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In this article we will discuss about the tests for estimation of proline in plants.
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 chromophore (red colour) and read at 520 nm.
Materials:
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i. Acid Ninhydrin: Warm 1.25 g ninhydrin in 30 ml glacial acetic acid and 20 ml 6M phosphoric acid, with agitation until dissolved. Store at 4°C and use within 24 h.
ii. 3% Aqueous Sulphosalicyclic Acid
iii. Glacial Acetic Acid
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iv. Toluene
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v. Proline.
Test Procedure:
1. By homogenizing 0.5 g of plant material in 10 ml of 3% aqueous sulphosalicyclic acid, the extract was made.
2. The homogenate is filtered through Whatman No. 2 filter paper.
3. 2 ml of filtrate is taken in a test tube and 2 ml of glacial acetic acid and 2 ml acid-ninhydrin are added in a sequence.
4. The mixture is heated it in the boiling water-bath for 1 h.
5. The reaction is stopped by placing the tube in ice-bath.
6. 4 ml toluene is added to the reaction mixture and stir well for 20-30 sec.
7. The toluene layer is separated and warm to room temperature.
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8. The red colour intensity is measured at 520 nm.
9. A series of standard with pure proline in a similar way is made and prepare a standard curve. 10. Thus the amount of proline in the test sample is determined from the standard curve.
Calculation:
Express the proline content on fresh-weight-basis as:
µ moles per g tissue = µg proline/ml x ml toluene/115.5 × 5/g sample
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where 115.5 is the molecular weight of proline.
