User:Zachary I. Mendel/Notebook/Zacks Notebook/2013/10/08

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Objective

To observe and measure ADA turnover kinetics in the absence of an inhibitor. This work will be the basis of our comparison to ADA-AuNP turnover studies.

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 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

Notes

Table 1. Preparation of 0.0123M stock solution

Mass of adenosine (g) 0.0165
Volume of buffer(mL)5


Table 2. Preparation of 40μM adenosine solution

Volume of stock(mL) 0.0098
Volume of buffer(mL)2.9902

Figure 1. Corrected Absorbance spectra of the conversion of adenosine to inosine by ADA
Image:Adakinetics10082013zem.png

Table 3. Molar Absorptivity of Adenosine at 260nm and Inosine at 250nm

Wavelength Adenosine Inosine
2501333311937.5
26016866.77312.5


Figure 2. Monitoring concentration versus time of the conversion of adenosine to inosine using ADA as a catalyst.
Image:ConcvstimeadentoinoADAcatalystzem.png


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