Protocol for: Free Proline estimation in plants
Abstract or Description:
ESTIMATION OF FREE PROLINE CONTENT IN PLANT TISSUES
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
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
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.
- Take the 0.5 g plant
homogenized in 5 ml of 3% sulphosalycylic acid using pre washed mortar and
- Filter the homogenate through Whatman No. 1 filter
paper and collect filtrate will be used for the estimation of proline
- Take 2 ml of extract in test tube and add 2 ml of
glacial acetic acid and 2 ml of ninhydrin reagent
- Heat reaction mixture in a boiling water bath at
for 1hour. Brick red colour will develops
- After cooling the reaction mixtures, 4 ml of
toluene is added and then transferred to a separating funnel
thorough mixing, the chromospheres containing toluene is separated and its
absorbance read at 520 nm in spectrophotometer against toluene blank
- Prepare standard curve of proline by taking 5 to
100 µg ml-1 concentration.
- 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