User:Javier Vinals Camallonga/Notebook/Javier Vinals notebook/2013/10/09

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Objective

We are running the same procedure as yesterday only with an inhibitor, EHNA.

Description

  1. Make a 40μM solution of adenosine in buffer (50mM phosphate buffer, pH 7.4)
  2. Go to Dr. Hartings lab for enzyme kinetics measurements.
    1. Add 3mL of adenosine solution to the cuvette
    2. Start your kinetics measurement
      1. 1ms integration (on front panel)
      2. 10 scan average (on front panel)
      3. Set "Save the first available scan every" to 15 seconds (after clicking File>Save)
      4. Set "Stop after this amount of time" to 10 minutes (after clicking File>Save)
      5. Set "File Type" to Tab Delimited
      6. Give the files a directory and a name
      7. Click accept
      8. Just before 1 minute add 1ul of EHNA

Data

EHNA stock

  1. 5mg EHNA in 1mL DMSO ---> 15.9mM EHNA
  2. (1.9μL)(15.9mM EHNA)=(10mL)(C2). C2 ---> 3μM EHNA

The reaction samples will contain roughly 1nM EHNA

After we collected the data, we worked up the data and we found the concentration of inosine and adenosine, based on beer's law, and the peaks 260 and 250, from which we know epsilon, b and the absorbance. After putting the files and working it up, we obtained the graph of Adenosine to Inosine.

"Figure 1. Adenosine to Inosine"

"Table 1. System of equation to calculate concentration of Adenosine"


Adenosine Absorbance at time 0 molar absorptivity Initial concentration
at 2600.435140253.1016*10^-5
at 2500.34311058.79313.1016*10^-5


"Table 2. System of equation to calculate concentration of Inosine"


Inosine Absorbance at time 0 molar absorptivity Initial concentration
at 26005254.513890
at 2500110070


Then after this, we set up a system of equations to find out the concentration at different points during the time the reaction took, and sketched a graph.

"Figure 2. Change in concentrations from Adenosine to Inosine"




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