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Estimation of Proline

Proline is a basic amino acid found in high percentage in basic protein. Free proline is said to play a role in plants under stress conditions. Though the molecular mechanism has not yet been established for the increased level of proline, one of the hypotheses refers to breakdown of protein into amino acids and conversion to proline for storage. Many workers have reported a several-fold increase in the proline content under physiological and pathological stress conditions. Hence, the analysis of proline in plants has become routine in pathology and physiology division of agricultural sciences. Principle During selective extraction with aqueous sulphosalicylic acid, proteins are precipitated as a complex. Other interfering materials are also presumably removed by absorption to the protein-sulphosalicylic acid complex. The extracted proline is made to react with ninhydrin in acidic conditions (pH 1.0) to form the chlorophore (red color) and read at 520nm. Materials Acid Ninhydrin Warm 1.25g ninhydrin in 30mL glacial acetic acid and 20mL 6M phosphoric acid, with agitation until dissolved. Store at 4C and use within 24h. 3% Aqueous Sulphosalicyclic Acid Glacial Acetic Acid Toluene Proline Procedure 1. Extract 0.5g of plant material by homogenizing in 10mL of 3% aqueous sulphosalicylic acid. 2. Filter the homogenate through Whatman No. 2 filter paper. 3. Take 2mL of filtrate in a test tube and add 2mL of glacial acetic acid and 2mL acid ninhydrin. 4. Heat it in the boiling water bath for 1h. 5. Terminate the reaction by placing the tube in ice bath. 6. Add 4mL toluene to the reaction mixture and stir well for 20-30sec. 7. Separate the toluene layer and warm to room temperature. 8. Measure the red color intensity at 520nm. 9. Run a series of standard with pure proline in a similar way and prepare a standard curve. 10. Find out the amount of proline in the test sample from the standard curve. Calculation Express the proline content on fresh-weight-basis as follows: g proline/mL x mL toluene 5 moles per g tissue x = 115.5 g sample where 115.5 is the molecular weight of proline.

Standards
Dissolve 0.1g proline in 100ml distilled water Make 10 concentrations by taking 1-10ml from proline solution and making volume upto 100ml by distilled water. Now take 1ml from each above concentrations. Add 1ml sulphosalicylic acid to this. Add 2ml
of glacial acetic acid and 2mL acid ninhydrin. Do the same process as from step 4. A sample of only distilled water is also run doing the same process as for proline samples. For stardards blank is distilled water. For samples blank is toluene.