User:Matt Hartings/Notebook/AU Biomaterials Design Lab/2013/09/17

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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.


In order to obtain good results, we need our buffers to be as free of oxygen (oxygen is ... an oxidizing agent, so we need to try to remove it from our experiment) as we can get them. I will prepare the buffers and reagents and will show you, group by group, how I did this.


  1. Take a spectrum of the buffer we are using
  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. Note gently add all reagents together (slowly pipette) in order to reduce the amount of oxygen added to the system
    3. Take a spectrum of this sample
  3. Add 1uL 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 - It may take a few additions before you see any change from the original
    2. 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
    3. 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.


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.

In order to prepare for tomorrow and have a better understanding of what we're doing today see the following references.

This reference highlights some of the changes that we'll be observing in the spectra. Note, specifically, figure 4. We won't be using this exact experimental technique, tho.

This reference goes more into detail into the kind of experiment we will be performing tomorrow. Note specifically the Redox Titrations portion of the Materials and Methods section.



3.0533g Tris and 1.4677g NaCl in 500mL water. pH set to 7.5 with 3M HCl

Final concentration: 50.4mM Tris 50.2mM NaCl

Buffer degassed by bubbling nitrogen through it for 3 hours

Sodium Dithionite

19.8mg in 10mL degassed buffer --> 11.3mM

2.5mL of this solution was diluted to 25mL with degassed buffer to make a final stock concentration of 1.13mM. This solution was degassed an extra 30 minutes.


35.1mg in 10mL degassed buffer --> 10.7mM

2.5mL of this solution was diluted to 25mL with degassed buffer to make a final stock concentration of 1.07mM. This solution was degassed an extra 30 minutes.

Horseradish Peroxidase

6.9mg of HRP in 10.0mL degassed buffer --> 17uM

After the student experiments, we observed that there were no changes taking place, I went back and took a couple of spectra on my own.


  1. 250uL of HRP stock solution and 750uL of concentrated (~10mM) Dithionite stock solution
  2. 500uL of HRP stock solution and 500uL of concentrated (~10mM) Dithionite stock solution
  3. 750uL of HRP stock solution and 250uL of concentrated (~10mM) Dithionite stock solution
  4. 250uL of HRP stock solution and 750uL of concentrated (~10mM) K3[Fe(CN)4] stock solution

Here are the spectra


Obviously, we were adding too low of a concentration during the lab session today. After everyone left, I did a quick set of spectra with more concentrated stocks. I found that the iron in the protein, as is, is likely all 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. These features are best to monitor as there is no dithionite or ferricyanide absorbance in that range. This data is corroborated in the linked reference. I'm not sure what's going on at higher ferricyanide concentrations, but, with lower concentrations (600:1 ferricyanide to HRP) I observed, but didn't save, data that showed spectra similar to what we observed with only HRP in solution.