User:Daniel-Mario Larco/Notebook/AU Biodesign Lab - 09/03/2013/2013/10/09

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Objective

To observe and measure ADA turnover kinetics in the presence of 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 and 1uL of inhibitor 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 30ul of 0.01u/mL ADA

Data

  1. Buffer
    1. 0.7318 g of NaH2PO4·H2O and 5.2520g Na2HPO4·7H2O in 500mL water --> 50mM Phosphate buffer pH 74
  2. Adenosine deaminase (ADA) stock
    1. 1.1mg (24.0units in 1 mg) in 25mL of buffer --> 1.1 units/mL ADA
  3. ADA for experiments
    1. 100uL of stock ADA and 900uL buffer --> 0.11 units/mL ADA

Results

The spectra revealed a shift in the position of the peak. THe peak moved from 260nm to 250nm which is an indication of the formation of Inosine from Adenosine. Here is a graph that illustrates this shift over time.

From our past data, we know that the molar absorptivity at 260nm for Adenosine and Inosine are 14025 and 5254 respectively and the molar absorptivity of inosine at 250nm is 11007 From today's observed that, the molar absorptivity of adenosine at 250nm was 11058.8. Using these values we were able to calculate the concentrations of adenosine and inosine over time using a system of equations. The change in concentration from the initial to final concentrations was put on a scatter plot over time. Here is the resulting graph:

The rate of change from adenosine to inosine was relatively the same as without the inhibitor which indicates that perhaps the amount of inhibitor added was not sufficient.


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