PROTOCOL: Extraction and determination of proline

Initiating Author Name:

Petronia Carillo & Yves Gibon
Author Affiliations:
Petronia Carillo - Department of Life Science, II University of Naples, via Vivaldi 43, 81100,
Caserta, Italy
Yves Gibon - INRA, UMR 619, Biologie du Fruit, 33883 Villenave dOrnon, France

Overview
This protocol describes how to assay the amino acid proline with a ninhydrin-based simple, fast and
harmless method using a microplate reader or a cuvette spectrophotometer.

Background
Overproduction of proline is a widespread response observed in plants experiencing various
stresses, in particular osmotic stresses. The determination of this amino acid is therefore very useful
to assess the physiological status and more generally to understand stress tolerance in plants. Here
we describe a simple, fast and relatively harmless ninhydrin-based method, which is suitable for the
high throughput determination of free proline content using a microplate reader. Ninhydrin (2,2dihydroxyindane-1,3-dione, CAS number 485-47-2) is extensively used to assay amino acids. At
neutral pH, it destroys each primary -amino acid and also reacts with the released NH3 to form a
deep purple chromogen referred to as Ruhemann's Purple, which has a maximum absorption at
about 570 nm. At low pH, Ruthermanns purple is also yielded, but it quickly looses an amine
residue, which results into colourless derivatives. The reaction with proline and other imino acids
such as hydroxyproline or pipecolic acid yields a yellow-orange product at neutral pH, as the
cyclised N-group is not released. At low pH, the chromogen is red, with a peak of absorbance at
520 nm. It is important to note that ornithine and to a lesser extent lysine also yield a red chromogen
at low pH, which result from one ninhydrin molecule binding two amino acids. Nevertheless, in
plants under stress, levels of these amino acids are usually much lower than proline levels. In order
to decrease background noise (e.g., high levels of anthocyanins that may interfere with the
quantification), the chromogen is classically extracted using harmful solvents such as benzene
(Troll and Lindsey, 1955) or toluene (Bates et al., 1973). We have removed this step to make the
assay safer and easier, considering that interferences due to other compounds are usually low,
especially in plants under osmotic stress.
CAUTION: Ninhydrin is irritant to skin and respiratory system, thus requiring adequate
precautions, in particular the wearing of gloves. This colorimetric assay is quantitative and provides
reliable data about proline content. The sensitivity is of about 1 nmole and the linearity is in a range
of 1-100 nmoles.

Materials/Equipment

Proline
Ninhydrin
Glacial acetic acid
Ethanol (98%)

Screw-cap tubes (1.5 ml tubes or 96-microplate tubes)

Block heater or water bath
Centrifuge (for microtubes or for microplates)
Polystyrene spectrophotometer 1.5 ml-cuvette or polystyrene flat-bottom 96-well
microplates (flat bottom)
Cuvette spectrophotometer or microplate reader equipped with a monochromator or a 520
nm filter

Units, terms, definitions

Proline content - mol g-1 FW

Procedure
Plant material and extraction
Proline is very soluble and can be readily extracted by heating explants or aliquots of ground plant
material for 20 min in pure ethanol as well as in water. Proline can also be extracted together with
total amino acids, pigments, soluble sugars by heating plant material twice with 80% ethanol and
once with 50% ethanol as described by Cross et al. (2006), which results into a 70:30 ethanol:water
mixture (v/v). Proline and total amino acids may also be extracted using a cold extraction procedure
by mixing 20-50 mg fresh weight aliquots with 0.4-1 ml of ethanol:water (40:60 v/v). The resulting
mixture is left overnight a 4C, and then centrifuged at 14000 g (5 min). The cold extraction
procedure can be repeated on the pellet and supernatants pooled and used for the analyses. The first
extraction, however, already allows a recovery > 93% (Carillo et al. 2008)
Proline determination
Solutions (store at -20C):
Extract: 20 to 50 times diluted fresh weight (w/v), typically in a 70:30 ethanol:water mixture (v/v)
(Hummel et al., 2009).
Standards: proline solutions ranging from 0.04 to 1 mM, in the same medium as the one used for
the extraction.
Reaction mix: ninhydrin 1% (w/v) in acetic acid 60% (v/v), ethanol 20% (v/v). Protect from light.
CAUTION For the following steps gloves are highly recommended
MICROPLATE READER PROTOCOL:
In screw-cap tubes process as following:

pipette 100 l of reaction mix

pipette up to 50 l ethanolic extract or 50 l of 1-0.4-0.2-0.1-0.04 mM proline standard
prepared in 70:30 ethanol:water (v/v)
seal the tubes, mix and heat at 95C (either in block heater or water bath) for 20 min
after cooling at room temperature, spin down quickly (1 min, 2500 rpm)
transfer e.g. 100 l of the mixture to a microplate well
read at 520 nm (microplate reader)

NB: screw-cap tubes in 96-well format enable the use of a pipetting robot and of an automatic
capper/decapper.
CUVETTE SPECTROPHOTOMETER PROTOCOL:
In 1.5 ml screw-cap tubes, pipette as following:

1000 l of reaction mix

pipette up to 500 l ethanolic extract or 100 l of 5-2-1-0.5-0.2 mM proline standard
completed with up to 400l of ethanol:water (40:60 v/v)
seal the tubes, mix and heat at 95C in the block heater for 20 min
centrifuge (1 min, 10000 rpm)
transfer tube contents to a 1.5 ml cuvette
read at 520 nm

Calculations:
As shown in figure 1, the relationship between the amount of proline and the corresponding
absorbance is linear in the 1-50 nmol range in the microplate reader (note that linearity may vary
according to reader features) and in the 1-100 nmol range in a cuvette spectrophotometer.
The following equation is used to calculate the amount of proline in the extracts:
Proline in nmol.mg -1 FW or in mol.g-1 FW =
(Absextract blank)/slope*Volextract/Volaliquot*1/FW
Where: Absextract is the absorbance determined with the extract, blank (expressed as absorbance) and
slope (expressed as absorbancenmol-1) are determined by linear regression, Volextract is the total
volume of the extract, Volaliquot is the volume used in the assay, FW (expressed in mg) is the amount
of plant material extracted. It is assumed that Absextract is within the linear range.
In plant tissues, proline typically ranges from 0.5 (unstressed) to 50 (stressed) mol.g -1 fresh
weight.

Calibration curve for proline

3

Absorbance

y = 0.055x + 0.078
2

R2 = 0.999

1
y = 0.031x + 0.041
R2 = 0.999
0
0

10

20

30

40

50

Proline (nmoles)

Figure 1. Calibration curve obtained with the microplate procedure ( ). Note that with the cuvette
procedure ( ) the linear range is approximately the same.

Literature references
Bates LS, Waldren RP, Teare ID (1973) Rapid determination of free proline for water stress studies.
Plant Soil 39: 205-207.
Carillo P, Mastrolonardo G, Nacca F, Parisi D, Verlotta A, Fuggi A (2008) Nitrogen metabolism in
durum wheat under salinity: accumulation of proline and glycine betaine. Functional Plant Biology
35: 412426.
Cross JM, von Korff M, Altmann T, Bartzetko L, Sulpice R, Gibon Y, Palacios N, Stitt M (2006)
Variation of Enzyme Activities and Metabolite Levels in 24 Arabidopsis Accessions Growing in
Carbon-Limited Conditions. Plant Physiology 142: 1574-1588.
Hummel I, Pantin F, Sulpice R, Piques M, Rolland G, Dauzat M, Christophe A, Pervent M,
Bouteill M, Stitt M, Gibon Y, Muller B. (2010) Arabidopsis plants acclimate to water deficit at
low cost through changes of carbon usage: an integrated perspective using growth, metabolite,
enzyme, and gene expression analysis. Plant Physiology 154: 357-372.
Troll W, Lindsey J. (1955) A photometric method for the determination of proline. Journal of
Biological Chemistry 215: 655-660.

Health, safety & hazardous waste disposal considerations

Ninhydrin is irritant to skin and respiratory system, thus requiring adequate precautions, in
particular the wearing of gloves.

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