PREPARATION OF HIGHLY ACTIVE HUMAN ERYTHROCYTE GLUTATHIONE S-TRANSFERASE P1-1 USING AFFINITY MEMBRANES, AND PROPERTIES OF THE PURIFIED ENZYME

Human glutathione S-transferase P1-1 (GSTP1-1) is an important enzyme in clinical diagnostics since its overexpression in solid tumours and lymphomas largely contributes to multiple drug resistance of cancer cells. Small amounts of the enzyme can also be isolated from erythrocytes. However, known isolation methods are disadvantageous and do not allow to obtain the purified enzyme in good yield and with high specific activity (≥ 100 U/mg protein). In order to elaborate more effective purification procedure for the erythrocyte GSTP1-1, we have for the first time synthesized and investigated glutathione-containing affinity membranes based on chemically modified cellulose paper. The membranes have been shown to successfully replace conventional glutathione-agarose affinity gels in the enzyme purification. To isolate and purify GSTP1-1 from erythrocyte hemolysate, a novel two-step method has been developed involving preliminary hemoglobin removal on a small anion exchange column and further affinity chromatography on the cartridge with glutathione-containing membranes. In terms of activity yield (76.5%), purification factor (23589-fold), and specific activity of the purified enzyme (104.5 U/mg), the method is notably superior to previously published procedures. Gel electrophoretic and MALDI-TOF mass spectrometric analyses reveal apparent homogeneity (≥ 95%) of the obtained preparation. Steady-state kinetic parameters have been determined for the purified GSTP1-1 in the conjugation reaction between glutathione and 1-chloro-2,4-dinitrobenzene at pH 6.5 and 25 °С: for the former substrate, K_m = 0.19 mМ, k_cat = 47.8 s^–1; for the latter, K_m = 0.68 mМ, k_cat = 54.3 s^–1. The results of the present work may be useful for screening new enzyme inhibitors with possible antitumour activity. The affinity membranes may also find application in isolating native and recombinant glutathione S-transferase (GST) isoforms from various sources, as well as fusion proteins with GST tag. 

1. Hayes J. D, Flanagan J. U., Jowsey I. R., “Glutathione transferases”, Annual Review of Pharmacology and Toxicology, 2005, vol. 45, pp. 51–88.

2. Laborde E., “Glutathione transferases as mediators of signaling pathways involved in cell proliferation and cell death”, Cell Death & Differentiation, 2010, vol. 17, no. 9, pp. 1373–1380.

3. Oakley A., “Glutathione transferases: a structural perspective”, Drug Metabolism Reviews, 2011, vol. 43, no. 2, pp. 138–151.

4. Townsend D. M., Tew K. D., “The role of glutathione-S-transferase in anti-cancer drug resistance”, Oncogene, 2003, vol. 22, no. 47, pp. 7369–7375.

5. Fabrini R., Bocedi A., Del Grosso E., Morici L., Federici G., Palleschi A., Ricci G., “Erythrocyte glutathione transfer-ase: a novel biomarker to check environmental pollution hazardous for humans”, Biochemical And Biophysical Research Communications, 2012, vol. 426, no. 1, pp. 71–75.

6. Schröder K. R., Hallier E., Peter H., Bolt H. M., “Dissociation of a new glutathione S-transferase activity in human erythrocytes”, Biochemical Pharmacology, 1992, vol. 43, no. 8, pp. 1671–1674.

7. Hirrell P. A., Collins M. F., Nimmo I. A., Strange R. C., “The human glutathione S-transferases. Studies on the kinetic, stability and inhibition characteristics of the erythrocyte enzyme”, Biochimica et Biophysica Acta, 1987, vol. 913, no. 1, pp. 92–96.

8. Awasthi Y. C., Singh S. V., “Purification and characterization of a new form of glutathione S-transferase from human erythrocytes”, Biochemical and Biophysical Research Communications, 1984, vol. 125, no. 3, pp. 1053–1060.

9. Howie A. F., Hayes J. D., Beckett G. J., “Purification of acidic glutathione S-transferases from human lung, placenta and erythrocyte and the development of a specific radioimmunoassay for their measurement”, Clinica Chimica Acta, 1988, vol. 177, no. 1, pp. 65–76.

10. Hamed R. R., Maharem T. M., Abdel-Meguid N., Sabry G. M., Abdalla A.-M., Guneidy R. A., “Purification and biochemical characterization of glutathione S-transferase from Down syndrome and normal children erythrocytes: a comparative study”, Research In Developmental Disabilities, 2011, vol. 32, no. 5, pp. 1470–1482.

11. Marcus C. J., Habig W. H., Jakoby W. B., “Glutathione transferase from human erythrocytes. Nonidentity with the enzymes from liver”, Archives of Biochemistry and Biophysics, 1978, vol. 188, no. 2, pp. 287–293.

12. Desideri A., Caccuri A. M., Polizio F., Bastoni R., Federici G., “Electron paramagnetic resonance identification of a highly reactive thiol group in the proximity of the catalytic site of human placenta glutathione transferase”, Journal of Biological Chemistry, 1991, vol. 266, no. 4, pp. 2063–2066.

13. Vander Jagt D. L., Wilson S. P., Heidrich J. E., “Purification and bilirubin binding properties of glutathione S-transferase from human placenta”, FEBS Letters, 1981, vol. 136, no. 2, pp. 319–321.

14. Battistoni A., Mazzetti A. P., Petruzzelli R., Muramatsu M., Federici G., Ricci G., Lo Bello M., “Cytoplasmic and periplasmic production of human placental glutathione transferase in Escherichia coli”, Protein expression and purification, 1995, vol. 6, no. 5, pp. 579–587.

15. Kolm R. H., Stenberg G., Widersten M., Mannervik B., “High-level bacterial expression of human glutathione trans-ferase P1-1 encoded by semisynthetic DNA”, Protein expression and purification, 1995, vol. 6, no. 3, pp. 265–271.

16. Zou H., Luo Q., Zhou D., “Affinity membrane chromatography for the analysis and purification of proteins”, Journal of Biochemical and Biophysical Methods, 2001, vol. 49, no. 1-3, pp. 199-240.

17. Gilevich S. N., Zavizion E. Yu., “Affinity membranes on the basis of chemically modified cellulose for selective solid-phase protein extraction”, Trudy XXIII Mezhdunarodnoi nauchno-tekhnicheskoi konferentsii “Khimicheskie reaktivy, reagenty i protsessy malotonnazhnoi khimii”, 27–29 oktiabria 2010 goda, Minsk [Proc. XXIII Int. Conf. “Chemical reactants, reagents and processes of low-tonnage chemistry”, October 27–29, 2010, Minsk], Belaruskaia navuka, Minsk, BY, 2011, pp. 305–319.

18. Gilevich S. N., Zavizion E. Yu., “Preparation and hemoglobin-binding properties of immobilized metal affinity membranes based on chemically modified chromatographic paper”, Sbornik dokladov IV Mezhdunarodnoi konferentsii “Khimiya, struktura i funktsiya biomolekul”, 17–19 oktiabria 2012 g., Belarus’, Minsk [Collection of reports IV International conference “Chemistry, structure and function of biomolecules”, 17–19 October 2012, Belarus, Minsk], Minsk, BY, 2012, pp. 46–47.

19. Forde G. M., “Preparation, analysis and use of an affinity adsorbent for the purification of GST fusion protein”, Methods in Molecular Biology, 2008, vol. 421, pp. 125–136.

20. Burton S. C., Harding D. R. K., “High-density ligand attachment to brominated allyl matrices and application to mixed mode chromatography of chymosin”, Journal of Chromatography A, 1997, vol. 775, no. 1–2, pp. 39–50.

21. Ke Y. M., Chen C. I., Kao P. M., Chen H. B., Huang H. C., Yao C. J., Liu Y. C., “Preparation of the immobilized metal affinity membrane with high amount of metal ions and protein adsorption efficiencies”, Process biochemistry, 2010, vol. 45, no. 4, pp. 500–506.

22. Scoble J. A., Scopes R. K., “Assay for determining the number of reactive groups on gels used in affinity chromatography and its application to the optimisation of the epichlorohydrin and divinylsulfone activation reactions”, Journal of Chromatography A, 1996, vol. 752, no. 1–2, pp. 67–76.

23. Friedman M., “Applications of the ninhydrin reaction for analysis of amino acids, peptides, and proteins to agricultural and biomedical sciences”, Journal of Agricultural and Food Chemistry, 2004, vol. 52, no. 3, pp. 385–406.

24. Tsuruga M., Matsuoka A., Hachimori A., Sugawara Y., Shikama K., “The molecular mechanism of autoxidation for human oxyhemoglobin”, Journal of Biological Chemistry, 1998, vol. 273, no. 15, pp. 8607–8615.

25. Habig W. H., Pabst M. J., Jakoby W. B., “Glutathione S-transferases. The first enzymatic step in mercapturic acid formation”, Journal of Biological Chemistry, 1974, vol. 249, no. 22, pp. 7130–7139.

26. Bisswanger H., Practical Enzymology, 2nd ed., Wiley-Blackwell, Weinheim, DE, 2011.

27. Laemmli U. K., “Cleavage of structural proteins during the assembly of the head of bacteriophage T4”, Nature, 1970, vol. 227, no. 5259, p. 680–685.

28. Bradford M. M., “A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding”, Analytical Biochemistry, 1976, vol. 72, no 1–2, pp. 248–254.

29. Credou J., Volland H., Danob J., Berthelot T., “A one-step and biocompatible cellulose functionalization for covalent antibody immobilization on immunoassay membranes”, Journal of Materials Chemistry B, 2013, vol. 1, no. 26, pp. 3277–3286.

30. Huang Y., Misquitta S., Blond S. Y., Adams E., Colman R. F., “Catalytically active monomer of glutathione S-transferase pi and key residues involved in the electrostatic interaction between subunits”, Journal of Biological Chemistry, 2008, vol. 283, no. 47, pp. 32880–32888.

31. Fabrini R., Bocedi A., Camerini S., Fusetti M., Ottaviani F., Passali F. M., Topazio D., Iavarone F., Francia I., Castagnola M., Ricci G., “Inactivation of human salivary glutathione transferase P1-1 by hypothiocyanite: a post-translational control system in search of a role”, PLoS One, 2014, vol. 9, no. 11, e112797, doi:10.1371/journal.pone.0112797.

32. Fabrini R., De Luca A., Stella L., Mei G., Orioni B., Ciccone S., Federici G., Lo Bello M., Ricci G., “Monomer-dimer equilibrium in glutathione transferases: a critical re-examination”, Biochemistry, 2009, vol. 48, no. 43, pp. 10473–10482.

33. Wheatley J. B., Hughes B., Bauer K., Schmidt D. E., “Study of chromatographic parameters for glutathione S-tran- sferases on an high-performance liquid chromatography affinity stationary phase”, Journal of Chromatography A, 1994, vol. 676, no. 1, pp. 81–90.

34. Chen L. H., Choi Y. S., Park Jung W., Kwon J., Wang R. S., Lee T., Ryu S. H., Park Joon W., “Effect of linker for immobilization of glutathione on BSA-assembled controlled pore glass beads”, Bulletin of the Korean Chemical Society, 2004, vol. 25, no. 9, pp. 1366–1370.

35. Mannervik B., Guthenberg C., “Glutathione Transferase (Human Placenta)”, Methods in Enzymology, 1981, vol. 77, pp. 231–235.

36. Thumser A. E., Ivanetich K. M., “Kinetic mechanism of human erythrocyte acidic isoenzyme ρ”, Biochimica et Biophysica Acta, 1993, vol. 1203, no. 1, pp. 115–120.

37. Strange R. C., Johnson P. H., Lawton A., Moult J. A., Tector M. J., Tyminski R. J., Cotton W., “Studies on the variability of glutathione S-transferase from human erythrocytes”, Clinica Chimica Acta, 1982, vol. 120, no. 2, pp. 251–260.

38. Forman H. J., Zhang H., Rinna A., “Glutathione: overview of its protective roles, measurement, and biosynthesis”, Molecular Aspects of Medicine, 2009, vol. 30, no 1–2, pp. 1–12.

39. Turk S., Erkmen G. K., Dalmizrak O., Ogus I. H., Ozer N., “Purification of glutathione S-transferase pi from erythrocytes and evaluation of the inhibitory effect of hypericin”, Protein Journal, 2015, vol. 34, no. 6, pp. 434–443.