Aim: Aim of the present study was to carry out the partial purification and biochemical characterization of glutathione S-transferase (GST) from the somatic tissue of ruminal amphistome parasite, Gastrothylax crumenifer (Gc) infecting Indian water buffalo (Bubalus bubalis).

Materials and Methods: The crude somatic homogenate of Gc was subjected to progressive ammonium sulfate precipitation followed by size exclusion chromatography in a Sephacryl S 100-HR column. The partially purified GST was assayed spectrophotometrically, and the corresponding enzyme activity was also recorded in polyacrylamide gel. GST isolated from the amphistome parasite was also exposed to variable changes in temperature and the pH gradient of the assay mixture.

Results: The precipitated amphistome GST molecules showed maximum activity in the sixth elution fraction. The GST subunit appeared as a single band in the reducing polyacrylamide gel electrophoresis with an apparent molecular weight of 26 kDa. The GST proteins were found to be fairly stable up to 37°C, beyond this the activity got heavily impaired. Further, the GST obtained showed a pH optima of 7.5.

Conclusion: Present findings showed that GST from Gc could be conveniently purified using gel filtration chromatography. The purified enzyme showed maximum stability and activity at 4°C.

Keywords: Bubalus bubalis, Gastrothylax crumenifer, glutathione S-transferase, purification, somatic tissue.

1. Toledo, R. and Fried, B. (2014) Advances in Experimental Medicine and Biology 766 (Digenetic Trematodes). Springer Publications, Springer Science+Business Media, New York. p365-392.

2. Saifullah, M.K., Ahmad, G. and Abidi, S.M.A. (2013) Immunodetection of coproantigens for the diagnosis of amphistomosis in naturally infected Indian Water Buffalo, Bubalus bubalis. Vet. Parasitol., 191: 66-72. [Crossref] [PubMed]

3. Chauhan, V.D., Patel, P.V., Hasnani, J.J., Pandya, S.S., Pandey, S., Pansuriya, D.V. and Choudhary, V. (2015) Study on hematological alterations induced by amphistomosis in buffaloes. Vet. World., 8(3): 417-420. [Crossref] [PubMed] [PMC]

4. Kumar, B., Maharana, B.R., Prasad, A., Joseph, J.P., Patel, B. and Patel, J.S. (2016) Seasonal incidence of parasitic diseases in bovines of south western Gujarat (Junagadh), India. J. Parasit. Dis., 40(4): 1342-1346. [Crossref] [PubMed] [PMC]

5. Dixit, A.K., Das, G. and Baghel, R.P.S. (2017) Gastrointestinal helminthosis: Prevalence and associated determinants in goats of Jabalpur, India. J. Parasit. Dis., 41(2): 414-416. [Crossref] [PubMed]

6. Sorathiya, L.M., Fulsoundar, A.B., Rao, T.K.S. and Kumar, N. (2017) Prevalence and risk factors for gastrointestinal parasitism in traditionally maintained goat flocks of South Gujarat. J. Parasit. Dis., 41(1): 137-141. [Crossref] [PubMed]

7. Sheehan, D., Meade, G., Foley, V.M. and Dowd, C.A. (2001) Structure, function and evolution of glutathione transferases: Implications for classification of non-mammalian members of an ancient enzyme superfamily. Biochem. J., 360: 1-16. [Crossref] [PubMed] [PMC]

8. Lu, J. and Holmgren, A. (2014) The thioredoxin antioxidant system. Free Radic. Biol. Med., 66: 75-87. [Crossref] [PubMed]

9. Mannervik, B. (1985) The isoenzymes of glutathione transferases. Adv. Enzymol. Relat. Areas Mol. Biol., 57: 357-417. [Crossref]

10. Callahan, H.L., Crouch, R.K. and James, E.R. (1988) Helminth anti-oxidant enzymes: A protective mechanism against host oxidants? Parasitol. Today, 4(8): 218-225. [Crossref]

11. Brophy, P.M. and Pritchard, D.I. (1992) Immunity to helminths: Ready to tip the biochemical balance? Parasitol. Today, 8(12): 419-422. [Crossref]

12. Brophy, P.M. and Barret, J. (2009) Glutathione-S-transferase in helminthes. Parasitology, 100: 345-349. [Crossref]

13. Brophy, P.M. and Pritchard, D.I. (1994) Parasitic helminth glutathione S-transferases: Update on their potential for immuno and chemotherapy. Exp. Parasitol., 79(1): 89-96. [Crossref] [PubMed]

14. Liebau, E., Wildenburg, G., Brophy, P.M., Walter, R.D. and Henkle, D.K. (1996) Biochemical analysis, gene structure and localization of the 24 kDa glutathione S-transferase from Onchocerca volvulus. Mol. Biochem. Parasitol., 80: 27-39. [Crossref]

15. Brophy, P.M., Ben-Smith, A., Brown, A., Behnke, J.M. and Pritchard, D.I. (1994) Glutathione S-transferase from the gastrointestinal nematode Heligmosomoides polygyrus and mammalian liver compared. Comp. Biochem. Physiol. B, 109(4): 585-592. [Crossref]

16. Barrett, J. (1998) Cytochrome P450 in parasitic protozoa and helminths. Comp. Biochem. Physiol. C, 121(1-3): 181-183. [Crossref]

17. Bourke, C.D., Nausch, N., Rujeni, N., Appleby, L.J., Trottein, F., Midzi, N., Mduluza, T. and Mutapi, F. (2014) Cytokine responses to the anti-schistosome vaccine candidate antigen glutathione-S-transferase vary with host age and are boosted by praziquantel treatment. PLoS Negl. Trop. Dis., 8(5): e2846. [Crossref]

18. Li, L.G., Wang, Z.Q., Liu, R.D., Yang, X., Liu, L.N., Sun, G.G., Jiang P., Zhang, X., Zhang, G.Y. and Cui, J. (2015) Trichinella spiralis: Low vaccine potential of glutathione S-transferase against infections in mice. Acta Trop., 146: 25-32. [Crossref]

19. Mbanefo, E.C., Kumagai, T., Kodama, Y., Kurosaki, T., Furushima-Shimogawara, R., Cherif, M.S., Mizukami, S., Kikuchi, M., Huy, N.T., Ohta, N., Sasaki, H. and Hirayama, K. (2015) Immunogenicity and anti-fecundity effect of nanoparticle coated glutathione S-transferase (SjGST) DNA vaccine against murine Schistosoma japonicum infection. Parasitol. Int., 64(4): 24-31. [Crossref] [PubMed]

20. Diemert, D.J., Freire, J., Valentene, V., Fraga, C.G., Taller, F., Grahek, S., Campbell, D., Amar, J., Maria, V.P., Martin, E., Maria, F.G. and Maria, E.B. (2017) Safety and immunogenicity of the Na-GST-1 hookworm vaccine in Brazilian and American adults. PLoS Negl. Trop. Dis., 11(5): e0005574. [Crossref]

21. Bradford, M.M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein dye binding. Anal. Biochem., 72: 248-254. [Crossref]

22. Spector, T. (1978) Refinement of the coommassie blue method of protein quantitation. Anal. Biochem., 86(1): 142-146. [Crossref]

23. Peterson, E.A. and Sober, H.A. (1962) In: Colowick, S.P. and Kaplan, N.O., editor. Methods in Enzymology. 3rd ed., Vol. 5. Academic Press Inc., New York.

24. Habig, W.H., Pabst, M.J. and Jakoky, W.B. (1974) The first enzymatic step in mercapturic acid formation. J. Biol. Chem., 249(22): 7130-7139.

25. Laemmli, U.K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 227(5259): 680-685. [Crossref]

26. Ricci, G.M., Lo Bello, A.M., Caccuri, F., Galiazzo, F. and Federici, G. (1984) Detection of glutathione transferase activity on polyacrylamide gels. Anal. Biochem. 143(2): 226-230. [Crossref]

27. Precious, W.Y. and Barrett, J. (1989) Xenobiotic metabolism in helminths. Parasitol. Today, 5(5): 156-160. [Crossref]

28. Marchewka, Z., Piwowar, A., Ruzik, S. and Dlugosz, A. (2017) Glutathione S-transferases class Pi and Mi and their significance in oncology. Postepy Hig. Med. Dosw., 71: 541-550. [Crossref]

29. Mateen, S., Moin, S., Khan, A.Q., Zafar, A. and Fatima, N. (2016) Increased reactive oxygen species formation and oxidative stress in rheumatoid arthritis. PLoS One, 11(4): e0152925. [Crossref]

30. Ragab, S.M., Badr, E.A. and Ibrahim, A.S. (2016) Evaluation of glutathione-S-transferase P1 polymorphism and its relation to bone mineral density in Egyptian children and adolescents with beta-thalassemia major. Mediterr. J. Hematol. Infect. Dis., 8(1): e2016004. [Crossref]

31. Shu, K.H., Wang, C.H., Wu, C.H., Huang, T.M., Wu, P.C., Lai, C.H., Tseng, L.J., Tsai, P.R., Connolly, R. and Wu, V.C. (2016) Urinary pi- glutathione S-transferase predicts advanced acute kidney injury following cardiovascular surgery. Nat. Sci. Rep., 6: 26335. [Crossref] [PubMed] [PMC]

32. Balatonyi, B., Gasz, B., Kovacs, V., Lantos, J., Jancso, G., Marczin, N. and Roth, E. (2013) The role of the inhibition of glutathione-S-transferase in the protective mechanisms of ischemic post conditioning. Can. J. Physiol. Pharmacol., 91(8): 625-632. [Crossref] [PubMed]

33. Phulukdaree, A., Khan, S., Moodley, D. and Chuturgoon, A.A. (2012) GST polymorphisms and early-onset coronary artery disease in young South African Indians. S. Afr. Med. J. 102(7): 627-630. [Crossref]

34. de Mendonca, E., Salazar-Alcala, E. and Fernandez-Mestre, M. (2014) Role of genes GSTM1, GSTT1, and MnSOD in the development of late-onset Alzheimer disease and their relationship with APOE*4. Neurologia, 31(8): 535-542. [Crossref] [PubMed]

35. Kim, J.G., Ahn, C.S., Kim, S.H., Bae, Y.A., Kwon, N.Y., Kang, I., Yang, H.J., Sohn, W.M. and Kong, Y. (2016) Clonorchis sinensis omega-class glutathione transferases play major roles in the protection of the reproductive system during maturation and the response to oxidative stress. Parasit. Vectors, 9(1): 337. [Crossref]

36. Srinivasan, L., Mathew, N., Karuna, T. and Muthuswamy, K. (2011) Biochemical studies on glutathione S-transferase from the bovine filarial worm Setaria digitata. Parasitol. Res., 109(1): 213-219. [Crossref] [PubMed]

37. O'Leary, K.A., Hathaway, K.M. and Tracy, J.W. (1992) Schistosoma mansoni: single-step purification and characterization of glutathione S-transferase isoenzyme 4. Exp. Parasitol., 75(1): 47-55. [Crossref]

38. Smith, D.B. and Johnson, K.S. (1988) Single-step purification of polypeptides expressed in Escherichia coli as fusions with glutathione S-transferase. Gene, 67(1): 31-40. [Crossref]

39. Panaccio, M., Wilson, L.R., Crameri, S.L., Wijffels, G.L. and Spithill, T.W. (1992) Molecular characterization of cDNA sequences encoding glutathione-S-transferases of Fasciola hepatica. Exp. Parasitol., 74(2): 232-237. [Crossref]

40. Asojo, O.A. and Ceccarelli, C. (2014) Structure of glutathione S-transferase 1 from the major human hookworm parasite Necator americanus (Na-GST-1) in complex with glutathione. Acta Crystallogr. F. Struct. Biol. Commun., 70: 1162-1166. [Crossref] [PubMed] [PMC]

41. Fleitas, A.L., Randall, L.M., Moller, M.N. and Denicola, A. (2016) Purification of a recombinant glutathione transferase from the causative agent of hydatidosis, Echinococcus granulosus. Biochem. Mol. Biol. Educ., 44(1): 28-33. [Crossref] [PubMed]

42. Plancarte, A., Rendon, J.L. and Landa, A. (2004) Purification, characterization and kinetic properties of the Taenia solium glutathione S-transferase isoform 26.5 kDa. Parasitol. Res., 93(2): 137-144. [Crossref] [PubMed]

43. Gupta, S., Singh, A., Yadav, M., Singh, K. and Rathaur, S. (2007) MALDI mass sequencing and characterization of filarial glutathione-S-transferase. Biochem. Biophys. Res. Commun., 356(2): 381-385. [Crossref] [PubMed]

44. Ahmad, R., Srivastava, A.K. and Walter, R.D. (2008) Purification and biochemical characterization of cytosolic glutathione S-transferase from filarial worms Setaria cervi. Comp. Biochem. Physiol., 151(3): 237-245. [Crossref] [PubMed]

45. Walker, J., Crowley, P., Moreman, A.D. and Barrett, J. (1993) Biochemical properties of cloned glutathione S-transferases from Schistosoma mansoni and Schistosoma japonicum. Mol. Biochem. Parasitol., 61(2): 255-264. [Crossref]

46. Salvatore, L., Wijffels, G., Sexton, J.L., Panaccio, M., Mailer, S., McCauley, I. and Spithill, T.W. (1995) Biochemical analysis of recombinant glutathione-S-transferase of Fasciola hepatica. Mol. Biochem. Parasitol., 69(2): 281-288. [Crossref]

47. Torres-Rivera, A. and Landa, A. (2008) Cooperative kinetics of the recombinant glutathione transferase of Taeniasolium and characterization of the enzyme. Arch. Biochem. Biophys. 477(2): 372-378. [Crossref] [PubMed]