User:Daniel-Mario Larco/Notebook/AU Biodesign Lab - 09/03/2013/2013/10/01

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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.

Description

Three sets of measurements will be performed today.

  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)

Here is the UV-Vis absorption spectrum for HRP, Luminol and H2O2 obtained. Note that the absorption of HRP is greater than 1 which is why we made a new solution diluted by a factor of 1/20 with the buffer solution.


  1. Chemiluminescence of luminol oxidation reaction initiated by 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. Measure light produced as result of reaction, integrated over a specific time range
    6. Integrate area under the curve
  1. 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.
  • Matt Hartings Note: We will be doing Step 1 (while I'm teaching my other class) and Step 3 (after I get back from class) today. We'll do step 2 tomorrow.

Data

Stock Solution

  1. Buffer
    1. 0.6175g Tris in 1L, pH set to 8 with HCl ---> 5.1mM
  2. HRP
    1. 1.7mg in 50mL buffer (MW ~ 44,000) ---> 0.77uM
  3. Luminol
    1. Dissolve 13.4mg luminol in 300uL of DMSO
    2. Add to 50mL buffer ---> 1.51mM
  4. H2O2
    1. 177uL 30% H2O2 into 50mL buffer ---> Should be 45mM
    2. Check this concentration! The molar absorptivity of H2O2 at 240nm is 40,000 M-1cm-1

Results

There were some complications with the spectra as none of them appeared to be working. We used 1/20 dilutions of the HRP, Luminol and H2O2 and it still did not work possibly because the hydrogen peroxide to luminol ratio was too high.

We used the data from Trisha's group for our analysis and the results show a decrease of HRP


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