User:Mary Mendoza/Notebook/CHEM572 Exp. Biological Chemistry II/2013/04/19

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Procedure for Adenosine and Inosine Spectrum

  • To obtain spectrum of 40 μM adenosine, dilute 600 μL of 200 μM adenosine stock solution in 2400 μL 0.05 M phosphate buffer.
  • Stock solution of 200 μM inosine (MW 268.2 g/mol) was prepared. A spectrum was taken using the same dilution mentioned above.

Protocol for Assay

  • The reagents were added in the following sequence:
    • 1.78 mL of phosphate buffer
    • 600 μL of 200 μM adenosine (final concentration 50 μM)
  • This was allowed to mix through venting and placed on the chamber for absorbance reading.
    • 300 μL of phosphate buffer (or inhibitor dissolved in phosphate buffer)
    • 300 μL of dimethyl sulfoxide (DMSO) (or inhibitor dissolved in DMSO)
  • The cuvette contents were mixed by venting and placed on the chamber for absorbance reading.
    • 20 μL of adenosine deaminase (ADA) was added to catalyze the reaction
  • The kinetics of the reaction was taken using the UVProbe software.
  • Cuvette Mixture no Inhibitor

Image:CuvetteMixtureNoInhibitor.png

  • Cuvette Mixture with Inhibitor

Image:CuvetteSolnInhibitorHisto.png

  • Inhibitors Ran today:
  1. 50 μM 3-methylacetylsalicylic acid (ZINC 00001221)(Aldrich S376647-1G)
  2. 50 μM Methyl (4-hydroxy-3-methoxyphenyl)acetate (Aldrich PH003466-1MG)
  3. 50 μM [3,4-bis(acetyloxy)benzoic acid] (Aldrich PH00962-150MG)


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