User:Zachary I. Mendel/Notebook/Zacks Notebook/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)
  2. 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
  3. 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

Notes

Making 1/9 dilution of luminol

1/100 HRP

Figure 1. Corrected Absorbance Spectra of preliminary kinetics

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

  • 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

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


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