User:Mary Mendoza/Notebook/CHEM572 Exp. Biological Chemistry II/2013/02/13

Trial 2 & 3 of ADA Kinetic Assay

• The agenda for this laboratory period are the completion of trials 2 and 3 of ADA Kinetic Assay and obtain the K_m and V_max of the enzymatic reaction.
• UV2550 Shimadzu Spectrophotometer was baseline with phosphate buffer. The temperature control of the CPS controller was set at 25°C.
• Baseline was performed by preparing two cuvettes filled with phosphate buffer, one placed on the blank slot and the other at cell slot 1.
• After baseline, an assay containing 150 μM adenosine was collected using the UV Probe. Trial 2 was complete by running an assay with the 200 μM adenosine.
• The concentration of each sample was obtained from the calculations below:

• During the previous laboratory data collection of 100 μM trial 2, an error was encountered by the opening of the cell chamber. Thus, it was decided to repeat the run for 100 μM.
• Ran kinetics on 2.56 uM solution (20uL ADA, 600uL 12.8 uM adenosine, and 2.38 mL pH7.4, 0.05M phosphate buffer) for 5 minutes at 265 nm.
  • dAbs was obtained and plotted vs. time in excel.
• Ran kinetics on 6.4 uM solution (20uL ADA, 600uL 32 uM adenosine, and 2.38 mL pH 7.4, 0.05M phosphate buffer) for 5 minutes at 265 nm.
  • dAbs was obtained and plotted vs. time in excel.
• Ran kinetics on 16 uM solution (20uL ADA, 600uL 80 uM adenosine, and 2.38 mL pH 7.4, 0.05M phosphate buffer) for 5 minutes at 265 nm.
  • dAbs was obtained and plotted vs. time in excel.
• Ran kinetics on 40 uM solution (20uL ADA, 600uL 200 uM adenosine, and 2.38 mL pH 7.4, 0.05M phosphate buffer) for 5 minutes at 265 nm.
  • dAbs was obtained and plotted vs. time in excel.
• Ran kinetics on 100 uM solution (20uL ADA, 600uL 500 uM adenosine, and 2.3 8mL pH 7.4, 0.05M phosphate buffer) for 5 minutes at 265 nm.
  • dAbs was obtained and plotted vs. time in excel.
• Ran kinetics on 150 uM solution (20uL ADA, 600uL 750 uM adenosine, and 2.3 8mL pH 7.4, 0.05M phosphate buffer) for 5 minutes at 265 nm.
  • dAbs was obtained and plotted vs. time in excel.
• Ran kinetics on 200 uM solution (20uL ADA, 600uL 1000 uM adenosine, and 2.3 8mL pH 7.4, 0.05M phosphate buffer) for 5 minutes at 265 nm.
  • dAbs was obtained and plotted vs. time in excel.
• Average velocities were obtained.
• The completion of trials 2 & 3 were followed by the calculation for the K_m and V_max. Data was exchanged with Catherine Koenigsknecht and Michael Nagle.
• The excel worksheet of the finalized data is attached below.

Kinetics Assay