), and the optical density of supernatant was read at 645nm and 663nm wavelengths in a spectrophotometer. Proline concentration was estimated by the method of Bates et al. (1973). Fresh leaves (250 mg) were homogenized in 10 ml of 3% aqueous sulphosalicylic acid and homogenate was centrifuged at 8000 × g for 15 min. A total of 2 ml aliquot of the supernatant was added with equal volume of glacial acetic acid and acid ninhydrin (prepared by dissolving 1.25 g ninhydrin in 30 ml acetic acid and 20 ml of 6N H3PO4) and incubated at 100°C for 1 h. The complete reaction was terminated by keeping mixture in ice bath and chromophore was extracted with 4 ml of toluene. This mixture shook vigorously for 15-20s and the chromatophore-containing toluene was drawing out from the aqueous phase and absorbance was determined spectrophotometrically at 520 nm with toluene for a blank. Proline content was calculated by following formula:
\begin{equation} \frac{[(µg\ proline/ml\ \times ml\ toluene)/115.5\ \ µg/µmole]\ \ }{[(g\ sample)/5]}=µmole\ proline/g\ of\ fresh\ weight\ material\nonumber \\ \end{equation}