User:Helen L. Slucher/Notebook/CHEM 571/2013/10/01

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Objective

Monitor the kinetics and yield of the horseradish peroxidase-catalyzed oxidation of luminol.

Description

  1. UV-Vis Absorbance of reactants, catalysts, and products
    1. horseradish peroxidase (Use stock solution)
    2. luminol (use stock solution)
    3. 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)
  2. Kinetics of luminol oxidized by changes in absorption spectrum, reaction carried out in stopped flow mixer
    1. Add HRP/Luminol stock solution to stopped flow mixer
    2. Add H2O2 stock solution to stopped flow mixer
    3. equilibrate mixer tubes with sample.
    4. Initiate Mixing
    5. Using luminol and 3-aminophthalic acid spectra as endpoints, determine the kinetics of 3-aminophthalic acid synthesis.

Data

  • Image:Screen shot 2013-10-12 at 6.07.13 PM.png
  1. Series 1: HRP Stock
  2. Series 2: 0.126mM Luminol
  3. Series 3: H2O2 Stock
  • Image:Screen shot 2013-10-12 at 6.52.44 PM.png

Notes

  • Stock
  1. Buffer = 5.1 mM
  2. HRP = 0.77 uM
  3. Luminol = 1.51 mM
  4. H2O2 = 45 mM
  • Luminol dilution solution: 0.126 mM


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