- To obtain a spectrum of 40 μM adenosine and inosine
- A run of 40 μM adenosine with 50 μM varying inhibitors were conducted using the UV 2500 Shimadzu spectrophotometer.
- Three runs of each inhibitor were taken; the average of the runs were taken. The standard deviation were calculated and added as error bars.
- Produce histogram
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
- Cuvette Mixture with Inhibitor
- 50 μM 3-methylacetylsalicylic acid (ZINC 00001221)(Aldrich S376647-1G)
- 50 μM Methyl (4-hydroxy-3-methoxyphenyl)acetate (Aldrich PH003466-1MG)
- 50 μM [3,4-bis(acetyloxy)benzoic acid] (Aldrich PH00962-150MG)