A Specific and Sensitive Enzymatic Assay for the Quantitation of L-Proline
Because proline accumulates rapidly in response to several stress conditions such as drought and excess salt, increased intracellular levels of free proline are considered a hallmark of adaptive reactions in plants, particularly in response to water stress. Proline quantitation is easily achievable...
| Main Authors: | , |
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| Format: | Article |
| Language: | English |
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Frontiers Media S.A.
2020-10-01
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| Series: | Frontiers in Plant Science |
| Subjects: | |
| Online Access: | https://www.frontiersin.org/articles/10.3389/fpls.2020.582026/full |
| _version_ | 1818883198590910464 |
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| author | Giuseppe Forlani Dietmar Funck |
| author_facet | Giuseppe Forlani Dietmar Funck |
| author_sort | Giuseppe Forlani |
| collection | DOAJ |
| description | Because proline accumulates rapidly in response to several stress conditions such as drought and excess salt, increased intracellular levels of free proline are considered a hallmark of adaptive reactions in plants, particularly in response to water stress. Proline quantitation is easily achievable by reaction with ninhydrin, since under acidic conditions peculiar red or yellow reaction products form with this unique cyclic amino acid. However, little attention has been paid to date to cross-reaction of ninhydrin with other amino acids at high levels, or with structurally related compounds that may also be present at significant concentrations in plant tissues, possibly leading to proline overestimation. In vitro at high pH values, δ1-pyrroline-5-carboxylate reductase, the enzyme catalyzing the second and last step in proline synthesis from glutamate, was early found to catalyze the reverse oxidation of proline with the concomitant reduction of NAD(P)+ to NAD(P)H. Here we characterized this reverse reaction using recombinant enzymes from Arabidopsis thaliana and Oryza sativa, and demonstrated its utility for the specific quantification of L-proline. By optimizing the reaction conditions, fast, easy, and reproducible measurement of L-proline concentration was achieved, with similar sensitivity but higher specificity than the commonly used ninhydrin methods. |
| first_indexed | 2024-12-19T15:29:51Z |
| format | Article |
| id | doaj.art-30f332ae13ad4944b31d4577e548d319 |
| institution | Directory Open Access Journal |
| issn | 1664-462X |
| language | English |
| last_indexed | 2024-12-19T15:29:51Z |
| publishDate | 2020-10-01 |
| publisher | Frontiers Media S.A. |
| record_format | Article |
| series | Frontiers in Plant Science |
| spelling | doaj.art-30f332ae13ad4944b31d4577e548d3192022-12-21T20:15:46ZengFrontiers Media S.A.Frontiers in Plant Science1664-462X2020-10-011110.3389/fpls.2020.582026582026A Specific and Sensitive Enzymatic Assay for the Quantitation of L-ProlineGiuseppe Forlani0Dietmar Funck1Department of Life Science and Biotechnology, University of Ferrara, Ferrara, ItalyLaboratory of Plant Physiology and Biochemistry, Department of Biology, University of Konstanz, Konstanz, GermanyBecause proline accumulates rapidly in response to several stress conditions such as drought and excess salt, increased intracellular levels of free proline are considered a hallmark of adaptive reactions in plants, particularly in response to water stress. Proline quantitation is easily achievable by reaction with ninhydrin, since under acidic conditions peculiar red or yellow reaction products form with this unique cyclic amino acid. However, little attention has been paid to date to cross-reaction of ninhydrin with other amino acids at high levels, or with structurally related compounds that may also be present at significant concentrations in plant tissues, possibly leading to proline overestimation. In vitro at high pH values, δ1-pyrroline-5-carboxylate reductase, the enzyme catalyzing the second and last step in proline synthesis from glutamate, was early found to catalyze the reverse oxidation of proline with the concomitant reduction of NAD(P)+ to NAD(P)H. Here we characterized this reverse reaction using recombinant enzymes from Arabidopsis thaliana and Oryza sativa, and demonstrated its utility for the specific quantification of L-proline. By optimizing the reaction conditions, fast, easy, and reproducible measurement of L-proline concentration was achieved, with similar sensitivity but higher specificity than the commonly used ninhydrin methods.https://www.frontiersin.org/articles/10.3389/fpls.2020.582026/fullproline measurementchemical assayenzymatic assayspecificitysensitivity |
| spellingShingle | Giuseppe Forlani Dietmar Funck A Specific and Sensitive Enzymatic Assay for the Quantitation of L-Proline Frontiers in Plant Science proline measurement chemical assay enzymatic assay specificity sensitivity |
| title | A Specific and Sensitive Enzymatic Assay for the Quantitation of L-Proline |
| title_full | A Specific and Sensitive Enzymatic Assay for the Quantitation of L-Proline |
| title_fullStr | A Specific and Sensitive Enzymatic Assay for the Quantitation of L-Proline |
| title_full_unstemmed | A Specific and Sensitive Enzymatic Assay for the Quantitation of L-Proline |
| title_short | A Specific and Sensitive Enzymatic Assay for the Quantitation of L-Proline |
| title_sort | specific and sensitive enzymatic assay for the quantitation of l proline |
| topic | proline measurement chemical assay enzymatic assay specificity sensitivity |
| url | https://www.frontiersin.org/articles/10.3389/fpls.2020.582026/full |
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