User:Carly M. Montanero/Notebook/CHEM-571/2013/09/17

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Objective

Today we are going to determine the amount of reagent that is required to fully oxidize or fully reduce horseradish peroxidase. For HRP oxidation, we will be using potassium ferricyanide, K3[Fe(CN)6]. K3[Fe(CN)6] has a standard reduction potential of 424mV (ref1 and ref2) vs NHE. For HRP reduction, we will be using sodium dithionite, which has a reduction potential of -460mV vs NHE. We will be monitoring oxidation and reduction through changes in the UV-Vis spectrum of HRP. In order to do this we will also have to account for the absorbance of the K3[Fe(CN)6, which has an absorption feature at 420nm (for Fe2+, ε = 4.7 M-1cm-1). This is being done in preparation for our experiments tomorrow where we will be determining the redox potential of HRP.

From Dr. Hartings

Procedure

We used the procedure from Dr. Hartings notebook.

Figures

HRPdithionite.jpg

HRPferricyanide.jpg

RatiosHRP.jpg

Notes

  • The concentration of sodium dithionite was 1.13mM. The solution was degassed for 3 hours.
  • The concentration of potassium ferricyanide was 1.07mM. The solution was degassed for 3 hours and 30 minutes.
  • The concentration of horseradish peroxidase is 17 μM.
  • The concentration of buffer was 50.4mM Tris 50.2mM NaCl. The solution was degassed for 3 hours.
  • The oxidation results were not as expected. Dr. Hartings suspects that the concentration of sodium dithionite was too low.
  • We observed similarly poor results for the reduction.