User:Moira M. Esson/Notebook/CHEM-571/2013/09/18

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Objectives

  1. Perform redox titration on HRP to determine the standard redox potential of the protein
    • Note: Dr. Hartings the course instructor performed the reduction of HRP, which was unsuccessful on 09/17/2013 prior to the lab. All info can be found here.
  2. Take UV/vis sample at the minimum redox potential value.


Redox Titration

  • For the general protocol of the HRP redox titration and the sample preparation please refer here


Table 1. Specific volume of samples used during redox titration.

Sample number Volume of HRP Sample(mL)
11
21.05
31.055
41.056
51.059
61.064
71.074
81.084
  • Sample 1 contained solely HRP. The additional volumes were sodium dithionite.


Figure 1. Absorbance Spectra of the redox titration of HRP
Image:FullyoxidizedreducedHRPzem.png

  • The wavelength maximum of fully oxidized HRP was 402.44nm.
  • The wavelength of fully reduced HRP was 433.74nm.
  • The sample with the addition of 64μL sodium dithionite was the fully oxidized sample.
  • The sample with the addition of 84uL sodium dithionite was the fully reduced sample.
  • Note: The presence of oxygen in the sample(as the titration was not performed in a glove box) could be the cause of such a large volume addition of the reducing agent to be present in the sample that was fully oxidized.


Figure 2. Redox titration of HRP
Image:PercentreducedHRPzem.png
Table 2. Redox titration of HRP (looking at percent oxidized)

% Oxidized Potential
-2.633063185-0.5122
0-0.5803
3.260060544-0.4781
30.97191805-0.4045
82.708593160.0146
98.04493512-0.3879
98.62977455-0.355
100-0.3519


  • Equation used to calculate the percent oxidized was:

(Abs.partially oxidized 402.44nm-Abs. fully reduced 402.44nm)/(Abs.fully oxidized-Abs.fully reduced)*100
Table 2. Redox titration of HRP % reduced data

% Reduced Potential
-2.736716560.0146
-0.472156173-0.355
0-0.3519
0.156439504-0.3879
56.60826937-0.4045
94.88970062-0.4045
96.52628268-0.4781
100-0.5803


Notes

The use of the midpoint cell potential of unmodified HRP will be used in further experiments to compare the effect of Au nanoparticles.



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