Objectives
- Monitor the kinetics and yield of horseradish peroxidase catalyzed oxidation of luminol
- Measure UV-Vis absorbance of luminol, HRP, and Luminol+HRP+H2O2
- Monitor chemiluminscence of luminol oxidation using stop-flow techniques
- Measure kinetics of reaction using stop-flow techniques and absorbance
UV-vis
Stock solutions prepared before class:
- Luminol: 13.5mg luminol in 300μL DMSO added to 50mL 5mM Tris pH=8
- Buffer: 5mM Tris pH=8
- HRP: 1.7μM HRP in 50mL 5mM Tris pH=8
- H2O2: 117μL of 30%H2O2 in 50mL 5mM buffer.
Samples Run:
- 1/20 dilution of lumino1
- HRP stock solution
- 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
- 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:
- Add the desired HRP/Luminol stock solution to the stop flow mixer.
- Add H2O2 stock solution to the stop flow mixer.
- 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)
- Initiate Mixing
- Take a absorbance spectrum of the sample every 30 seconds for 10 minutes in order to monitor the reaction
- Using the luminol and 3-aminophthalic acid spectra as endpoints, determine the kinetics of the 3-aminophthalic acid synthesis
Sample preparation of
Figure 2. Absorbance spectra of the oxidation of luminol by HRP.
Figure 3. Absorption spectra of the production of 3-aminopthalate by the oxidaiton of luminol by HRP.
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