User:Helen L. Slucher/Notebook/CHEM 571/2013/09/17: Difference between revisions
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== | ==Objective== | ||
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 [http://en.wikipedia.org/wiki/Potassium_ferricyanide potassium ferricyanide], K<sub>3</sub>[Fe(CN)<sub>6</sub>]. K<sub>3</sub>[Fe(CN)<sub>6</sub>] has a standard reduction potential of 424mV ([http://pubs.acs.org/doi/abs/10.1021/ja00902a014 ref1] and [http://pubs.acs.org/doi/abs/10.1021/ja01018a013 ref2]) vs [http://en.wikipedia.org/wiki/NHE NHE]. For HRP reduction, we will be using [http://en.wikipedia.org/wiki/Sodium_dithionite 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 K<sub>3</sub>[Fe(CN)<sub>6</sub>, which has an absorption feature at 420nm (for Fe<sup>2+</sup>, ε = 4.7 M<sup>-1</sup>cm<sup>-1</sup>). This is being done in preparation for our experiments tomorrow where we will be determining the redox potential of HRP. | |||
==Description== | |||
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. | |||
<u>oxidation</u> | |||
# Take a spectrum of the buffer we are using | |||
# Make a 1mL HRP solution in your cuvette with a final concentration of between 5 and 10uM | |||
## Allow this sample to diffuse over 5 minutes | |||
## Note gently add all reagents together (slowly pipette) in order to reduce the amount of oxygen added to the system | |||
## Take a spectrum of this sample | |||
# Add 1uL of the K<sub>3</sub>[Fe(CN)<sub>6</sub>] solution to your cuvette with HRP | |||
## Allow 5 minutes for the sample to equilibrate | |||
## Take a spectrum | |||
# Repeat the previous step until the HRP spectrum is unchanged from the time before | |||
## Note - It may take a few additions before you see any change from the original | |||
## 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 | |||
## 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 K<sub>3</sub>[Fe(CN)<sub>6</sub>], you will be changing your HRP concentration. | |||
# Note the amount of K<sub>3</sub>[Fe(CN)<sub>6</sub>] required to fully oxidize your sample. Determine the ratio of concentration of K<sub>3</sub>[Fe(CN)<sub>6</sub>]/HRP in the fully oxidized sample. | |||
<u>reduction</u> | |||
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. | |||
[http://www.sciencedirect.com/science/article/pii/S0003269704009054 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. | |||
[http://pubs.acs.org/doi/abs/10.1021/bi9816857 This reference] goes more into detail into the kind of experiment we will be performing tomorrow. Note specifically the <i>Redox Titrations</i> portion of the Materials and Methods section. | |||
Revision as of 18:18, 16 September 2013
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ObjectiveToday 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. DescriptionIn 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. oxidation
reduction 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.
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