User:Helen L. Slucher/Notebook/CHEM 571/2013/09/17

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Objective

Determine the amount of reagent that is required to fully oxidize or fully reduce horseradish peroxidase.

Description

Oxidation

  1. Take a spectrum of the buffer
  2. Make a 1mL HRP solution in your cuvette with a final concentration of between 5 and 10uM
    1. Allow this sample to diffuse over 5 minutes
    2. Take a spectrum of this sample
  3. Add 2uL of the K3[Fe(CN)6] solution to your cuvette with HRP
    1. Allow 5 minutes for the sample to equilibrate
    2. Take a spectrum
  4. Repeat the previous step until the HRP spectrum is unchanged from the time before
    1. Note - the best place to note the change is from the Soret peak, i.e. the absorption feature near 400nm. Upon oxidation, this peak should diminish and shift to lower wavelengths
    2. Note - In order to best monitor changes, it will be best to input your data promptly into excel to monitor changes. You will also want to normalize each spectrum to concentration (divide by the concentration). (Upon each addition of K3[Fe(CN)6], you will be changing your HRP concentration.
  5. Note the amount of K3[Fe(CN)6] required to fully oxidize your sample. Determine the ratio of concentration of K3[Fe(CN)6]/HRP in the fully oxidized sample.

Reduction

  1. Follow along with the procedure for oxidation and, instead, use sodium dithionite for the reduction. Upon reduction the Soret peak will increase in intensity and shift to higher wavelengths.

Data

  • Screen shot 2013-10-12 at 4.18.54 PM.png
  • Screen shot 2013-10-12 at 4.13.47 PM.png
  1. Series 1: 2
  2. Series 2: 4
  3. Series 3: 6
  4. Series 4: 8
  5. Series 4: 9
  6. Series 5: 10
  7. Series 6: 12
  8. Series 7: 14
  9. Series 8: 16
  • Screen shot 2013-10-12 at 4.13.58 PM.png
  1. Series 1: 2
  2. Series 2: 4
  3. Series 3: 6
  4. Series 4: 8
  5. Series 5: 10
  6. Series 6: 12

Notes

  • Starting concentration of HRP = 17.25 uM
  • 580 uL HRP + 420 uL buffer = 1 mL total volume
  • The iron in the protein was likely in the +3 oxidation state. The protein isn't fully reduced until around 2000 equivalents of sodium dithionite are added. This change is best monitored by the absorption features between 500 and 700 nm (i.e. the Q-band). In the oxidized state (ferry-horseradish peroxidase), the spectrum has features at 500nm and 643 nm. When reduced, these features shift to 553nm with a shoulder at 583nm.
  • Screen shot 2013-10-12 at 4.33.37 PM.png
  • Screen shot 2013-10-12 at 4.33.47 PM.png