User:Javier Vinals Camallonga/Notebook/Javier Vinals notebook/2013/09/18

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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:

  1. 50mM Tris
  2. 50mM NaCl

and the following redox mediators (in order to stabilize the solution potential)

  1. 20uM duroquinone (tetramethyl-1,4-benzoquinone) (for 20mM should measure 32.8mg in 10mL)
  2. 10uM pyocyanine (we didn't receive this chemical)
  3. 10uM 2-hydroxy-1,4-naphthoquinone (for 10mM should measure 17.415mg in 10mL)
  4. 10uM anthraquinone-2-sulfonate (for 10mM should measure 31.026mg in 10mL)
  5. 2uM benzyl viologen (1,1'-Dibenzyl-4,4'bipyridinium Dichloride Hydrate) (for 2mM should measure 8.187mg)
  6. 1uM phenylsafranine (for 1mM should measure 3.228mg)
  7. 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.

Sodium Dithionite

6.3419g in 25.0mL --> 1.46M

Degassed for 2 hours


All of the dyes were placed in the same 10mL volumetric

  1. Duroquinone: 33.3mg in 10mL --> 20.3mM
  2. 2-hydroxy-1,4-naphthoquinone 18.0mg in 10mL --> 10.3mM
  3. anthraquinone-2-sulfonate 30.8mg --> 9.93mM
  4. benzyl viologen 8.7mg --> 2.1mM
  5. phenylsafranine 2.6mg --> 0.81mM
  6. 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.

MRH2013-09-18 10.48.07.jpg

The dye solution has a blue-ish tint to it.


Measured 13.2mg in 10mL (dye solution) --> 33uM

We run 8 different samples in the Uv-Vis, and observed the reduction and oxidation of the different samples that we had.

"Image 1. Absorbance of fully reduced and fully oxidized HRP with Dithionite"

Spectra of fully oxi and fully red HRP with dithionite.png

"Table 1. Volume and concentrations of the samples run"

Volume(mL) Concentration(uM)
1 33
1.05 31.4285714
1.055 31.2796209
1.06 31.1320755
1.065 30.9859155
1.070 30.8411215
1.075 30.6976744
1.09 30.2752294

The 100% oxidation we found for our sample is 0.00663976. The 100% reduction we found for our sample is 0.00948221. The fact that the sample was not covered, and therefore there was oxygen present in the sample might be the result of the addition of such high volumes of the reducing agent. We calculated the percent reduction at the wavelength 438.76nm and percent oxidation at the wavelength 402.8nm.

"Table 2. Percent oxidized and reduced"

HRP 50 '55 '60 65 70 75 90
oxi 0.01284848 0.01250398 0.01246803 0.01082557 0.0125847 0.01063554 0.00856678 0.00620872
red 0.00187879 0.00213172 0.00195013 0.00469001 0.00232333 0.00502594 0.00820847 0.011361
% oxi 1 0.94811559 0.94270124 0.69533387 0.96027266 0.66671386 0.35514235 0
% red 0 0.02667416 0.00752356 0.29647308 0.04688148 0.33190047 0.66753215 1
Cell potential (mV) 19.2 -41.4 -130.1 -312.6 -333.8 -371 -433.5 -560

"Image 2. Reduction by Dithionite"

Percent oxi and red Javier Vinals.png