The procedure was taken from Dhea Patel's notebook entry.
The assay runs were continued from last week's, 02/05/2013, calculated values.
The objective of performing the assay is to obtain the rate of the enzymatic reaction of adenosine into inosine by ADA.
Baseline 200-400nm using pH 7.4, 0.05M phosphate buffer
Temperature control: 25°C
Phosphate buffer in reference cell
Ran kinetics on 250uM solution (20uL ADA, 600uL 1250 uM adenosine, and 2.38mL pH 7.4, 0.05M phosphate buffer) for 5 minutes at 265 nm.
absorbance exceeded 4.00, so the assay was stopped and a new solution was prepared.
Ran kinetics on 2.56uM solution (20uL ADA, 600uL 12.8 uM adenosine, and 2.38mL 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 6.4uM solution (20uL ADA, 600uL 32 uM adenosine, and 2.38mL 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 16uM solution (20uL ADA, 600uL 80 uM adenosine, and 2.38mL pH 7.4, 0.05M phosphate buffer) for 5 minutes at 265 nm.
dAbs was obtained and plotted vs. time in excel
The following table describes the volumes and concentrations of the components added to the cuvette.
Note: Since the 250 μM adenosine assay exhibited a high absorbance, this concentration was replaced with two other adenosine concentrations, 150 and 200 μM.
The stock solution for 150 μM adenosine was made by diluting 150 μL of 5 mM adenosine into 850 ng/μL of 0.05 M phosphate buffer. The 200 μM adenosine was made by diluting 200 μL of 5 mM adenosine into 800 μL of 0.05 M phosphate buffer.