User:Moira M. Esson/Notebook/CHEM-571/2013/10/01

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  1. Monitor the kinetics and yield of horseradish peroxidase catalyzed oxidation of luminol
    1. Measure UV-Vis absorbance of luminol, HRP, and Luminol+HRP+H2O2
    2. Monitor chemiluminscence of luminol oxidation using stop-flow techniques
    3. Measure kinetics of reaction using stop-flow techniques and absorbance


Stock solutions prepared before class:

  1. Luminol: 13.5mg luminol in 300μL DMSO added to 50mL 5mM Tris pH=8
  2. Buffer: 5mM Tris pH=8
  3. HRP: 1.7μM HRP in 50mL 5mM Tris pH=8
  4. H2O2: 117μL of 30%H2O2 in 50mL 5mM buffer.

Samples Run:

  1. 1/20 dilution of lumino1
  2. HRP stock solution
  3. Reaction of HRP+Luminol stock+H2O2 (prepared solution by taking 1/3mL of each sample and allowing to react for ~5min)

Sample notes:

  • Performed 1/20 dilution of 13.5mg luminol in 300μL DMSO added to 50mL 5mM Tris pH=8. (The luminol stock solution).
    • Make a 10mL stock solution of 1/20 dilute luminol stock solution.
  • Prepared stock solution of hydrogen peroxide was too dilute to measure (characteristic peak at 432 nm was not present)

Figure 1. Corrected Absorbance Spectra of preliminary kinetics
HRP diluteluminol h2o2 zem.png

  • The H2O2 stock solution appears to be too dilute to produce a spectrum.
  • A 1/20 dilution of luminol was used in order to produce an absorbance peak with a value below 1.

Kinetics of luminol oxidized by HRP

General Protocol:

  1. Add the desired HRP/Luminol stock solution to the stop flow mixer.
  2. Add H2O2 stock solution to the stop flow mixer.
  3. Equilibrate mixer tubes with sample
    • Note: For the steps described above, no air bubbles can be present in the sample. Air bubbles will affect the kinetics data(oxygen will act as an oxidant and produce unreliable data)
  4. Initiate Mixing
  5. Take a absorbance spectrum of the sample every 30 seconds for 10 minutes in order to monitor the reaction
  6. Using the luminol and 3-aminophthalic acid spectra as endpoints, determine the kinetics of the 3-aminophthalic acid synthesis

Data collection:

  • The 1/20 dilution of luminol was used for the reaction mixture
  • A 1/20 dilution of hydrogen peroxide stock solution was used based on other groups findings
  • Stock solution of HRP was used
  • The sample which was run was completely oxidized upon mixture of the reaction. This may be due to the ratio of luminol to the concentrated HRP. Another group's data will be analyzed and the kinetics procedure will be repeated again tomorrow.

Results: The below results were obtained from Trisha's group

  • A diluted solution of luminol with a concentration of .126mM was used with the stock solutions of HRP and hydrogen peroxide

Figure 2. Absorbance spectra of the oxidation of luminol by HRP focusing on the 350nm region Kineticsmonitoringzem10012013.png

  • The wavelength maximum occurs at 348.99nm and experiences a growth in signal. This indicates the production of 3-aminophthalic acid.

Figure 3. Absorption spectra of the production of 3-aminopthalate by the oxidaiton of luminol by HRP focusing on the 420nm region Kinematicsincreaseprdtzem10012013.png

  • The wavelength maximum occurs at 415.42nm. During the course of the ten minutes, a signal reduction occurred, indicating the oxidation of luminol.