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.
- 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)
- Chemiluminescence of luminol oxidation reaction initiated by 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
- Measure light produced as result of reaction, integrated over a specific time range
- Integrate area under the curve
- 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.
- 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
- Buffer
- 0.6175g Tris in 1L, pH set to 8 with HCl ---> 5.1mM
- HRP
- 1.7mg in 50mL buffer (MW ~ 44,000) ---> 0.77uM
- Luminol
- Dissolve 13.4mg luminol in 300uL of DMSO
- Add to 50mL buffer ---> 1.51mM
- H2O2
- 177uL 30% H2O2 into 50mL buffer ---> Should be 45mM
- 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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