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Free Proline estimation in plants

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Abstract or Description:





Proline (abbreviated as Pro or P) is a α-amino acid, one of the twenty DNA-encoded amino acids found in proteins. It is unique among the 20 protein-forming amino acids in that the α-amino group is secondary. The more common L form has S stereochemistry.

          In plants proline is synthesized from glutamic acid through a pathway catalyzed by pyrroline-5-carboxylate synthetase (1) and pyrroline-5-carboxylate reductase.

Its accumulation under various abiotic stresses (heat, cold, drought, moisture and salinity) in important crop plants considered as a tolerance mechanism. It is suggested to act as an osmolyte/compatible as well as a source of nitrogen during recovery from stress. Compatible solutes act a as chemical chaperone, which protects proteins during various abiotic stresses.  Proline estimation is based on the formation of brick red colored praline-ninhydrin complex in acidic medium. This complex is soluble in toluene and thus can be separated from aqueous phase. This ensures that there is no interference with other amino acids, which also form blue colored complex with ninhydrin. The toluene soluble brick-red colored complex absorbs at 520 nm.




Free proline content in the leaves is determined following the method off (Bates et al., 1973). The protocol is based on the formation of red colored formazone by proline with ninhydrin in acidic medium, which is soluble in organic solvents like toluene.



Instruments and glassware

Test tubes, test tube stand, micro-pipettes (20-200 µl, 100-1000 µl and 5 ml), Whatman No. 1 filter papers, visible range spectrophotometer.



Glacial acetic acid (Analytical grade)

Sulphosalycylic acid (3%): Three gram of sulphosalycylic acid was dissolved in 100 ml of distilled water.

Orthophosphoric acid (6 N): Required volume of orthophosphoric acid (38.1 ml) was taken and volume was made to 100 ml, using distilled water to get 6 N orthophosphoric acid.

Acid ninhydrin: Ninhydrin (1.25 g) was dissolved in a blend of 30 ml of glacial acetic acid and 20 ml of 6 N orthophosphoric acid.



  1. Take the 0.5 g plant tissue and homogenized in 5 ml of 3% sulphosalycylic acid using pre washed mortar and pestle
  2. Filter the homogenate through Whatman No. 1 filter paper and collect filtrate will be used for the estimation of proline content.
  3. Take 2 ml of extract in test tube and add 2 ml of glacial acetic acid and 2 ml of ninhydrin reagent
  4. Heat reaction mixture in a boiling water bath at 100 oC for 1hour. Brick red colour will develops
  5. After cooling the reaction mixtures, 4 ml of toluene is added and then transferred to a separating funnel
  6.  After thorough mixing, the chromospheres containing toluene is separated and its absorbance read at 520 nm in spectrophotometer against toluene blank
  7. Prepare standard curve of proline by taking 5 to 100 µg ml-1 concentration.
  8. Free proline content in sample is estimated by referring to a standard curve made from known concentrations of proline by taking following formula.

FW = Fresh weight of leaf tissue

A   = absorbance at 520 nm

D    = Initial dilution

115.5   = Molecular weight of proline



Bates, L.S., Waldran, R.P. and Teare, I.D. (1973). Rapid determination of free proline for water stress studies. Plant Soil 39: 205-208.


Citation: Shekhar C Bisht, VPKAS, Almora, India
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Posted By: shekharcbisht on 2/25/2011 10:30:31 AM
Dol Kar (Jan 14, 2014, 6:52 AM)
Please elaborate the equqtion used to calculate total protein. Thanks
fkaz007 (May 29, 2011, 3:01 AM)
Hi, the formula is not displayed in the above text. Can you please update it.
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