User:Carly M. Montanero/Notebook/CHEM-571/2013/10/08

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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.

From Dr. Hartings.

Procedure

Creating the Adenosine Stock

  1. Add 0.1071 g of adenosine to a 10 ml volumetric flask. Fill with 50 mM phosphate buffer at pH 7.4. This created a stock solution of 0.04 M.
  2. Add 9.98 μL of the 0.04 M adenosine stock to a 10 mL volumetric flask. Fill with 50 mM phosphate buffer at pH 7.4. This created a final solution of 40 μM.

Enzyme Kinetics Measurement

  1. Add 3 mL of 40 μM adenosine solution to the cuvette.
  2. Start kinetics measurement:
  • 1 ms integration
  • 10 scan average
  • Set "Save the first available scan every" to 15 seconds
  • Set "Stop after this amount of time" to 10 minutes
  • Set "File Type" to Tab Delimited
  • Just before 1 minute, add 30 μL of 0.01 units/mL ADA.

Figures

Cmj 10.09.13 ADAnoinhibitor.png

Notes

Adenosine deaminase (ADA) stock

  • 1.1mg (24.0 units in 1 mg) in 25mL of buffer --> 1.1 units/mL ADA

ADA for experiments

  • 100uL of stock ADA and 900uL buffer --> 0.11 units/mL ADA