Fluorometric assays for coproporphyrinogen oxidase and protoporphyrinogen oxidase |
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| Authors: | P Labbe J M Camadro H Chambon |
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| Affiliation: | 1. School of Environmental Science and Engineering, Kochi University of Technology (KUT), Tosayamada, Kochi 782-8502, Japan;2. Carnegie Institution for Science, 260 Panama St. Stanford, CA 94305, USA;3. Laboratory of Aquatic Natural Products Chemistry, Graduate School of Agricultural & Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan;4. Japan Science and Technology Agency (JST), 5 Sanbancho, Chiyoda, Tokyo 102-0075, Japan;1. BASF Belgium Coordination Center – Innovation Center Gent, Technologiepark 101, 9052 Ghent, Belgium;2. Rothamsted Research, Harpenden, Hertfordshire AL5 2JQ, UK;3. Laboratory of Functional Plant Biology, Department of Biology, Ghent University, K. L. Ledeganckstraat 35, 9000 Ghent, Belgium;1. Department of Chemistry and Biochemistry, Santa Clara University, Santa Clara, CA, 95053, USA;2. Department of Mechanical Engineering, Santa Clara University, Santa Clara, CA, 95053, USA;3. Department of Mechanical and Aerospace Engineering, Princeton University, Princeton, NJ, 08544, USA;4. KTH Mechanics, Stockholm, SE-10044, Sweden;1. Department of Biochemistry and Molecular Biology, Federal University of Paraná, P.O. Box 19046, Central Polytechnic, Curitiba 81531-980, Paraná, Brazil;2. Department of Chemistry, Federal University of Paraná, Cx.P. 19061, Central Polytechnic, Curitiba 81531-980, Paraná, Brazil |
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| Abstract: | We describe fluorometric assays for two enzymes of the heme pathway, coproporphyrinogen oxidase and protoporphyrinogen oxidase. Both assays are based on measurement of protoporphyrin IX fluorescence generated from coproporphyrinogen III by the two consecutive reactions catalyzed by coproporphyrinogen oxidase and protoporphyrinogen oxidase. Both enzymatic activities are measured by recording protoporphyrin IX fluorescence increase in air-saturated buffer in the presence of EDTA (to inhibit ferrochelatase that can further metabolize protoporphyrin IX) and in the presence of dithiothreitol (that prevents nonenzymatic oxidation of porphyrinogens to porphyrins). Coproporphyrinogen oxidase (limiting) activity is measured in the presence of a large excess of protoporphyrinogen oxidase provided by yeast mitochondrial membranes isolated from commercial baker's yeast. These membranes are easy to prepare and are stable for at least 1 year when kept at -80 degrees C. Moreover they ensure maximum fluorescence of the generated protoporphyrin (solubilization effect), avoiding use of a detergent in the incubation medium. The fluorometric protoporphyrinogen oxidase two-step assay is closely related to that already described (J.-M. Camadro, D. Urban-Grimal, and P. Labbe, 1982, Biochem. Biophys. Res. Commun. 106, 724-730). Protoporphyrinogen is enzymatically generated from coproporphyrinogen by partially purified yeast coproporphyrinogen oxidase. The protoporphyrinogen oxidase reaction is then initiated by addition of the membrane fraction to be tested. However, when very low amounts of membrane are used, low amounts of Tween 80 (less than 1 mg/ml) have to be added to the incubation mixture to solubilize protoporphyrin IX in order to ensure optimal fluorescence intensity. This detergent has no effect on the rate of the enzymatic reaction when used at concentrations less than 2 mg/ml. Activities ranging from 0.1 to 4-5 nmol protoporphyrin formed per hour per assay are easily and reproducibly measured in less than 30 min. |
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