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Petronia Carillo
Università degli Studi della Campania "Luigi Vanvitelli
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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 d’Ornon, 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
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
Procedure
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 4°C, 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
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:
Where: Absextract is the absorbance determined with the extract, blank (expressed as absorbance) and
slope (expressed as absorbance∙nmol-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) µmol.g -1 fresh
weight.
Calibration curve for proline
y = 0.055x + 0.078
2 R2 = 0.999
Absorbance
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.
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.
Troll W, Lindsey J. (1955) A photometric method for the determination of proline. Journal of
Biological Chemistry 215: 655-660.
Ninhydrin is irritant to skin and respiratory system, thus requiring adequate precautions, in
particular the wearing of gloves.