User:Nancy T. Miles/Notebook/N.Miles Lab 2013-09-03/2013/09/17
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To determine the amount of reagent required to fully oxidize, using potassium ferricyanide, or reduce, using sodium hithionite horseradish peroxidase
The following procedures for this experiment were obtained from User:Matt_Hartings/Notebook/AU_Biomaterials_Design_Lab/2013/09/17
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.
The following data are from the experiment conducted by Dr. Hartings
3.0533g Tris and 1.4677g NaCl in 500mL water. pH set to 7.5 with 3M HCl
Final concentration: 50.4mM Tris 50.2mM NaCl
Buffer degassed by bubbling nitrogen through it for 3 hours
19.8mg in 10mL degassed buffer --> 11.3mM
2.5mL of this solution was diluted to 25mL with degassed buffer to make a final stock concentration of 1.13mM. This solution was degassed an extra 30 minutes.
35.1mg in 10mL degassed buffer --> 10.7mM
2.5mL of this solution was diluted to 25mL with degassed buffer to make a final stock concentration of 1.07mM. This solution was degassed an extra 30 minutes.
6.9mg of HRP in 10.0mL degassed buffer --> 17uM
After the student experiments, we observed that there were no changes taking place, I went back and took a couple of spectra on my own.
Here are the spectra