Abstract
Proline, a stress marker, is routinely quantified by a protocol that essentially uses hazardous toluene. Negative impacts of toluene on human health prompted us to develop a reliable alternate protocol for proline quantification. Absorbance of the proline-ninhydrin condensation product formed by reaction of proline with ninhydrin at 100 °C in the reaction mixture was significantly higher than that recorded after its transfer to toluene, revealing that toluene lowers sensitivity of this assay. λ max of the proline-ninhydrin complex in the reaction mixture and toluene were 508 and 513 nm, respectively. Ninhydrin in glacial acetic acid yielded higher quantity of the proline-ninhydrin condensation product compared to ninhydrin in mixture of glacial acetic acid and H3PO4, indicating negative impact of H3PO4 on proline quantification. Further, maximum yield of the proline-ninhydrin complex with ninhydrin in glacial acetic acid and ninhydrin in mixture of glacial acetic acid and H3PO4 was achieved within 30 and 60 min, respectively. This revealed that H3PO4 has negative impact on the reaction rate and quantity of the proline-ninhydrin complex formed. In brief, our proline quantification protocol involves reaction of a 1-ml proline sample with 2 ml of 1.25 % ninhydrin in glacial acetic acid at 100 °C for 30 min, followed by recording absorbance of the proline-ninhydrin condensation product in the reaction mixture itself at 508 nm. Amongst proline quantification protocols known till date, our protocol is the most simple, rapid, reliable, cost-effective, and eco-friendlier.
Similar content being viewed by others
References
Alia, Pardha-Saradhi P (1991) Proline accumulation under heavy metal stress. J Plant Physiol 138:554–558
Alia, Pardha-Saradhi P (1993) Suppression in mitochondrial electron transport is the prime cause behind stress induced proline accumulation. Biochem Biophys Res Commun 193:54–58
Alia, Pardha-Saradhi P, Mohanty P (1991) Proline enhances primary photochemical activities in isolated thylakoid membranes of Brassica juncea by arresting photoinhibitory damage. Biochem Biophys Res Commun 181:1238–1244
Alia, Prasad KVSK, Pardha Saradhi P (1995) Zinc induced changes in the levels of free radicals and proline in Brassica juncea and Cajanus cajan. Phytochemistry 39:45–47
Alia, Pardha-Saradhi P, Mohanty P (1997) Involvement of proline in protecting thylakoid membranes against free radical-induced photodamage. J Photochem Photobiol B: Biol 38:253–257
Anastas P, Eghbali N (2010) Green chemistry: principles and practice. Chem Soc Rev 39:301–312
Arora S, Pardha-Saradhi P (1995) Light induced enhancement in proline levels in Vignaradiata exposed to environmental stresses. Aust J Plant Physiol 22:383–386
Bates LS, Waldren RP, Teare ID (1973) Rapid determination of free proline for water stress studies. Plant Soil 39:205–207
Chinard FP (1952) Photometric estimation of proline and ornithine. J Biol Chem 199:91–95
Cingoz GS, Verma SK, Gurel E (2014) Hydrogen peroxide-induced antioxidant activities and cardiotonic glycoside accumulation in callus cultures of endemic Digitalis species. Plant Physiol Biochem 82:89–94
Iqbal N, Umar S, Khan NA (2015) Nitrogen availability regulates proline and ethylene production & alleviates salinity stress in mustard (Brassica juncea). J Plant Physiol 178:84–91
Kavi Kishor PB, Hong Z, Miao GH, Hu CAA, Verma DPS (1995) Overexpression of [delta]-pyrroline-5-carboxylate synthetase increases proline production and confers osmotolerance in transgenic plants. Plant Physiol 108:1387–1394
Kimura Y (1967) A study of glutamine interference in the acidified ninhydrin reaction for proline. Anal Chim Acta 37:240–245
Lee C-W, Dai Y-T, Chien C-H, Hsu D-J (2006) Characteristics and health impacts of volatile organic compounds in photocopy centers. Environ Res 100:139–149
Magne C, Larher F (1992) High sugar content of extracts interfere with colorimetric determination of proline and free amino acids. Anal Biochem 200:115–118
Main KM, Mortensen GK, Kaleva MM, Boisen KA, Damgaard IN, Chellakooty M, Schmidt IM, Suomi A-M, Virtanen HE, Petersen JH, Andersson A-M, Toppari J, Skakkebaek NE (2006) Human breast milk contamination with phthalates and alterations of endogenous reproductive hormones in infants three months of age. Environ Health Perspect 114:270–276
McNary JE, Jackson EM (2007) Inhalation exposure to formaldehyde and toluene in the same occupational and consumer setting. Inhal Toxicol 19:573–576
Pardha-Saradhi P, Alia, Vani B (1993) Inhibition of mitochondrial electron transport is the prime cause behind proline accumulation during mineral deficiency in Oryza sativa. Plant Soil 156:465–468
Pardha-Saradhi P, Alia, Arora S, Prasad KVSK (1995) Proline accumulates in plants exposed to UV radiation and protect them against UV induced peroxidation. Biochem Biophys Res Com 209:1–5
Ranjit SL, Manish P, Penna S (2015) Early osmotic, antioxidant, ionic, and redox responses to salinity in leaves and roots of Indian mustard (Brassica juncea L.). Protoplasma. doi:10.1007/s00709-015-0792-7
Rienth M, Romieu C, Gregan R, Walsh C, Torregrosa L, Kelly MT (2014) Validation and application of an improved method for the rapid determination of proline in grape berries. J Agric Food Chem 62:3384–3389
Sanchita, Singh R, Mishra A, Dhawan SS, Shirke PA, Gupta MM, Sharma A (2015) Physiological performance, secondary metabolite and expression profiling of genes associated with drought tolerance in Withania somnifera. Protoplasma. doi:10.1007/s00709-015-0771-z
Schweet RS (1954) The quantitative determination of proline and pipecolic acid with ninhydrin. J Biol Chem 208:603–614
Shabnam N, Pardha-Saradhi P, Sharmila P (2014) Phenolics impart Au3+-stress tolerance to cowpea by generating nanoparticles. PLoS One 9, e85242
Sharmila P, Pardha-Saradhi P (2002) Proline accumulation in heavy metal stressed plants: an adaptive strategy. In: Prasad MNV, Strzalka K (eds) Physiology and biochemistry of metal toxicity and tolerance in plants. Kluwer Acad Publ, Dordrecht, pp 179–199
Sheldon RA (2012) Fundamentals of green chemistry: efficiency in reaction design. Chem Soc Rev 41:1437–1451
Sivakumar P, Sharmila P, Pardha-Saradhi P (1998) Proline suppresses Rubisco activity in higher plants. Biochem Biophys Res Commun 252:428–432
Sivakumar P, Sharmila P, Pardha-Saradhi P (2000) Proline alleviates salt stress induced enhancement in ribulose 1,5-bisphosphate oxygenase activity. Biochem Biophys Res Commun 279:512–515
Sivakumar P, Sharmila P, Pardha-Saradhi P (2001) Proline suppresses Rubisco activity by dissociating smaller subunits from holoenzyme. Biochem Biophys Res Commun 282:236–241
Szabados L, Savoure A (2009) Proline, a multifunctional amino acid. Trends Plant Sci 15:89–97
Troll W, Lindsley JA (1955) A photometric method for the determination of proline. J Biol Chem 215:655–660
Yetiman AE, Kesmen Z (2015) Identification of acetic acid bacteria in traditionally produced vinegar and mother of vinegar by using different molecular techniques. Int J Food Microbiol 204:9–16
Zhang C-S, Lu Q, Verma DPS (1995) Removal of feedback inhibition of Δ1-pyrroline-5-carboxylate synthetase, a bifunctional enzyme catalyzing the first two steps of proline biosynthesis in plants. J Biol Chem 270:20491–20496
Author information
Authors and Affiliations
Corresponding author
Ethics declarations
Conflict of interest
The authors declare that they have no competing interests.
Additional information
Handling Editor: Peter Nick
Rights and permissions
About this article
Cite this article
Shabnam, N., Tripathi, I., Sharmila, P. et al. A rapid, ideal, and eco-friendlier protocol for quantifying proline. Protoplasma 253, 1577–1582 (2016). https://doi.org/10.1007/s00709-015-0910-6
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00709-015-0910-6