User:Alexander Cvitan/Notebook/Experimental Biological Chemistry Lab/2013/09/17

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Objective

  • Complete the procedure as outlined by Dr.Hartings, here<br.>
  • Find out the amount of potassium ferricyanide and sodium dithionite it will take to fully oxidize and reduce a sample of horseradish peroxidase.

Procedure

  • It was suspected that the stock solutions we were using during lab today weren't concentrated enough in order to complete our objectives. As a result, we will be using data put together by Dr.Hartings presented in the link above.<br.>
  • All measurements outlined in the procedure were performed using micropipettes. <br.>

Data

  • The graphs represented below represent the data that was collected from our experiment. Based off of the result because all of the lines overlap it proves that not enough oxidation or reduction reagent was added to cause any measurable change.<br.>

Sept 17 oxid zoom out.pngSept 17 oxid zoom in.png<br.> Sept 17 reduc zoom out.png Sept 17 Reduc zoom in.png<br.>

PLEASE NOTE THAT THE DATA REPRESENTED BELOW WAS COMPLETED BY DR.HARTINGS

Buffer

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

Sodium Dithionite

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.

K3[Fe(CN)6]

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.

Horseradish Peroxidase

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.

Samples

  1. 250uL of HRP stock solution and 750uL of concentrated (~10mM) Dithionite stock solution
  2. 500uL of HRP stock solution and 500uL of concentrated (~10mM) Dithionite stock solution
  3. 750uL of HRP stock solution and 250uL of concentrated (~10mM) Dithionite stock solution
  4. 250uL of HRP stock solution and 750uL of concentrated (~10mM) K3[Fe(CN)4] stock solution

Here are the spectra

  • The first graph represents the larger view of the spectra including all peaks.<br.>

20130917 MRH HRP Redox 1.png

  • This graph represented a zoomed in view of our peak on interest.<br.>

20130917 MRH HRP Redox 2.png