The o-Phenylenediamine-Horseradish Peroxidase System:: Enzyme Kinetics in the General Chemistry Laboratory
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[1] Department of Chemistry, Indiana University
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- Localización: Journal of chemical education, ISSN 0021-9584, Vol. 76, Nº 5 (May), 1999, págs. 642-642
- Idioma: inglés
- Texto completo no disponible (Saber más ...)
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Resumen
We have developed an enzyme kinetics laboratory experiment that can easily be incorporated into an undergraduate chemistry program. The purpose of the experiment is to measure the kinetic parameters in the oxidative coupling reaction of o-phenylenediamine (OPD) to 2,3-diaminophenazine (DAP), a reaction catalyzed by the enzyme horseradish peroxidase (HRP). Horseradish peroxidase is a protein with a mass of 34 kDa found in the root of the horseradish plant. Students follow the kinetics of the reaction by monitoring the change in intensity of the colored product at 450 nm. Several kinetic runs are performed with varying OPD concentration and fixed HRP concentration and the kinetic parameters are determined by straight-line plots of the Michaelis-Menten equation. Vernier Software Colorimeters interfaced to personal computers via the LabVIEW programming language were used in the experiments. Alternatively, conventional UV-vis spectrophotometers linked to strip-chart recorders could be used for measuring the kinetic runs. The following ranges of values for kinetic parameters were obtained: vmax = 6.5 ± 1.5 x 10-3 mM s-1, KM = 0.6 ± 0.2 mM, k0 = 1.15 ± 0.15 x 103 s-1. Variations including the effects of HRP concentration and inhibitors on the kinetics are discussed.
