To begin, prepare the serum and enzyme mixtures along with their respective controls. In the microplate reader, set up a kinetic read protocol to measure the absorbance at 260 nanometers with the shortest reading interval for 30 minutes. Next, thaw all the frozen samples on ice and dilute each thawed sample 1 to 10 with 1x PBS pre-warmed to 37 degrees Celsius in LoBind microcentrifuge tubes.
To prepare a five millimolar adenosine stock, add 66.8 milligrams of adenosine to 50 milliliters of 1x PBS. After diluting it to a 250 micromolar solution, pre-warm the adenosine to 37 degrees Celsius in an incubator. To a 96-well ultraviolet compatible microplate, add 160 microliters of the adenosine followed by 40 microliters of each diluted protein sample in triplicates.
Measure the absorbance of each well at 260 nanometers for 30 minutes at 37 degrees Celsius. For data analysis, export the data to a spreadsheet, then determine the slope of the linear region of the absorbance data as a function of time for the first several minutes. Subtract the slope of the negative control consisting of pooled human serum or PBS from the slopes of the enzyme plus serum mixtures and the enzyme plus PBS mixtures respectively.
Finally, normalize all the adjusted slopes to the original slope at the zero hour time point to obtain the fraction of activity remaining using the equation, and plot the fraction of activity remaining versus time. Wildtype HsADA1 activity decreased more rapidly in pooled human serum compared to 1x PBS over five days. The absorbance decline curves for wildtype HsADA1 showed a steeper initial slope at day zero compared to day five.
Negative control samples showed negligible absorbance change compared to enzyme samples.
