We're going to perform a redox titration on HRP in order to determine the standard potential of this protein.
We will use a Pine Instruments Honeycomb Spectroelectrochemical cell coupled to a WaveNow USB Potentiostat. The redox reaction will be monitored using a galvanic cell setting. UV Vis spectra will be recorded on an Ocean Optics Jaz Spectrometer. We will specifically use the Q-band to observe redox state following with my results from yesterday.
Following the procedure in this reference, we will add DTT in 1uL increments and observe both the UVVis spectrum and the open circuit potential of the SpecEchem cell. We will ultimately plot %oxidized or %reduced versus voltage read.
The degassed buffer will contain:
- 50mM Tris
- 50mM NaCl
and the following redox mediators (in order to stabilize the solution potential)
- 20uM duroquinone (tetramethyl-1,4-benzoquinone) (for 20mM should measure 32.8mg in 10mL)
10uM pyocyanine (we didn't receive this chemical)
- 10uM 2-hydroxy-1,4-naphthoquinone (for 10mM should measure 17.415mg in 10mL)
- 10uM anthraquinone-2-sulfonate (for 10mM should measure 31.026mg in 10mL)
- 2uM benzyl viologen (1,1'-Dibenzyl-4,4'bipyridinium Dichloride Hydrate) (for 2mM should measure 8.187mg)
- 1uM phenylsafranine (for 1mM should measure 3.228mg)
- 1uM indigo-carmine (for 1mM should measure 4.66mg)
Our HRP concentration will be roughly 30uM (in order to better observe the Q-bands). Also, the cuvette path length is shorter than 1cm, so we'll need a higher concentration to observe spectral changes.
The final concentration of DTT should be 2000X the HRP concentration. This comes out to 60mM. We should perform 1uL additions. The initial volume in the cuvette will be 1.1mL (1mL is initially added to the cuvette and 0.1mL used to fill the honeycomb). (the sodium dithionite stock solution should be ~10M unfortunately the max solubility is 1.4M).
The buffer that was made yesterday will be used today with more degassing.
6.3419g in 25.0mL --> 1.46M
Degassed for 2 hours
All of the dyes were placed in the same 10mL volumetric
- Duroquinone: 33.3mg in 10mL --> 20.3mM
- 2-hydroxy-1,4-naphthoquinone 18.0mg in 10mL --> 10.3mM
- anthraquinone-2-sulfonate 30.8mg --> 9.93mM
- benzyl viologen 8.7mg --> 2.1mM
- phenylsafranine 2.6mg --> 0.81mM
- indigo-carmine 4.6mg --> 0.99mM
These dyes were diluted by a factor of 1000 (25uL into 25mL). This solution was then degassed for 1 hour.
The dye solution has a blue-ish tint to it.
Measured 13.2mg in 10mL (dye solution) --> 33uM
In order to fully reduce the 33uM HRP solution, we had to add sodium dithionite until we noticed no more changes in reduction peak and potential.
The 8 samples used were:
- 1mL 33uM HRP
- 1mL 33uM HRP + 50uL Dithionite
- 1mL 33uM HRP + 55uL Dithionite
- 1mL 33uM HRP + 60uL Dithionite
- 1mL 33uM HRP + 65uL Dithionite
- 1mL 33uM HRP + 70uL Dithionite
- 1mL 33uM HRP + 75uL Dithionite
- 1mL 33uM HRP + 90uL Dithionite
Using this information, we were able to calculate the new concentration of HRP in the solutions, which will be used to normalize the absorbances of UV-Vis
With our data now normalized, the percent oxidized and reduced were calculated for each of the concentrations.
Here is the graph of the absorbance of the different sample versus. the wavelength.
The percent oxidized was calculated at 402.8 nm.
The percent reduced was calculated at 438.76 nm.
Here are the results of the calculations and also the corresponding cell potential recorded at that concentration.
|Cell potential (mV)
These results allowed to plot the percent reduced versus. cell potential and percent oxidized versus. cell potential.