ACETONE PRECIPITATION OF PROTEINS FOR DETERMINATION OF THE OF ENZYMES ACTIVITY IN MEDICINAL PLANTS
UDC 577.151: 577.13
Abstract
Plant phenolic compounds are widely used in medicine and agriculture. The study of the mechanisms of their biosynthesis and accumulation is a complex task. It is also true for the determination of the activity of the key plant enzymes such as shikimate dehydrogenase. At the same time, the isolation of proteins and enzymes from plant is complicated by the presence of phenolic compounds and pigments. The aim of the research was to evaluate the feasibility of the acetone precipitation of proteins of leaf extracts of plants to determine the activity of shikimate dehydrogenase. The object of the study was the leaves of sea buckthorn (H. rhamnoides) and silky dogwood (C. sericea). The proteins were precipitated from the crude extract with cold (-20 °C) acetone in varying ratio followed by washing three times with acetone and final dissolution in buffer. The activity of shikimate dehydrogenase was determined at pH=10.0 and expressed in ncat per mg of protein. Protein fractions with different activity of shikimate dehydrogenase were isolated from the leaves of sea buckthorn (H. rhamnoides) and silky dogwood (C. sericea). It was shown that the protein fraction with a ratio of 1 : 1.5 of extract : acetone (60% saturation) is most convenient to work with, has a high enzyme activity and does not contain colored impurities. This method of protein isolation and pre-purification is fast, requires a small amount of sample, and suitable for screening studies of the activity of various enzymes in medicinal plants.
Downloads
Metrics
References
Chalker-Scott L., Fuchigami L.H. Low temperature stress physiology in crops. CRC press, 2018, pp. 67–80. DOI: 10.1201/9781351074186.
Tzin V., Galili G., Aharoni A. eLS. John Wiley & Sons, Ltd: Chichester, 2012. DOI: 10.1002/9780470015902.a0001315.pub2.
Ossipov V., Salminen J.P., Ossipova S., Haukioja E., Pihlaja K. Biochemical Systematics and Ecology, 2003, vol. 31(1), pp. 3–16. DOI: 10.1016/S0305-1978(02)00081-9.
Aksonov A.A., Krol' T.A., Baleyev D.N., Osipov V.I. XI mezhdunarodnogo simpoziuma «Fenol'nyye soyedineniya: fun-damental'nyye i prikladnyye aspekty». [XI International Symposium “Phenolic Compounds: Fundamental and Ap-plied Aspects”]. Moscow, 2022. (in Russ.).
Tahmasebi E.S., Rabiei Z., Vannozzi G.P., Akbari G.A. J. Agr. Sci. Tech., 2011, vol. 13, pp. 943–952.
Sairam R.K., Srivastava G.C. Plant Science, 2002, vol. 162(6), pp. 897–904. DOI: 10.1016/S0168-9452(02)00037-7.
Ossipov V., Bonner C., Ossipova S., Jensen R. Plant Physiology and Biochemistry, 2000, vol. 38(12), pp. 923–928. DOI: 10.1016/S0981-9428(00)01203-1.
Gavrilenko V.F., Ladygina M.Ye., Khandobina L.M. Bol'shoy praktikum po fiziologii rasteniy. [Large workshop on plant physiology]. Moscow, 1975, 392 p. (in Russ.).
Bradford M.M. Analytical biochemistry, 1976, vol. 72(1-2), pp. 248–254. DOI: 10.1016/0003-2697(76)90527-3.
R Core Team. R Foundation for Statistical Computing. Vienna, Austria, 2013. URL: http://www.R-project.org/.
RStudio Team. RStudio. PBC, Boston, MA, 2020. URL: http://www.rstudio.com/.
Doucette A., Crowell A. Proteomics Technologies and Applications. IntechOpen, 2019, pp. 23–39. DOI: 10.5772/intechopen.85547.
Hýsková V., Bělonožníková K., Šmeringaiová I., Kavan D., Ingr M., Ryšlavá H. Phytochemistr, 2021, vol. 190, 112881. DOI: 10.1016/j.phytochem.2021.112881.
Ryšlavá H., Doubnerová V., Muller K., Baťková P., Schnablová R., Liberda J., Čeřovská N. Collection of Czechoslo-vak Chemical Communications, 2007, vol. 72(10), pp. 1420–1434. DOI: 10.1135/cccc20071420.
Dixon M., Webb E.C. Advances in Protein chemistry, 1962, vol. 16, pp. 197–219. DOI: 10.1016/S0065-3233(08)60030-3.
Magsumov T., Ziying L., Sedov I. International Journal of Biological Macromolecules, 2020, vol. 160, pp. 880–888. DOI: 10.1016/j.ijbiomac.2020.05.260.
Fernandes V.N.A., Mangolin C.A., Neves A.F., Sousa Nogueira F.C., Zeni Neto H., Machado M.F.P.S. Advances in Horticultural Science, 2020, vol. 34(2), pp. 233–241.
![](http://journal.asu.ru/public/journals/1/article_12549_cover_ru_RU.jpg)
Copyright (c) 2023 chemistry of plant raw material
![Creative Commons License](http://i.creativecommons.org/l/by/4.0/88x31.png)
This work is licensed under a Creative Commons Attribution 4.0 International License.
This work is licensed under a Creative Commons Attribution 4.0 International License.
The authors, which are published in this journal, agree to the following conditions:
1. Authors retain the copyright to the work and transfer to the journal the right of the first publication along with the work, at the same time licensing it under the terms of the Creative Commons Attribution License, which allows others to distribute this work with the obligatory indication of the authorship of this work and a link to the original publication in this journal .
2. The authors retain the right to enter into separate, additional contractual agreements for the non-exclusive distribution of the version of the work published by this journal (for example, to place it in the university depository or to publish it in a book), with reference to the original publication in this journal.
3. Authors are allowed to post their work on the Internet (for example, in a university repository or on their personal website) before and during the review process of this journal, as this may lead to a productive discussion, as well as more links to this published work.