AU Biomaterials Design Lab:Protocols/ADA Activity Assay
Adenoise Deaminase Kinetic Assay
- 1) Use 5 ml of 0.1 mM adenosine in phosphate buffer, & 5 ml of 0.1 mM inosine in phosphate buffer (0.05 M potassium phosphate, pH 7.4)
Collect a spectrum of the adenosine, the inosine and the ADA solutions. Find the extinction coefficient of adenonise and inosine at 235 nm and 265 nm.
Which wavelength should you use for this assay?
Determination of Kinetic Parameters
Reagents and Supplies
- 0.05 M potassium phosphate, pH 7.4
- adenosine deaminase (your most pure sample)
- 3 mM adenosine (in KH2PO4 buffer)
2) Level of Adenosine Deaminase to Use in the Kinetic Assays Find the amount of enzyme to use in the kinetic assays by completing the following assays. Prepare 3 ml of assay solutions according to the following table and record the absorbance changes at 235nm. (The volumes of ADA given are typical but you may need to expand the range if your activity is dilute OR dilute your sample in ADA assay buffer if it's highly active.)
|Assay||Phosphate buffer [mL]||Adenosine [mL]||ADA solution [µL]|
- It will be necessary to find a suitable volume of your stock enzyme solution that produces a ΔA235/min of 0.02-0.03 under conditions of saturating levels of substrate as found in the above solutions. Construct a graph in your notebook of ΔA/min versus amount of enzyme (µl) used and determine the volume of enzyme to use for the remaining kinetic measurements. You may round the calculated volumes to a convenient volume (e.g., 10, 15, 20, etc. µl).
This graph should demonstrate that the reaction rate is linearly dependent on the level of enzyme at saturating levels of substrate.
3) Km's of Adenosine and 2'-Deoxyadenosine
- To determine Km and Vm for a particular substrate, the rate (or velocity) of the reaction is measured at various concentrations of the substrate.
(The amount of enzyme and the total volume of the assay solution must be held constant ±2% of the total volume. Note that in part 2 the total volume of the assay solution varied by 35 µl. This change represents less than 2% of the total solution and normal pipetting errors are expected to be about 3% due to multiple additions. Therefore, these small volume changes are within the experimental error and will not make a significant contribution to the total error.)
- Construct a chart in your notebook of the volumes of each component added to the reaction mixture so that the total volume is 3 ml. Measure the reaction rate with 0.005, 0.010, 0.020, 0.040, 0.080, 0.10, 0.30 ml of adenosine solution. Compare the reaction rates of the 0.1 ml and the 0.3 ml adenosine assays. If these rates are not within 10% of each other, try 0.35 ml of adenosine. A graph of rate (nmol/min) versus [adenosine] (µM) should show that an asymptote is being approached.
- From the plot of reaction rate versus substrate concentration (µM) estimate the Km for adenosine.
- With the data from Part 2, construct a graph of velocity (nmol/min) versus [substrate] (in µM) to demonstrate that a saturating level of substrate has been reached. Draw a smooth curve described by the data points. Estimate Vm and Km from this plot. The Vm is the asymptote that is being approached and is read from the y-axis. Extend a horizontal line at the half maximal velocity (Vm/2) and determine the concentration of substrate where the horizontal line intersects your plot.
- Prepare a graph of 1/velocity versus 1/[adenosine] . This graph can eventually be used to compare to the Vm and Km for ADA /AuNPs kinetics.
For comparison. Suggested table for your notebook: Assay [adenosine] in assay (µM) 1/[adenosine] (1/µM) deltaA/min rate (nmol/min) 1/rate (min/nmol)