Luminometric determination of oxidase activity in peroxisomal fractions of rat liver: glycolate oxidase |
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| Authors: | C Leupold A V?lkl H D Fahimi |
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| Affiliation: | 1. Department of Physiological Sciences, Federal University of Espírito Santo, Vitória, Espírito Santo, Brazil;2. Department of Pharmacy, University of Vila Velha, Espírito Santo, Brazil;1. Medical Proteomics Unit, Office for Research and Development, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand;2. Department of Immunology and Immunology Graduate Program, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand;3. Center for Research in Complex Systems Science, Mahidol University, Bangkok, Thailand |
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| Abstract: | The feasibility of using the H2O2-mediated chemiluminescence for determination of the activity of oxidases in peroxisomes of rat liver has been investigated. In an assay medium containing luminol, horseradish peroxidase, and azide with glycolate as substrate, a linear relationship is obtained between the amount of peroxisomal protein used and the luminescence signal. In comparison with other techniques available for measuring the activities of peroxisomal oxidases the luminometric approach described here is 5-10 times more sensitive than the spectrophotometric methods and 100 times more efficient than the polarographic determination of O2. Under the optimal assay conditions the glycolate oxidase activity can be determined in amounts as low as 0.5 micrograms peroxisomal protein. |
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