Re: Follow-up question about extracting proline from plant tissue

Hi Shatha,

Thanks for your follow up to my answer to your earlier question (1264602900.Bc). While the intent of your research isn’t made clear, I will assume you are assaying for proline in plant proteins.

There are several questions that reflect upon review of the paper you cited: http://www.plantphysiol.org/content/vol47/issue5/ This is a well thought out, well written paper.

Many of your questions are answered there, since the authors address anthocyanins… and were the thrust of our original answer.

While the proline derivatives will be toluene soluble, there is a partial solubility of most of these other derivatives and plant pigments in toluene. Lipophyllic/aqueous extractions are not exact, as almost all aqueous samples partially are soluble in lipophylls and vice versa. These are called “partial solubility impurities”, almost all extraction based purification procedures suffer from this phenomena. Quantitation by spectroscopy in this way may well be “on a spectral slope”. Lange, et. al. addresses this.

There are vastly more plant pigments in either extract than proline. To overcome that, a high molecular weight chromatography step(example: Sephadex G 100) may help. That step will partially purify the high mole weight globular proteins out from the small molecular weight pigments very early in the procedure to ensure that proteins were the true analytes being measured for proline content.

Hydrolysis of the partially purified proteins in boiling acid, reduce the volume followed by two experiments:

Use thin layer chromatography, then follow up with ninhydrin treatment of the plate. When the proline-ninhydrin derivative (yellow) is removed from the plate and re-dissolved, the proline may be quantitated by spectral means.

Or, extract the toluene phase, take it to total dryness, the residue re- dissolved and treat with ninhydrin. The residues may be spotted on a thin layer plate and run in an appropriately balanced polar-nonpolar solvent mixture. The proline-ninhydrin derivative (yellow in color) will separate quite well from the other components. When re-dissolved, the proline derivative is quantitated by spectral means.

The results should agree within experimental error.

Here are references, full text available on the net, that may be helpful. Thanks for your question!

Peter

H. Lange, W. Shropshire, Jr., and H. Mohr. An Analysis of Phytochrome- mediated Anthocyanin Synthesis.
Plant Physiol. 1971 47: 649-655. doi:10.1104/pp.47.5.649
http://www.plantphysiol.org/content/vol47/issue5/

http://www.jbc.org/content/199/1/91.full.pdf
http://www.jbc.org/content/184/2/607.full.pdf+html
http://www.cem.msu.edu/~reusch/VirtualText/proteins.htm
http://www.jbmb.or.kr/jbmb/jbmb_files/[37-3]0406021411_275.pdf