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Ethanol increases sensitivity of oxalate oxidase assays and facilitates direct activity staining in SDS gels

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Abstract

Oxalate oxidase, and H2O2-generating enzyme, has been characterized from several plants, and is widely used for clinical detection of oxalate. Using a germin-like oxalate oxidase from barley leaves, we have developed and optimized novel methods for measuring oxalate oxidase activity. As oxalate oxidase is SDS-tolerant, its activity can be detected directly in SDS-PAGE gels in the presence of ethanol. This ethanol-dependent method is a hundred times more sensitive than the current methods. Furthermore, ethanol also improves the sensitivity of oxalate oxidase assays performed in solution. We found at least a 10-fold increase in sensitivity in comparison to a current method. The assay in solution is, in addition, useful for detection of oxalate. This elevation in sensitivity may be due to the immobilization of the enzyme in protein precipitates as a result of the treatment with ethanol.

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Abbreviations

AOS:

active oxygen species

References

  • Bernier, F., G. Limieux, D. Pallotta. 1987. Gene families encode the major encystment-specific proteins ofPhysarum polycephalum plasmodia. Gene 59:265–277.

    Article  PubMed  CAS  Google Scholar 

  • Dumas, B., A. Sailland, J.-P. Cheviet, G. Freyssinet, K. Pallett. 1993. Identification of barley oxalate oxidase as a germin-like protein. C.R. Acad. Sci. Paris, 316:793–798.

    PubMed  CAS  Google Scholar 

  • Dumas, B., G. Freyssinet, K.E. Pallett. 1995. Tissue-specific expression of germin-like oxalate oxidase during development and fungal infection of barley seedlings. Plant Physiol. 107:1091–1096.

    PubMed  CAS  Google Scholar 

  • Elstner, E.F., H. Schempp, G. Preibisch, S. Hippeli, W. Osswald. 1994. Biological sources of free radicals, pp. 13–45 in:Free Radicals in the Environment, Medicine and Toxicology. (eds. H. Nohl, H. Esterbauer, C. Rice-Evans). Richelieu Press, London.

    Google Scholar 

  • Hansen, E.H., S.K. Winther, M. Gundstrup. 1994. Enzymatic assay of oxalate in urine by flow injection analysis using immobilized oxalate oxidase and chemiluminescence detection. Anal. Lett. 27:1239–1253.

    CAS  Google Scholar 

  • Harlow, E., D. Lane. 1988.Antibodies: A Laboratory Manual. Cold Spring Harbor Laboratory Press, New York.

    Google Scholar 

  • Laker, M.F., A.F. Hofmann, B.J.D. Meeuse. 1980. Spectrophotometric determination of urinary oxalate with oxalate oxidase prepared from moss. Clin. Chem. 26:827–830.

    PubMed  CAS  Google Scholar 

  • Lane, B.G., J.M. Dunwell, J.A. Ray, M.R. Schmitt, A.C. Cuming. 1993. Germin, a protein marker of early plant development, is an oxalate oxidase. J. Biol. Chem. 268:12239–12242.

    PubMed  CAS  Google Scholar 

  • Lane, B.G. 1994. Oxalate, germin, and the extracellular matrix of higher plants. FASEB J. 8:294–301.

    PubMed  CAS  Google Scholar 

  • Lathika, K.M., K.V. Inamdar, U. Tarachand, B.B. Singh, K.G. Raghavan. 1995. Determination of urinary oxalate using banana oxalate oxidase: Comparison with immobilized enzyme. Anal. Lett. 28:425–442.

    CAS  Google Scholar 

  • Pundir, C.S. 1991. Purification and properties of oxalate oxidase from sorghum leaves. Phytochem. 30:1065–1067.

    Article  CAS  Google Scholar 

  • Pundir, C.S., Satyapal, N.K. Kuchhal. 1993. Immunobilization of barley oxalate oxidase onto alkylamine glass for determing urinary oxalate. Clin. Chem. 39:1750–1751.

    PubMed  CAS  Google Scholar 

  • Saka Amini, M.A., J.J. Vallon. 1994. Comparison of performances and analytical applications of two immobilized oxalate oxidase sensors. Anal. Chim. Acta 299:75–79.

    Article  CAS  Google Scholar 

  • Sambrook, J., E.F. Fritsch, T. Maniatis. 1989.Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, New York.

    Google Scholar 

  • Satyapal, C.S. Pundir. 1993. Purification and properties of an oxalate oxidase from leaves of grain sorghum hybrid CSH-5. Biochim. Biophys. Acta 1161:1–5.

    PubMed  CAS  Google Scholar 

  • Zhang, Z., D.B. Collinge, H. Thordal-Christensen. 1995. Germin-like oxalate oxidase, a H2O2-producing enzyme, accumulates in barley attacked by the powdery mildew fungus. Plant J. 8:139–145.

    Article  CAS  Google Scholar 

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Author notes

  1. Ziguo Zhang

    Present address: Department of Plant Sciences, University of Oxford, UK

  2. Jing Yang

    Present address: Huazhong Agricultural University, Wuhan, P.R. China

Authors and Affiliations

  1. Plant Pathology Section, Department of Plant Biology, The Royal Veterinary and Agricultural University, Thorvaldsensvej 40, DK-1871 Frederiksberg C, Copenhagen, Denmark

    Ziguo Zhang, Jing Yang, David B. Collinge & Hans Thordal-Christensen

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  1. Ziguo Zhang
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  2. Jing Yang
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  3. David B. Collinge
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  4. Hans Thordal-Christensen
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Zhang, Z., Yang, J., Collinge, D.B. et al. Ethanol increases sensitivity of oxalate oxidase assays and facilitates direct activity staining in SDS gels. Plant Mol Biol Rep 14, 266–272 (1996). https://doi.org/10.1007/BF02671662

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