Kinetics Problem set 2009
Tony Carruthers
A student has purified a novel adenosine binding protein from rat cerebral cortex
neurons. He decides to perform an in vitro binding assay to determine the adenosine
binding properties of this novel neuronal protein.
He reconstitutes the binding protein at 4 nM and measures the time course of binding at
0.5, 1, 5 and 10 µM [adenosine]. The results are shown in Table 1.
He also measures adenosine binding in the presence of an adenosine antagonist
A2001 ([A2001] = 2 µM) to determine whether the antagonist simply displaces
adenosine from its binding site or prevents bound adenosine from transducing its effect.
The results are shown in Table 2.
Answer the following questions:
1) Is adenosine binding a first order process?
2) What is KD for adenosine binding to the adenosine binding protein? Use two different
methods to answer this question - one method must include the use of data when
binding is not at equilibrium, the second must use equilibrium binding data.
3) What are the first and second order rate constants describing adenosine dissociation
from (koff) and association (kon) with the adenosine binding protein respectively?
Watch your units carefully.
4) How do you think the inhibitor affects the binding protein? (Ask how does Kd(app)
change for adenosine binding to determine the type of inhibition produced by A2001)
5) What is Ki for A2001 interaction with the adenosine binding protein?
6) How does the inhibitor affect kon and koff?
7) How many adenosine binding sites are present per adenosine binding protein?
Hints:
It may be easier if you convert concentrations to µM to do the math.
Assume that binding at 5000 seconds has achieved complete equilibrium.
Use Kaleidagraph or Prism software for your analyses. These are available at:
http://inside.umassmed.edu/is/acs/graphing.aspx
Table 1 adenosine binding in the absence of antagonist
[adenosine]free
M
0.5e-6
a
1e-6
a
5E-06
a
1E-05
a
Kinetics Problem set 2009
Tony Carruthers
Table 1 adenosine binding in the absence of antagonist
time sec
[adenosine ]b
b
[adenosine ]b
b
M
[adenosine ]b
b
M
[adenosine ]b
b
M
M
0
5
10
20
0.00E+00
3.88E-11
7.54E-11
1.42E-10
0.00E+00
7.73E-11
1.49E-10
2.79E-10
0.00E+00
3.71E-10
6.91E-10
1.20E-09
0.00E+00
7.08E-10
1.26E-09
2.02E-09
40
60
5000
2.54E-10
3.42E-10
6.67E-10
4.90E-10
6.50E-10
1.14E-09
1.86E-09
2.23E-09
2.67E-09
2.77E-09
3.04E-09
3.20E-09
aThe
4 binding assays were performed at the [adenosine ]free shown above each
column.
bThe
concentration of bound adenosine that was measured is shown at each time point
for each [adenosine ]free used.
The concentration of adenosine binding protein was 4 nM.
Table 2 adenosine binding in the presence of 2 µM antagonist A2001
[adenosine]free
0.5e-6
1e-6
a
5E-06
a
1E-05
a
a
M
time sec
[adenosine ]b
b
M
[adenosine ]b
b
M
[adenosine ]b
b
M
[adenosine ]b
b
M
0
5
10
0.00E+00
1.95E-11
3.79E-11
0.00E+00
3.88E-11
7.54E-11
0.00E+00
1.90E-10
3.63E-10
0.00E+00
3.71E-10
6.91E-10
20
40
60
5000
7.18E-11
1.29E-10
1.76E-10
3.64E-10
1.42E-10
2.54E-10
3.42E-10
6.67E-10
6.59E-10
1.10E-09
1.40E-09
2.00E-09
1.20E-09
1.86E-09
2.23E-09
2.67E-09
aThe
4 binding assays were performed at the [adenosine ]free shown above each
column.
bThe
concentration of bound adenosine that was measured is shown at each time point
for each [adenosine ]free used.
The concentration of adenosine binding protein in all experiments was 4 nM.