The activity and catalytic properties of glutathione peroxidase at liver toxic disturbance

Abstract

The aim of this study was investigation of the functioning of glutathione peroxidase (GPx, EC
1.11.1.9.) under the condition of impaired liver function and purification of the enzyme by chromatographic
methods for the further investigation of its kinetic properties. The blood serum of patients with alcoholic hepatitis, undergoing in-patient treatment was used in the experiment. The control group included apparently healthy individuals with normal complete blood count and comprehensive biochemical profile results. Experimental toxic hepatitis (ETH) was modelled by oral administration of carbon tetrachloride to male albino Wistar rats (Rattus norvegicus). The control group included healthy animals kept under standard vivarium conditions. The activity of GPx was measured spectrophotometrically at a wavelength of 340 nm – based on a coupled enzymatic reaction with glutathione reductase. The purification of glutathione peroxidase obtained from healthy rat liver and the liver of rats with toxic hepatitis was performed by homogenisation of the material, gel-filtration on Sephadex G-25, ion-exchange chromatography on DEAE-cellulose, concentration of the enzyme fractions inan Amicon ultrafiltration cell, and gel chromatography on Toyopearl HW-65. The purity of the obtained enzyme preparation was evaluated using the electrophoretic method. The total protein content of the samples was determined according to the Lowry method. The study demonstrated an increase in the activity of the pathology markers in the blood serum of patients with alcoholic hepatitis and rats with ETH. The activity of GPx in the blood serum of patients with alcoholic hepatitis and in liver homogenate of rats with ETH also changed as compared to the control level. As a result of the experiment, enzyme preparations were obtained from the livers of healthy rats and rats with ETH. They had a specific activity of 1.46 and 3.64 U/mg, and a yield of 7.2 and 8.0% respectively. These preparations were then used to study the kinetic properties of GPx. It was determined that during toxic hepatitis GPx is less resistant to prolonged exposure to high temperatures as compared to the enzyme from the liver of a healthy animal. In addition, during pathology the optimum temperature for enzyme activity and inactivation constants also increased. The obtained results
allow us to suggest that the change in the activity of the enzyme under impaired liver function can be
caused by conformational changes of the enzyme molecule - under conditions of the increased generation of free radicals.

References
1. Kalinin A.V., Farmateka, 2005, No 1(97), pp. 48-55.
2. Gorbenko M.V., Popova T.N., Shul'gin K.K., Popov S.S., Eksperimental'naia i klinicheskaia
farmakologiia, 2013, Vol. 76, No 10, pp. 12-15.
3. Andreeshcheva E.M., Popova T.N., Artyukhov V.G., Matasova L.V., Bulletin of experimental
biology and medicine, 2004, No 4, pp. 399-402.
4. Davis B.J., Jones R.G., Farmer G.R., Ann. N. Y. Acad. Sci., 1964, Vol. 121, pp. 404-427.
DOI: 10.1111/j.1749-6632.1964.tb14213.x
5. Shevchenko A., Wilm H., Vorm O., Anal. Chem., 1996, Vol. 68, pp. 850-858. DOI:10.1021/ac950914h
6. Lowry О.Н., Rosebrough N.J., Farr A.L., Randall R.J., J. Biol. Chem., 1951, Vol. 193, pp. 265-275.
7. Glants S., Mediko-biologicheskaya statistika, M., Praktika, 1999, 459 p.
8. Kim J.H., Park S.H., Nam S.W., Choi Y.H., Int. J. Mol. Sci., 2012, Vol. 13, pp. 5740-5750. DOI: 10.3390/ijms13055740
9. Popov S.S., Pashkov A.N., Popova T.N., Semenikhina A.V. et al., Eksperimental'naia i klinicheskaia farmakologiia, 2007, Vol. 70, No 1, pp. 48-51.
10.Popov S.S., Pashkov A.N., Popova T.N., Zoloedov V.I. et al., Bulletin of Experimental
Biology and Medicine, 2007, No 8, pp. 170-173.