ISOLATION OF ALFALFA SEED TRYPSIN INHIBITOR USING AFFINITY CHROMATOGRAPHY
Keywords:alfalfa seeds, extraction, affinity chromatography, trypsin inhibitor, amino acid composition, molecular weight
A protein inhibitor of trypsin of the cultivar Eva alfalfa seeds was isolated and characterized. It has significant antiproteolytic activity and is promising for the creation of compositions that are designed to correct nutrition in various conditions, accompanied by increased activation of proteolytic enzymes. The stages of purification of the trypsin inhibitor from alfalfa seeds included: extraction of 0.05 M borate buffer (pH 7.6), fractionation of the protein component of the extract with ammonium sulfate, followed by dialysis of the fraction between 75% and 100% saturation and affinity chromatography on a biospecific sorbent trypsin - sepharose 4B. Calculations showed that from 100 g of alfalfa seeds of the cultivar Eva, 67.14 mg of trypsin inhibitor was obtained, the inhibitory activity of which is 27.6 IU / mg protein. Experimental data show that the obtained inhibitor reduces trypsin activity by 63.05% with a weight ratio of inhibitor: enzyme of 1: 1. It is known from literature that a soybean trypsin inhibitor reduces trypsin activity by 30%. The isolated trypsin inhibitor has significant inhibitory activity, the value of which is higher than the inhibitory activity of the plant trypsin inhibitor from soybeans. Affinity chromatography allowed us to obtain a pure, homogeneous trypsin inhibitor, which has significant antiproteolytic activity, which can be compared with the antiproteolytic activity of known plant trypsin inhibitors. From the obtained experimental data, it follows that both, the obtained inhibitor and the inhibitor, which is part of a commercial preparation, effectively reduce trypsin activity, slightly inhibit chymotrypsin activity, and do not affect α-amylase and protease activity. The above data indicate that the trypsin inhibitor of alfalfa seeds of the cultivar Eva is not a bifunctional inhibitor, since it does not affect the activity of amilolytic enzymes. The isolated inhibitor belongs to proteins with a high degree of hydrophobicity. The composition of amino acids, the side chains of which can take part in the formation of hydrogen bonds, is 25% of the total number of amino acid residues of the alfalfa seed trypsin inhibitor. For STI, this value is 23%.
Synovets, A.S., Levitsky, A.P. (1985). [Inhibitors of proteolytic enzymes in medicine]. Kiev: Zdorovia. (in Russian)
Krusir, G.V., Beltyukova, S.V., Liventsova, O.O., Prylutsky, V.P. (2019). Isolation and physicochemical properties of tomato seed protease. Pharmacom. 4:28–36.
Kislukhina, O.V. (2002). [Enzymes in food and feed production]. Moscow: DeLi print. (in Russian)
Marri, R., Grenner, D., Meyyes, P. [Human Biochemistry]. Moscow: Mir. (in Russian)
Mosolov, V.V., Valueva, T.A. (1993). [Plant proteins are inhibitors of proteolytic enzymes]. Moscow: VINITI. (in Russian).
Solomintsev, M.V., Mogilny, M.P. (2009). [Determination of the activity of inhibitors of proteolytic enzymes in food products] Izvestiya vuzov. Food technology, 1, 13–16. (in Russian).
Chanphai, P., Tajmir-Riahi, H.A. (2016). [Chitosan nanoparticles conjugate with trypsin and trypsin inhibitor]. Carbohydrate Polymers, 144, 346–352.
Коndratjuk, N., Pyvovarov, Y., Stepanova, Т., Matsuk, Y. (2018). Investigation of the films based on the uronate polysaccharides by the method of differential scanning calorimetry. Food Science and Technology, 12(3). https://doi.org/10.15673/fst.v12i3.1037
Kondratyuk, N. V., Pyvovarov, Ye. P., Padalka, A. M., others. (2017). [Rheological properties of food film-forming gels on the basis of uroconate polysaccharides] Progressive techniques and technologies of food production, restaurant business and trade: coll. science. Kharkiv: KhDUHT. 2 (26), 86-93. (in Ukrainian).
Chanphai P, Tajmir-Riahi HA. (2016) Trypsin and trypsin inhibitor bind PAMAM nanoparticles: Effect of hydrophobicity on protein-polymer conjugation. J Colloid Interface Sci. 2016 Jan 1;461:419-424. doi: 10.1016/j.jcis.2015.09.048.
Chanphai P, Agudelo D, Tajmir-Riahi HA. (2016) PEG and mPEG-anthracene conjugate with trypsin and trypsin inhibitor: hydrophobic and hydrophilic contacts. J Biomol Struct Dyn., 35(10):2257-2268. doi: 10.1080/07391102.2016.1214621.
Chanphai P, Kreplak L, Tajmir-Riahi HA. (2017) Aggregation of trypsin and trypsin inhibitor by Al cation. J Photochem Photobiol B. ;169:7-12. ttps://doi. org/ 10.1016/j.jphotobiol.2017.02.018.
Chanphai P, Tajmir-Riahi HA. (2016) Conjugation of chitosan nanoparticles with biogenic and synthetic polyamines: A delivery tool for antitumor polyamine analogues. Carbohydr Polym. 5;152:665-671. doi: 10.1016/j.carbpol.2016.06.113.
Chanphai. P., Tajmir-Riahi, H.A. (2017) Encapsulation of testosterone by chitosan nanoparticles. Int J Biol Macromol. 98:535-541. ttps://doi. org/ 10.1016/j.ijbiomac.2017.02.007.
Matias, L.L.R., Costa, R.O.A, Passos, T.S., Queiroz, J.L.C., Serquiz, A.C., Maciel, B.L.L., Santos, P.P.A., Camillo, C.S., Gonçalves, C., Amado, I.R., Pastrana, L., Morais, А. (2019). Tamarind Trypsin Inhibitor in Chitosan-Whey Protein Nanoparticles Reduces Fasting Blood Glucose Levels without Compromising Insulinemia: A Preclinical Study. Nutrients. 14;11(11):2770. https://doi. org/ 10.3390/nu11112770.
Cotabarrena, Ju., Broitmana, D. Ju., Quirogab, E., Obregóna, W.D. (2020). [The first thermostable trypsin inhibitor from Geoffroea decorticans seeds. A novel natural drug with potential application in biomedicine]. International Journal of Biological Macromolecules. 148, 869–879.
Cotabarren, J. D. Lufrano, M.G. Parisi, D. (2020). Biotechnological, biomedical, and agronomical applications of plant protease inhibitors with high stability: A systematic review. Plant Science, 292, 234–245. https://doi. org/10. 1016/j.plantsci.2019.110398
Krusir, G.V. (2010). [Forecasting of effective methods of stabilization of plant biocorrectors].Grain products and compound feeds. 2, 15–18. (in Ukrainian).
Brandt, J., Andersson, L.-O., Porath, J. (1975). [Covalent attachment of proteins to polysaccharide carriers by means of benzoquinone]. Biochimica et Biophysica Acta, 386, 196–202.
Agromonov, A. E., Shabarov, Yu. S. (1971). [Laboratory work in an organic practicum]. Moscow: MSU. (in Russian)
Perrin, D. D., Armarego, W. L. F., Dawn, R. P. (1966). Purification of Laboratory chemicals. Pergamon Press-Oxford.
Hartree E. F. (1972). [Determination of protein: A modification of the Lowry method that gives a linear photometric response]. Analytical Biochemistry, 48, 422 – 427.
Scopes R. (1985). [Methods of protein purification]. Moscow: Mir. (in Russian)
Osterman L.A . (1981). [Methods for the study of protein and nucleic acids: Electrophoresis and ultracentifugation (a practical guide)]. Moscow: Nauka. (in Russian).
Mosolov, V. V., Malova, E. L., Cheban, A. N. (1983). [Isolation of a specific inhibitor of serine proteinases of microorganisms from bean seeds]. Biochemistry, 48(10), 1680–1686
Copyright (c) 2021 Днипровский национальный университет имени Олеся Гончара
This work is licensed under a Creative Commons Attribution 4.0 International License.
- Authors reserve the right of attribution for the submitted manuscript, while transferring to the Journal the right to publish the article under the Creative Commons Attribution License. This license allows free distribution of the published work under the condition of proper attribution of the original authors and the initial publication source (i.e. the Journal)
- Authors have the right to enter into separate agreements for additional non-exclusive distribution of the work in the form it was published in the Journal (such as publishing the article on the institutional website or as a part of a monograph), provided the original publication in this Journal is properly referenced
- The Journal allows and encourages online publication of the manuscripts (such as on personal web pages), even when such a manuscript is still under editorial consideration, since it allows for a productive scientific discussion and better citation dynamics (see The Effect of Open Access).