Abstract

Enzyme inhibitors are widely used in experimental studies in various spheres to evaluate the mechanism of catalytic effect of enzymes, determine the nature of protein reactive groups, to identify the role of various enzymatic processes in metabolism. But inhibitors are not stable and thus, they need to be stabilized through immobilization on matrixes. The study of immobilization using the infrared spectroscopy ensures to prove the interaction between the inhibitor and polysaccharidic matrix. The results of infrared spectroscopy showed that the linking between the matrix and the inhibitor occurs by formation of intramolecular covalent linkings, electrostatic correlation between the charged groups of agar and inhibitor. The derived comparison curve VVOP shows the reduction in the intensity of the immobilized inhibitor (dietar y supplement) in the area of 3400 cm-1, that is consistent with the valent variations of the free group -OH that indicates on strengthening of the immobilized specimen hydrogen binding. Comparative study of pH-optimum of pancreatic α-amylase, native and immobilized inhibitor made it possible to conclude that pH-optimum of pancreatic α-amylase ispH 6.0, that of the native inhibitor α-amylase equals to 5.5, and the pH-optimum in the immobilized inhibitor considerably varies from 5.0 to 6.8 at the physiologic temperature (37 ± 1)ºC. Linearization methods of Michaelis-Menten equation by Lineweaver-Burk and Hanes were used to determine kinetic parameters of the dietary supplement inhibition. The kinetics of enzyme inhibition was assessed using the immobilized form of the inhibitor that resulted in the enzyme activity decrease at zero variations Km at the decreasing V max values which makes it possible to classify the inhibition to the linear uncompetitive type (catalyzed inhibition).

Keywords

pancreatic amylase inhibitor, hydrolysis kinetics, kinetic parameters of pancreatic amylase inhibition

REFERENCES

1. Kabachnyy P.I. Perspektivy sozdaniya lekarstvennykh sredstv gipoglikemicheskogo deystviya na osnove prirodnykh ingibitorov amiloliticheskikh fermentov: Lekarstvennye sredstva. Ekonomika, tekhnologiya i perspektivy polucheniya. Obzor [Perspectives to create medicinal products of glycemic action based on natural inhibitors of amylolytic enzymes: Medicinal products. Economics, technology and perspectives of production. Review]. Moscow: VNIISENTI of Minmedprom of the USSR, 1990, ed. 1. 23 p.
2. Krusir G.V. and Kushnir N.A. Prevalence of amylase inhibitors in crops. Zernovi produkty i kombikormy [Cereal products and combined feed], 2008, no. 4, pp. 15-20. (In Ukrainian).
3. Sidenius U., Olsen K., Svensson B., and Christensen U. Stopped-flow kinetic studies of the reaction of barley α-amylase/subtilisin inhibitor and the high pI barley α-amylase. FEBS Letters, 1995, March 20, vol. 361, iss. 2-3, pp. 250-254. DOI: 10.1016/0014-5793(95)00187-E.
4. Keleti T. Osnovy fermentativnoy kinetiki [Fundamentals of enzyme kinetics]. Moscow: Mir Publ., 1990. 350 p.
5. Dzyuba N.A. and Zemlyakova O.V. Features of immobilization α-amylase on protein’s and polysaccharide’s matrixes. Bulletin of the National Technical University "Kharkiv Polytechnic Institute": Mechanical-technological systems and complexes, 2015, no. 52 (1161), pp. 108-112. (In Ukrainian).
6. Krusir G.V. and Kushnir N.A. Sposib soderzhaniya ingibitora amilazy [A method of producing an inhibitor of amylase]. Patent Ukraine, no. 35845, 2008.
7. Agromonov A. E. and Shabarov Yu. S. Laboratornye raboty v organicheskom praktikume [Laboratory works in the organic practice]. Moscow: Khimiya Publ., 1974. 376 p.
8. Perrin D.D., Armarego W.L.F., and Perrin D.R. Purification of Laboratory chemicals, Oxford: Pergamon Press, 1966. 181 p.
9. Hartree E.F. Determination of protein: A modification of the lowry method that gives a linear photometric response. Analytical Biochemistry. 1972, August, vol. 48, iss. 2, pp. 422-427.
10. Krusir G.V. and Kushnir N.A. Biochemical characterization an inhibitor of α-amylase from boroshenets of oats. Proceedings of Odessa National Academy of Food Technologies, 2008, iss. 34, vol. 1, pp. 252-257. (In Ukrainian).
11. Kornish-Bouden E. Osnovy fermentativnoy kinetiki [Fundamentals of enzyme kinetics]. Moscow: Mir Publ., 1979. 280 p.
12. Dzyuba N. Identification of complexforming amylase - glutin by infrared spectroscopy. Commodities and markets, 2015, no. 2 (20), pp. 33-40. (In Ukrainian).
13. Murray R.K., Granner D.K., Mayes P.A., Rodwell V.W. Harper’s Biochemistry. Norwalk, San Mateo, 1988. (Russ. ed.: Marri R., Grenner D., Meyes P., Roduell V. Biokhimiya cheloveka: in 2 t. Tom 2. Moscow: Mir Publ., 2004. 414 p.).