Monitor the kinetics and yield of the horseradish peroxidase-catalyzed oxidation of luminol.
- UV-Vis Absorbance of reactants, catalysts, and products
- horseradish peroxidase (Use stock solution)
- luminol (use stock solution)
- 3-aminophthalic acid (product of reaction between luminol and H2O2 catalyzed by HRP. In order to take this measurement, react (in 1:1 ratio or with slight excess H2O2) luminol with H2O2 in presence of HRP. Allow the reaction to proceed for 5 minutes; take the spectrum)
- Kinetics of luminol oxidized by changes in absorption spectrum, reaction carried out in stopped flow mixer
- Add HRP/Luminol stock solution to stopped flow mixer
- Add H2O2 stock solution 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.
- Series 1: HRP Stock
- Series 2: 0.126mM Luminol
- Series 3: H2O2 Stock
- Buffer = 5.1 mM
- HRP = 0.77 uM
- Luminol = 1.51 mM
- H2O2 = 45 mM
- Luminol dilution solution: 0.126 mM