Objective
To monitor the kinetics and yield of the horseradish peroxidase-catalyzed oxidation of luminol. These experiments will be compared to future experiments with HRP-functionalized nanoparticles. These experiments are also meant to introduce researchers to stopped-flow techniques and rapid data collection.
From Dr. Hartings
Procedure
2. Chemiluminescence of Luminol Oxidation Reaction Initiated by Stopped Flow Mixer
- Add 64.9 μL HRP stock, 3.31 mL luminol stock, and 6.63 mL buffer to stopped flow mixer.
- Add 5.00 mL hydrogen peroxide stock and 5.00 mL buffer to stopped flow mixer.
- Equilibrate mixer tubes with sample.
- Initiate mixing.
- Measure light produced as result of reaction, integrated over a specific time range.
- Integrate area under the curve.
3. Kinetics of Luminol Oxidized by Absorption Spectrum Changes in Stopped Flow Mixer
- Add 245.13 μL HRP stock to a volumetric flask and dilute to 10 mL with buffer.
- Add 1.25 mL luminol stock to a volumetric flask and dilute to 10 mL with buffer.
- Add 2.00 mL of diluted HRP to stock and 10.00 mL of diluted luminol to stopped flow mixer.
- Add 5.00 mL hydrogen peroxide stock and 5.00 mL buffer to stopped flow mixer.
- Equilibrate mixer tubes with sample.
- Initiate mixing.
- Using luminol and 3-aminophthalic acid spectra as endpoints, determine the kinetics of 3-aminophthalic acid synthesis.
Figures
Notes
- The stock solution of HRP was 0.77 μM.
- The stock solution of luminol was 1.51 mM.
- The buffer solution was 5.1 mM Tris at pH = 8.
- The stock solution of hydrogen peroxide was 12.8 mM.
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