Estimation of proline
- 2. Proline - Introduction Proline is a proteinogenic aminoacid. Free proline accumulation is a common metabolic response of higher plants to water deficits, and salinity stress. Proline is highly water-soluble and acts as an osmolyte maintaining osmotic balance in plants subjected to abiotic stress.
- 3. Proline - Introduction Proline also protects the structure of membranes and cellular proteins. Many workers have reported several- fold increase in the proline content under physiological and pathological stress conditions. Hence, the analysis of proline is important for assessing the role of proline in physiological and pathological conditions of plants.
- 4. Principle Proline is extracted with sulphosalicylic acid. The extracted proline is made to react with acid Ninhydrin to form the red coloured chromophore. The absorbance is recorded at 520nm.
- 5. Materials Acid Ninhydrin: Warm 1.25g ninhydrin in 30ml glacial acetic acid and 20ml 6M phosphoric acid, with agitation until dissolved. Store at 4°C and use within 24 hours. 3% Aqueous Sulphosalicylicacid Glacial acetic acid Toluene
- 6. Standard Proline Stock solution- Weigh 100mg proline, dissolve in water and make upto 100ml in a volumetric flask. Working standard- Dilute 10ml of stock standard to 100ml in a volumetric flask. The concentration of this solution is 100μg/ml proline.
- 7. Extraction of proline Weigh 0.5g of plant material and homogenize in 10ml of 3% aqueous sulphosalicylic acid. Filter the homogenate through Whatman No. 2 filter paper and collect the filtrate.
- 8. Procedure Pipette out different aliquots (0.2, 0.4, 0.6, 0.8 and 1.0ml) of standard proline into different tubes. Take 2ml of filtrate (obtained from plant sample) in a separate test tube. Make up the solutions in all tubes to 2ml with water. A blank tube is taken and to it add 2ml of distilled water. Then add 2ml glacial acetic acid to all tubes. Mix well.
- 9. Procedure Add 2ml acid-ninhydrin reagent to all tubes and incubate in boiling water bath for 1 hour. Terminate the reaction by placing the tubes in ice bath. Add 4ml toluene to the reaction mixture and stir well for 20-30 seconds. Separate the toluene layer and bring to room temperature Measure the red colour intensity at 520 nm using toluene as blank. The colour is stable for one hour only. Find out the amount of proline in the test sample from the standard curve.
- 10. Observation and Calculation S.No Vol of Std (ml) Conc. of Std (μg) Vol. of water (ml) Vol of Glacial acetic acid (ml) Vol of acid Ninhydrin (ml) Keep in boiling water bath for 1 hour Vol of toluene (ml) OD at 520nm B S1 S2 S3 S4 S5 T1 T2
- 11. Result Express the proline content on fresh weight basis as µmoles per g tissue. Molecular weight of proline is 115.5
- 12. Answer the following questions Give the structure of proline. Proline is the only proteinogenic secondary amino acid which is a secondary amine, as the nitrogen atom is attached both to the α-carbon and to a chain of three carbons that together form a five-membered ring.
- 13. Answer the following questions Brief out the principle behind the proline estimation. Proline is extracted with sulphosalicylic acid. The extracted proline is made to react with acid Ninhydrin to form the red coloured chromophore. The absorbance is recorded at 520nm.
- 14. Answer the following questions What are the functions of proline during abiotic stress. Proline is known to act as an enzyme protectant during abiotic stress conditions. When supplied exogenously, proline has improved salt stress tolerance in various plant species. Under high-salt conditions, proline application enhances plant growth with increases in seed germination, biomass, photosynthesis, gas exchange, and grain yield.
- 15. Answer the following questions Why is proline called as a helix coil breaker in secondary structure of proteins? Proline and glycine are sometimes known as "helix breakers" because they disrupt the regularity of the α helical backbone conformation; however, both have unusual conformational abilities and are commonly found in turns.