Fluorescence quenching and measurement of glutathione in fresh vegetables

  • Yahong Chen
  • Quanzhong Wang
  • Lei Liu
  • Fengshou Tian
Original Paper


A new method for the determination of glutathione was established based on the principle of fluorescence quenching. The reaction mechanism has been studied by the measurement of fluorescence lifetime and based on the Stern–Volmer plot. The binding constant, K = 9.86 × 105 J mol−1 and the number of binding sites n = 1.12 were obtained against this reaction. The thermodynamic parameters were estimated. The data, ΔG = −34.19 KJ mol−1, ΔH = −182.2 KJ mol−1, and ΔS = −496.8 J K−1 mol−1 showed that the reaction was spontaneous and exothermic. The calibration curve was found to be linear between the fluorescence quenching (F 0 /F) and the concentration of glutathione with the range of 3.0 × 10−5–3.6 × 10−3 g/L. The detection limit was 4.5 μg/L and the relative standard derivative was 3.32% for 11 replicate determination of 6.0 × 10−4 g/L glutathione. This method can be used for the determination of glutathione in fresh vegetables with satisfactory results.


Fluorescence quenching Glutathione Enzyme-catalyzed 



This work was sponsored by the Natural Science Foundation of Henan Province of China (No. 172102310626) and supported by the Natural Science Foundation of Education Department of Henan Province of China (No. 16B416001).


  1. 1.
    P. Monostori, G. Wittmann, E. Karg, S. Turi, Deternination of glutathione and glutathione disulfide in biological sample: an in-depth review. J. Chromatogr. B 877, 3331–3346 (2009)CrossRefGoogle Scholar
  2. 2.
    K.C. Sekhar, R. Syed, M. Golla, Novel heteroaryl phosphonicdiamides PTPs inhibitors as anti-hyperglycemic agents. DARU J. Pharm. Sci. 22, 76 (2014)CrossRefGoogle Scholar
  3. 3.
    I. Rahaman, A. Kode, S.K. Biswas, Assay for quantitative determination of glutathione and glutathione disulfide levels using. Nat. Protoc. 1, 3159–3165 (2006)CrossRefGoogle Scholar
  4. 4.
    A. Musenga, R. Mandrioli, P. Bonifazi, Sensitive and selective determination of glutathioine in probiotic bacteria by capillary electrophotesis-laser induced fluotescence. Anal. Bioanal. Chem. 387, 917–924 (2007)CrossRefGoogle Scholar
  5. 5.
    D. Giustarini, P. Fanti, E. Matteucci, R. Rossi, Micro-method for the determination of glutathione in human blood. J. Chromatogr. B 964, 191–194 (2014)CrossRefGoogle Scholar
  6. 6.
    Y.H. Chen, F.S. Tian, R. Jiang, Spectrophotometric determination of glutathione based inhibitory effect on hemoglobin. Asian J. Chem. 22, 6007–6012 (2010)Google Scholar
  7. 7.
    J.P. Rafael, V. Antonio, S. Manuel, P.B. Dolores, Automatic kinetic method for the determination of reduced glutathione in blood. Anal. Chim. Acta 269, 273–279 (1992)CrossRefGoogle Scholar
  8. 8.
    Y.H. Chen, R.X. Cai, Highly sensitive fluorimetic determination of glutathione based inhibitory effect on multienzyme redox system. Spectrochim. Acta Part A 61, 3051–3055 (2005)CrossRefGoogle Scholar
  9. 9.
    J.C. Harfield, C. Batchelor-McAuley, R.G. Compton, Electrochemical determination of glutathione: a review. Analyst. 137, 2285–2296 (2012)CrossRefGoogle Scholar
  10. 10.
    J.G. Wang, H. Lü, Q.H. Sun, Method of electrochemical determination of glutathione. Acta Chim. Sin. 67, 415–419 (2009)Google Scholar
  11. 11.
    J.B. Raoof, R. Ojani, M. Kolbadinezhad, Voltammetric sensor for glutathione determination based on ferrocene-modified carbon paste electrode. J. Solid State Electrochem. 13, 1411–1416 (2009)CrossRefGoogle Scholar
  12. 12.
    X.C. Guo, P. Xie, J. Chen, X. Tuo, X.W. Deng, S.C. Li, D.Z. Yu, C. Zeng, Simultaneous quantitative determination of microcystin-LR and its glutathione metabolites in rat liver by liquid chromatography–tandem mass spectrometry. J. Chromatogr. B 963, 54–61 (2014)CrossRefGoogle Scholar
  13. 13.
    M. Dai, P. Xie, G.D. Liang, Simultaneous determination of microcystin-LR and its glutathione conjugate in fish tissues by liquid chromatography-tandem mass spectrometry. J. Chromatogr. B 862, 43–50 (2008)CrossRefGoogle Scholar
  14. 14.
    L.Y. Zhang, M.X. Sun, Fast determination of glutathione by capillary electrophoresis with fluorescence detection using β-cyclodextrin as modifier. J. Chromatogr. B 877, 4051–4054 (2009)CrossRefGoogle Scholar
  15. 15.
    W. Wang, H. Xin, H.L. Shao, W.R. Jin, Determination of glutathione in single human hepatocarcinoma cells by capillary electrophoresis with electrochemical detection. J. Chromatogr. B 889, 425–429 (2003)CrossRefGoogle Scholar
  16. 16.
    E.H. Hansen, Flow-injection enzymatic assays. Anal. Chim. Acta 216, 257–273 (1989)CrossRefGoogle Scholar
  17. 17.
    M. Sono, M.P. Roach, E.D. Coulter, Heme-containing oxygenases. Chem. Rev. 96, 2841–2887 (1996)CrossRefGoogle Scholar
  18. 18.
    Y. Saito, M. Mifune, S.J. Nakashima, Determination of hydrogen peroxide with N,N-diethylaniline and 4-aminoantipyrine by use of an anion-exchange resin modified with managanese tetrakis(sulfopheny) porphine as a substitute for peroxidase. Talanta 34, 667–669 (1987)CrossRefGoogle Scholar
  19. 19.
    Y.X. Ci, F. Wang, Mimesis of peroxidase by Mn-TMPyPin in the catalytic fluorescence peaction of the homovanillic acid-hydrogen peroxide system. Mikrochim. Acta 100, 63–68 (1990)CrossRefGoogle Scholar
  20. 20.
    Q.Z. Zhu, Q.G. Li, J.Z. Lu, Application of thiamine as afluorogenic substrate in the determination of hydrogen peroxidebased on the catalytic effect of hemin. Anal. Lett. 29, 1729–1740 (1996)CrossRefGoogle Scholar
  21. 21.
    L.Y. Mao, M. Zhu, H.X. Shen, Study on immobilized supramolecular inclusion compled of iron-porphyrin as an analogue for peroxide proteinase. Chem. J. Chin. Univ. 19, 442–445 (1998)Google Scholar
  22. 22.
    G.B. Jameson, F.S. Molinaro, J.I. Brauman, Models for the active site of oxygen-binding hemoproteins. Dioxygen binding properties and the structures of (2-methylimidazole)- meso-tetra(.alpha.,.alpha.,.alpha.,.alpha.-o-pivalamidophenyl)porphyrinatoiron(II)-ethanol and its dioxygen adduct. J. Am. Chem. Soc. 102, 3224–3237 (1980)CrossRefGoogle Scholar
  23. 23.
    T. Brittain, Molecular aspects of embryonic hemoglobin function. Mol. Aspects. Med. 23, 293–342 (2002)CrossRefGoogle Scholar
  24. 24.
    T.M. Larsen, T.C. Mueser, L.J. Parkhurst, Use of dual wavelength spectrophotometry and continuous enzymatic depletion of oxygen for determination of the oxygen binding constants of hemoglobin. Anal. Biochem. 197, 231–246 (1991)CrossRefGoogle Scholar
  25. 25.
    K. Zhang, R. Cai, D. Chen, Determination of hemoglobin based on its enzymatic activity for the oxidation of o-phenylenediamine with hydrogen peroxide. Anal. Chim. Acta 413, 109–113 (2000)CrossRefGoogle Scholar
  26. 26.
    Y. Chen, F. Tian, G. Zhang, High-sensitivity spectrofluorimetric determination of tiopronin based on inhibition of hemoglobin. Luminescence 26, 477–480 (2011)CrossRefGoogle Scholar
  27. 27.
    J.R. Lakowicz, Principles of Fluorescence Spectroscopy, 2nd edn. (Plenum Press, New York, 1986), p. 264Google Scholar
  28. 28.
    G.Z. Chen, X.Z. Huang, Z.Z. Zheng, Method of Fluorescence Analysis, 2nd edn. (Science Press, Beijing, 1991), pp. 115–116Google Scholar
  29. 29.
    M. Alain, B. Michel, D. Michel, How to illustrate ligand-protein binding in a class experiment: an elementary fluorescent assay. J. Chem. Educ. 63, 365–366 (1986)CrossRefGoogle Scholar
  30. 30.
    Q.Y. Xing, R.Q. Xu, Z. Zhou, The Fundament of Organic Chemistry, 2nd edn (Science Press, Beijing, 1993), p. 454Google Scholar
  31. 31.
    M. Zhang, Q.L. Lv, N.N. Yue, H.Y. Wang, Study of fluorescence quenching mechanism between quercetin and tyrosine-H2O2-enzyme catalyzed product. Spectrochim. Acta A 72, 572–576 (2009)CrossRefGoogle Scholar
  32. 32.
    P.D. Ross, S. Subramanian, Thermodynamics of protein association reactions: forces contributing to stability. Biochemistry 20, 3096–3102 (1981)CrossRefGoogle Scholar
  33. 33.
    R. Gotti, V. Andrisano, V. Cavrini, A. Bongini, Determination of glutathione in pharmaceuticals and cosmetics by HPLC with UV and fluorescence detection. Chromatography 39, 23–28 (1994)CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2017

Authors and Affiliations

  • Yahong Chen
    • 1
    • 2
  • Quanzhong Wang
    • 1
  • Lei Liu
    • 1
  • Fengshou Tian
    • 1
    • 2
  1. 1.Department of ChemistryZhoukou Normal UniversityZhoukouChina
  2. 2.Graduate Institute of Pharamaceutical ChemistryZhoukou Normal UniversityZhoukouChina

Personalised recommendations