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

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==Objectives==
==Objectives==
#Perform redox titration on HRP to determine the standard redox potential of the protein
#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 [[User:Moira_M._Esson/Notebook/CHEM-571/2013/09/17|09/17/2013]] prior to the lab. All info can be found [[User:Matt Hartings/Notebook/AU Biomaterials Design Lab/2013/09/17|here]].
#*Note: Dr. Hartings the course instructor performed the reduction of HRP, which was unsuccessful on [[User:Moira_M._Esson/Notebook/CHEM-571/2013/09/17|09/17/2013]] prior to the lab. All info can be found [[User:Matt Hartings/Notebook/AU Biomaterials Design Lab/2013/09/17|here]].
#Take UV/vis sample at the minimum redox potential value.
#Take UV/vis sample at the minimum redox potential value.
<br>
==Redox Titration==
*For the general protocol of the HRP redox titration and the sample preparation please refer [[User:Matt Hartings/Notebook/AU Biomaterials Design Lab/2013/09/18|here]]
<br>
Table 1. Specific volume of samples used during redox titration.
{| {{table}}
| align="center" style="background:#f0f0f0;"|'''Sample number'''
| align="center" style="background:#f0f0f0;"|'''Volume of HRP Sample(mL)'''
|-
| 1||1
|-
| 2||1.05
|-
| 3||1.055
|-
| 4||1.056
|-
| 5||1.059
|-
| 6||1.064
|-
| 7||1.074
|-
| 8||1.084
|}
*Sample 1 contained solely HRP. The additional volumes were sodium dithionite.
<br>
Figure 1. Absorbance Spectra of the redox titration of HRP
<br>
[[Image:FullyoxidizedreducedHRPzem.png]]
<br>
*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.
<br>
Figure 2. Redox titration of HRP
<br>
[[Image:PercentreducedHRPzem.png]]
<br>
Table 2. Redox titration of HRP (looking at percent oxidized)
<br>
{| {{table}}
| align="center" style="background:#f0f0f0;"|'''% Oxidized'''
| align="center" style="background:#f0f0f0;"|'''Potential'''
|-
| -2.633063185||-0.5122
|-
| 0||-0.5803
|-
| 3.260060544||-0.4781
|-
| 30.97191805||-0.4045
|-
| 82.70859316||0.0146
|-
| 98.04493512||-0.3879
|-
| 98.62977455||-0.355
|-
| 100||-0.3519
|}
<br>
*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
<br>
Table 2. Redox titration of HRP % reduced data
<br>
{| {{table}}
| align="center" style="background:#f0f0f0;"|'''% Reduced'''
| align="center" style="background:#f0f0f0;"|'''Potential'''
|-
| -2.73671656||0.0146
|-
| -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
|}
<br>
==Notes==
The use of the midpoint cell potential of unmodified HRP will be used in further experiments to compare the effect of Au nanoparticles. 


at 750 the data should be flat. (subtracted the average from around 750 from the negative values)


normalize the data for concentration(divide absorbance by concentration)
figure out what percentage something is oxidized or reduced(shifted to higher wavelengths)
percent oxidized= (abs.(max wavelength) partially oxidized-abs.(max wavelength)fully reduced)/(Abs.(max wavelength)fully oxidized-Abs.(wavlength)fully reduced)
*All of these should be the same wavelength!!!
plot percent oxidized/reduced versus cell potential
for cell potential use the minimum
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|}
|}


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__NOTOC__

Revision as of 18:01, 23 September 2013

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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)
1 1
2 1.05
3 1.055
4 1.056
5 1.059
6 1.064
7 1.074
8 1.084
  • Sample 1 contained solely HRP. The additional volumes were sodium dithionite.


Figure 1. Absorbance Spectra of the redox titration of HRP

  • 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

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
82.70859316 0.0146
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
-2.73671656 0.0146
-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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