Molecular and Cellular Biochemistry

, Volume 303, Issue 1–2, pp 45–51 | Cite as

Purification and kinetic properties of glutathione reductase from bovine liver



Glutathione reductase (GR, NADPH: oxidized glutathione oxidoreductase, EC catalyzes the reduction of oxidized glutathione (GSSG) to reduced glutathione (GSH) using NADPH as reducing cofactor. The aim of the present work was to purify and characterize GR from bovine liver. GR was purified using 2′, 5′ ADP-Sepharose 4B and DEAE-Sepharose Fast Flow columns. The enzyme has been purified 5456-fold and with a yield of 38.4%. The molecular and catalytic properties of bovine liver GR have been studied. Optimum temperature and pH was found to be 50°C and 7, respectively. The activation energy of the reaction catalyzed by the enzyme was 9.065 kcal/mole. The molecular weight of the enzyme was found to be 55 kDa by SDS-PAGE. Kinetic characterization of bovine liver GR was also investigated, KmNADPH 0.063 ± 0.008 mM and KmGSSG 0.154 ± 0.015 mM were determined. It is accepted that parallel lines observed in these double reciprocal plots obeys Ping Pong mechanism and we have showed this in our steady state study. According to our results of statistical analysis, the Ping Pong mechanism is a suitable model since the loss function is less than the other mechanisms. However, competitive inhibition by a product could be accepted in sequential mechanisms but not in a Ping Pong mechanism. In this study, kinetic data are consistent with a branching reaction mechanism previously proposed for GR from other sources by other studies.


Glutathione reductase Purification Kinetics Liver Branched and Ping Pong mechanism 



This work is a part of the project (02 G085) supported by Hacettepe University Scientific Research Unit


  1. 1.
    Williams CH Jr (1976) Flavin-containing dehydrogenases. In: Boyer PD (ed) The enzymes, vol 13. Academic Press, Inc., New York, pp 90–173Google Scholar
  2. 2.
    Schirmer RH, Krauth-Siegel RL, Schulz GE (1939) Glutathione reductase. In: Dolphin D, Poulson R, Avramovic O (eds) Glutathione: chemical, biochemical and medical aspects, Part A. Wiley, New York, pp 553–596Google Scholar
  3. 3.
    Bashir A, Perham RN, Scrutton NS, Berry A (1995) Altering kinetic mechanism and enzyme stability by mutagenesis of the dimer interface of glutathione reductase. Biochem J 312:527–533PubMedGoogle Scholar
  4. 4.
    Scott E, Duncan IW, Eksrand V (1963) Purification and properties of glutathione reductase of human erytrocytes. J Biol Chem 238:3928–3933PubMedGoogle Scholar
  5. 5.
    Carlberg I, Mannervik B (1975) Purification and characterization of the flavoenzyme glutathione reductase from rat liver. J Biol Chem 250:5475–5480PubMedGoogle Scholar
  6. 6.
    Gutterer JM, Dringen R, Hirrlinger J, Hamprecht B (1999) Purification of glutathione reductase from bovine brain, generation of an antiserum, and immunocytochemical localization of the enzyme in neural cells. J Neurochem 73:1422–1430PubMedCrossRefGoogle Scholar
  7. 7.
    Ng MC, Shichi H (1986) Purification and properties of glutathione reductases from bovine ciliary body. Exp Eye Res 43:477–489PubMedCrossRefGoogle Scholar
  8. 8.
    Carlberg I, Mannervik B (1981) Purification and characterization of glutathione reductase from calf liver. An improved procedure for affinity chromatography on 2′, 5′-ADP-Sepharose 4B. Anal Biochem 116:531–536PubMedCrossRefGoogle Scholar
  9. 9.
    Le Trang N, Bhargava KK, Cerami A (1983) Purification of glutathione reductase from gerbil liver in two steps. Anal Biochem 133:94–99PubMedCrossRefGoogle Scholar
  10. 10.
    Willmore WG, Storey KB (2006) Purification and properties of glutathione reductase from liver of the anoxia-tolerant turtle, Trachemys scripta elegans. Mol Cell Biochem 297:139–149PubMedCrossRefGoogle Scholar
  11. 11.
    Libreros-Minotta CA, Pardo JP, Mendoza-Hernandez G, Rendon JL (1992) Purification and characterization of glutathione reductase from Rhodospirillum rubrum. Arch Biochem Biophys 298:247–253PubMedCrossRefGoogle Scholar
  12. 12.
    McCallum MJ, Barrett J (1995) The purification and properties of glutathione reductase from the cestode Moniezia expansa. Int J Biochem Cell Biol 27:393–401PubMedCrossRefGoogle Scholar
  13. 13.
    Serrano A, Rivas J, Losada M (1984) Purification and properties of glutathione reductase from the Cyanobacterium anabaena sp. strain 7119. J Bacteriol 158:17–324Google Scholar
  14. 14.
    Bohme CC, Arscott LD, Becker K, Schirmer RH, Williams CH Jr (2000) Kinetic characterization of glutathione reductase from the malarial parasite Plasmodium falciparum. Comparison with the human enzyme. J Biol Chem 275:37317–37323PubMedCrossRefGoogle Scholar
  15. 15.
    Romero-Puertas MC, Corpas FJ, Sandalio LM, Leterrier M, Rodriguez-Serrano M, Del Rio LA, Palma JM (2006) Glutathione reductase from pea leaves: response to abiotic stress and characterization of the peroxisomal isozyme. New Phytol 170:43–52PubMedCrossRefGoogle Scholar
  16. 16.
    Garcia-Alfonso C, Martinez-Galisteo E, Llobell A, Barcena JA, Lopez-Barea J (1993) Horse-liver glutathione reductase: purification and characterization. Int J Biochem 25:61–68PubMedCrossRefGoogle Scholar
  17. 17.
    Madamanchi NR, Anderson JV, Alscher RG, Cramer CL, Hess JL (1992) Purification of multiple forms of glutathione reductase from pea (Pisum sativum L.) seedlings and enzyme levels in ozone-fumigated pea leaves. Plant Physiol 100:138–145PubMedCrossRefGoogle Scholar
  18. 18.
    Massey V, Williams CH Jr (1965) On the reaction mechanism of yeast glutathione reductase. J Biol Chem 240:4470–4480PubMedGoogle Scholar
  19. 19.
    Staal GE, Veeger C (1969) The reaction mechanism of glutathione reductase from human erythrocytes. Biochim Biophys Acta 185:49–62PubMedGoogle Scholar
  20. 20.
    Mannervik B (1973) A branching reaction mechanism of glutathione reductase. Biochem Biophys Res Commun 53:1151–1158PubMedCrossRefGoogle Scholar
  21. 21.
    Lopez-Barea J, Lee CY (1979) Mouse-liver glutathione reductase. Purification, kinetics, and regulation. Eur J Biochem 98:487–499PubMedCrossRefGoogle Scholar
  22. 22.
    Öğüş H, Özer N (1999) On the kinetics of human erythrocyte glutathione disulfide reductase: does the enzyme really play ‘Ping-Pong’? Turk J Biol 23:143–151Google Scholar
  23. 23.
    Montero S, Arriaga de D, Busto F, Soler F (1990) A study of the kinetic mechanism followed by glutathione reductase from mycelium of Phycomyces blakesleeanus. Arch Biochem Biophys 278:52–59PubMedCrossRefGoogle Scholar
  24. 24.
    Acan NL, Tezcan EF (1989) Sheep brain glutathione reductase: Purification and general properties. FEBS Lett 250:72–74PubMedCrossRefGoogle Scholar
  25. 25.
    Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein dye binding. Anal Biochem 72:248–254PubMedCrossRefGoogle Scholar
  26. 26.
    Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227(5259):680–685PubMedCrossRefGoogle Scholar
  27. 27.
    Merril H, Goldman D, Sedman SA, Ebert MH (1981) Ultrasensitive stain for proteins in polyacrylamide gels shows regional variation in CSF proteins. Science 211:1437–1438PubMedCrossRefGoogle Scholar
  28. 28.
    Dringen R, Gutterer JM (2002) Glutathione reductase from bovine brain. Methods Enzymol 348:281–288PubMedCrossRefGoogle Scholar
  29. 29.
    Ogus H, Özer N (1991) Human jejunal glutathione reductase: purification and evaluation of the NADPH- and glutathione-induced changes in redox state. Biochem Med Metab Biol 45(1):65–73PubMedCrossRefGoogle Scholar
  30. 30.
    Lopez-Barea J (1981) Mouse-liver glutathione reductase: inactivation by NADPH or two allelic variants. Rev Esp Fisiol 37(3):249–254PubMedGoogle Scholar
  31. 31.
    Erat M, Sakiroglu H, Ciftci M (2003) Purification and characterization of glutathione reductase from bovine erythrocytes. Prep Biochem Biotechnol 33(4):283–300PubMedCrossRefGoogle Scholar
  32. 32.
    Segel IH (1975) Enzyme Kinetics, 3rd edn. (Chapter 9 & 11). Wiley, Toronto, 273 ppGoogle Scholar
  33. 33.
    Icen A (1967) Glutathione reductase of human eyrtrocytes, purification and properties. Scand J Clin Lab Invest Suppl 96:1–67PubMedGoogle Scholar
  34. 34.
    Ramos-Martinez JI, Rodriguez Torres AM (1985) Glutathione reductase of mantle tissue from sea mussel Mytilus edulis Purification and characterization of two seasonal enzymatic forms. Comp Biochem Physiol B 80:355–360CrossRefGoogle Scholar
  35. 35.
    Shigeoka S, Onishi T, Nakano Y, Kitaoka S (1987) Characterization and physiological function of glutathione reductase in Euglena gracilis. Biochem J 242:511–515PubMedGoogle Scholar
  36. 36.
    De Lamotte F, Vianey-Liaud N, Duviau MP, Kobrehel K (2000) Glutathione reductase in wheat grain. 1. Isolation and characterization. J Agric Food Chem 48:4978–4983PubMedCrossRefGoogle Scholar
  37. 37.
    Ulusu NN, Tandogan B (2006) Purification and kinetics of sheep kidney cortex glucose-6-phosphate dehydrogenase. Comp Biochem Physiol B Biochem Mol Biol 143(2):249–255PubMedCrossRefGoogle Scholar
  38. 38.
    Patel MP, Marcinkeviciene J, Blanchard JS (1998) Enterococcus faecalis glutathione reductase: purification, characterization and expression under normal and hyperbaric O2 conditions. FEMS Microbiol Lett 166(1):155–163PubMedCrossRefGoogle Scholar
  39. 39.
    Rescigno M, Perham RN (1994) Structure of the NADPH-binding motif of glutathione reductase: efficiency determined by evolution. Biochemistry 33(19):5721–5727PubMedCrossRefGoogle Scholar
  40. 40.
    Rakauskiene GA, Cenas NK, Kulys JJ (1989) A ‘branched’ mechanism of the reverse reaction of yeast glutathione reductase. An estimation of the enzyme standard potential values from the steady-state kinetics data. FEBS Lett 243(1):33–36PubMedCrossRefGoogle Scholar
  41. 41.
    Serafini MT, Romeu A (1989) Steady-state kinetic studies of glutathione reductase. Rev Esp Fisiol 45(2):199–202PubMedGoogle Scholar
  42. 42.
    Chung YC, Hurlbert RE (1975) Purification and properties of the glutathione reductase of chromatium vinosum. J Bacteriol 123:203–211PubMedGoogle Scholar
  43. 43.
    Cleland WW (1963) The kinetics of enzyme-catalyzed reactions with two or more substrates or products. III. Prediction of initial velocity and inhibition patterns by inspection. Biochim Biophys Acta 67:188–96PubMedCrossRefGoogle Scholar

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© Springer Science+Business Media, LLC 2007

Authors and Affiliations

  1. 1.Department of Biochemistry, Faculty of MedicineHacettepe UniversityAnkaraTurkey

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