User:Matt Hartings/Notebook/Photosynthesis/2013/04/02

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Mb Catalysis in Methanol

Running tests on myoglobin catalysis in methanol today. Catalysis is on the conversion of o-phenylenediamine (OPD) into 2,3-diaminophenazine (DAPN)

OPD catalysis reaction.png


Spectra of the compounds in the figure below are taken from: Qi et al "Myoglobin as mimetic enzyme and its analytical application in determination of H2O2" Wuhan University Journal of Natural Sciences 2006, 11, 3, p677

OPDReferenceSpectra.png

Stock solutions used

  1. 16.4mg of o-phenylenediamine in 15.2mL of methanol
  2. 1.134 ml 30% H2O2 +8.866 ml Methanol
  3. 8.9mg of (5.7mg myoglobin +0.4861g KCl lyophilized from 20mL of 10mM citrate pH 3) in 1 mL of methanol.

Samples were made by

  1. 2.7 mL of the OPD solution
  2. 0.3 mL of the Mb solution
  3. Start the scan
    1. 1,000 us integration time
    2. Average over 500 scans
    3. Run for 10 minutes (600,000,000 us)
  4. Add 1mL of the H2O2 solution
    1. Added after scan reached 1%
  5. Kinetics scans were run at 5, 15, 25, 35, and 45C. Several before and after spectra were also taken, as were the dark and reference signal spectra.
  6. Absorbance measurements were calculated using the following equation: A = -log[(SampleSignal-DarkSignal)/(ReferenceSignal-DarkSignal)]

Shown below are kinetics traces and absorbance scans for the different temperatures

MRH20130402 5C absorbance.png MRH20130402 5C Kinetics.png

MRH20130402 15C absorbance.png MRH20130402 15C Kinetics.png