Skip to main content
SpringerLink
Log in

Electron spin resonance study on the formation of ascorbate free radical from ascorbate: The effect of dehydroascorbic acid and ferricyanide

  • Published:
Protoplasma Aims and scope Submit manuscript

Summary

Ascorbate free radical is considered to be a substrate for a plasma membrane redox system in eukaryotic cells. Moreover, it might be involved in stimulation of cell proliferation. Ascorbate free radical can be generated by autoxidation of the ascorbate dianion, by transition metal-dependent oxidation of ascorbate, or by an equilibrium reaction of ascorbate with dehydroascorbic acid. In this study, we investigated the formation of ascorbate free radical, at physiological pH, in mixtures of ascorbate and dehydroascorbic acid by electron spin resonance spectroscopy. It was found that at ascorbate concentrations lower than 2.5 mM, ascorbate-free radical formation was not dependent on the presence of dehydroascorbic acid. Removal of metal ions by treatment with Chelex 100 showed that autoxidation under these conditions was less than 20%. Therefore, it is concluded that at low ascorbate concentrations generation of ascorbate free radical mainly proceeds through metal-ion-dependent reactions. When ascorbate was present at concentrations higher than 2.5 mM, the presence of dehydroascorbic acid increased the ascorbate free-radical signal intensity. This indicates that under these conditions ascorbate free radical is formed by a disproportionation reaction between ascorbate and dehydroascorbic acid, having aK equil of 6 × 10−17 M. Finally, it was found that the presence of excess ferricyanide completely abolished ascorbate free-radical signals, and that the reaction between ascorbate and ferricyanide yields dehydroascorbic acid. We conclude that, for studies under physiological conditions, ascorbate free-radical concentrations cannot be calculated from the disproportionation reaction, but should be determined experimentally.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

Abbreviations

AFR:

ascorbate free radical

DHA:

dehydroascorbic acid

EDTA:

ethylenediaminetetraacetic acid

DTPA:

diethylenetri-aminepentaacetic acid

TEMPO:

2,2,6,6-tetramethylpiperidinoxy

References

  • Alcain FJ, Buron MI, Rodriguez-Aguilera JC, Villalba JM, Navas P (1990) Ascorbate free radical stimulates the growth of a human promyelocytic leukemia cell line. Cancer Res 50: 5887–5891

    Google Scholar 

  • Beyer RE (1994) The role of ascorbate in antioxidant protection of biomembranes: interaction with vitamin E and coenzyme Q. J Bioenerg Biomembr 26: 349–358

    Google Scholar 

  • Biaglow JE, Manevich Y, Uckun F, Held KD (1997) Quantitation of hydroxyl radicals produced by radiation and copper-linked oxidation of ascorbate by 2-deoxy-D-ribose method. Free Radie Biol Med 22: 1129–1138

    Google Scholar 

  • Bielski BHJ (1982) Chemistry of ascorbic acid radicals. In: Scib PA, Tolbert BM (eds) Ascorbic acid: chemistry, metabolism, and uses. American Chemical Society, Washington, DC, pp 81–100

    Google Scholar 

  • Bode AM, Cunningham L, Rose RC (1990) Spontaneous decay of oxidized ascorbic acid (dehydro-L-ascorbic acid) evaluated by high-pressure liquid chromatography. Clin Chem 36: 1807–1809

    Google Scholar 

  • Buettner GR (1990) Ascorbate oxidation: UV absorbance of ascorbate and ESR spectroscopy of the ascorbyl radical as assays for iron. Free Radie Res Commun 10: 5–9

    Google Scholar 

  • — (1988) In the absence of catalytic metals ascorbate does not autoxidize at pH 7: ascorbate as a test for catalytic metals. J Biochem Biophys Methods 16: 27–40

    Google Scholar 

  • — (1993) The pecking order of free radicals and antioxidants: lipid peroxidation, α-tocopherol, and ascorbate. Arch Biochem Biophys 300: 535–543

    Google Scholar 

  • —, Jurkiewicz BA (1993) Ascorbate free radical as a marker of oxidative stress: an EPR study. Free Radie Biol Med 14: 49–55

    Google Scholar 

  • —, Oberley LW, Leuthauser SW (1978) The effect of iron on the distribution of superoxide and hydroxyl radicals as seen by spin trapping and on the superoxide dismutase assay. Photochem Photobiol 28: 693–695

    Google Scholar 

  • Del Castillo-Olivares A, Esteban del Valle A, Marquez J, Nunez de Castro I, Medina MA (1995) Ehrlich cell plasma membrane redox system is modulated through signal transduction pathways involving cGMP and Ca2+ as second messengers. J Bioenerg Biomembr 27: 605–611

    Google Scholar 

  • — — — — — (1996) Effects of protein kinase C and phosphoprotein phosphatase modulators on Ehrlich cell plasma membrane redox system activity. Biochim Biophys Acta 1313: 157–160

    Google Scholar 

  • Iyanagi T, Yamazaki I, Anan KF (1985) One-electron oxidationreduction properties of ascorbic acid. Biochim Biophys Acta 806: 255–261

    Google Scholar 

  • Laroff GP, Fessenden RW, Schuler RH (1972) The electron spin resonance spectra of radical intermediates in the oxidation of ascorbic acid and related substances. J Am Chem Soc 94: 9062–9073

    Google Scholar 

  • Lumper L, Schneider W, Staudinger H (1967) Untersuchungen zur Kinetik der mikrosomalen NADH:semidehydroascorbat-oxydoreductase. Hoppe Seylers Z Physiol Chem 348: 323–328

    Google Scholar 

  • Mason RP (1984) Assay of in situ radicals by electron spin resonance. Methods Enzymol 105: 416–422

    Google Scholar 

  • Medina MA, Schweigerer L (1993) A plasma membrane redox system in human retinoblastoma cells. Biochem Mol Biol Int 29: 881–887

    Google Scholar 

  • Munoz E, Blazquez MV, Ortiz C, Gomez-Diaz C, Navas P (1997) Role of ascorbate in the activation of NF-KB by tumour necrosis factor-alpha in T-cells. Biochem J 325: 23–28

    Google Scholar 

  • Navas P, Villalba JM, Córdoba F (1994) Ascorbate function at the plasma membrane. Biochim Biophys Acta 1197: 1–13

    Google Scholar 

  • Rodriguez-Aguilera JC, Navas P (1994) Extracellular ascorbate stabilization: enzymatic or chemical process? J Bioenerg Biomembr 26: 379–384

    Google Scholar 

  • —, Navarro F, Arroyo A, Villalba JM, Navas P (1993) Transplasma membrane redox system of HL-60 is controlled by cAMP. J Biol Chem 268: 26346–26349

    Google Scholar 

  • Rose RC, Bode AM (1993) Biology of free radical scavengers: an evaluation of ascorbate. FASEB J 7: 1135–1142

    Google Scholar 

  • Sano S, Miyake C, Mikami B, Asada K (1995) Molecular characterization of monodehydroascorbate radical reductase from cucumber highly expressed inEscherichia coli. J Biol Chem 270: 21354–21361

    Google Scholar 

  • Schweinzer E, Goldenberg H (1992) Ascorbate-mediated transmembrane electron transport and ascorbate uptake in leukemic cell lines are two different processes. Eur J Biochem 206: 807–812

    Google Scholar 

  • — — (1993) Monodehydroascorbate reductase activity in the surface membrane of leukemic cells: characterization by a ferricyanide-driven redox cycle. Eur J Biochem 218: 1057–1062

    Google Scholar 

  • Vera JC, Rivas CI, Zhang RH, Farber CM, Golde DW (1994) Human HL-60 myeloid leukemia cells transport dehydroascorbic acid via the glucose transporters and accumulate reduced ascorbic acid. Blood 84: 1628–1634

    Google Scholar 

  • Villalba JM, Canalejo A, Rodriguez-Aguilera JC, Buron MI, Morré DJ, Navas P (1993) NADH-ascorbate free radical and -ferncyanide reductase activities represent different levels of plasma membrane electron transport. J Bioenerg Biomembr 25: 411–417

    Google Scholar 

  • Von Foerster G, Weis W, Staudinger H (1965) Messung der Elektronenspinresonanz an Semidehydroascorbinsäure. Liebigs Ann Chem 690: 166–169

    Google Scholar 

  • Wells WW, Xu DP (1994) Dehydroascorbate reduction. J Bioenerg Biomembr 26: 369–377

    Google Scholar 

  • Winkler BS, Orselli SM, Rex TS (1994) The redox couple between glutathione and ascorbic acid: a chemical and physiological perspective. Free Radie Biol Med 17: 333–349

    Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Sylvius Laboratory, Department of Molecular Cell Biology, Leiden University, Wassenaarseweg 72, 2333, AL Leiden, The Netherlands

    Martijn M. Van Duijn, Jolanda Van der Zee & Peter J. A. Van den Broek

Authors
  1. Martijn M. Van Duijn
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Jolanda Van der Zee
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Peter J. A. Van den Broek
    View author publications

    You can also search for this author in PubMed Google Scholar

Rights and permissions

Reprints and permissions

About this article

Cite this article

Van Duijn, M.M., Van der Zee, J. & Van den Broek, P.J.A. Electron spin resonance study on the formation of ascorbate free radical from ascorbate: The effect of dehydroascorbic acid and ferricyanide. Protoplasma 205, 122–128 (1998). https://doi.org/10.1007/BF01279302

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF01279302

Keywords

Search

Navigation