Module 7. Measuring DHFR Catalytic Activity – Effect of enzyme concentration of reaction rate. 1.
Introduction Enzymes are biocatalysts that speed up chemical reactions. The catalytic activity of an enzyme is the number of substrate
molecules converted to product per second per 1 molecule of enzyme.
𝑐𝑎𝑡𝑎𝑙𝑦𝑡𝑖𝑐 𝑎𝑐𝑡𝑖𝑐𝑖𝑡𝑦 𝑘𝑐𝑎𝑡 =
# 𝑠𝑢𝑏𝑠𝑡𝑟𝑎𝑡𝑒 𝑚𝑜𝑙𝑒𝑐𝑢𝑙𝑒𝑠 𝑐𝑜𝑛𝑣𝑒𝑟𝑡𝑒𝑑 𝑡𝑜 𝑝𝑟𝑜𝑑𝑢𝑐𝑡
1 𝑠𝑒𝑐𝑜𝑛𝑑 ∗ 1 𝑚𝑜𝑙𝑒𝑐𝑢𝑙𝑒 𝑜𝑓 𝑒𝑛𝑧𝑦𝑚𝑒
Note in the above equation that the units for catalytic activity will be 1/s. You could of course use a different unit of time,
for example minutes. In this case the units of catalytic activity would be 1/min.
In this module you will determine the catalytic activity for the enzyme DHFR.
You will also examine how enzyme concentration affects your results. The enzyme is a catalyst that speeds up the process
of converting substrate to product. The more enzyme molecules in the reaction vessel, the faster the reaction proceeds.
This is analogous to a factory: the more employees working, the more pieces of leather converted to shoes per hour (or
per minute, or per second). The more enzymes “working” in your tube, the more molecules of substrate are converted to
product per unit time.
To determine the catalytic activity, you will need to know the number of substrate molecules converted to product
molecules per unit time (see the equation above). To do this, you will mix the enzyme with substrate and then see how
fast the substrate is converted to product.
In a typical experiment, you will measure the change (decrease) in substrate concentration or the increase in product
concentration over time. In this experiment you will measure the disappearance of substrate and cofactor by recording the
decrease in Absorbance at 340 nm over time. In your data file, the Y-AXIS will be Absorbance340 nm and the X-AXIS will
be time. Pay attention to the units of the X-axis, be sure to write them down. The slope of the initial linear decrease will
represent the change in absorbance over time (ΔAbs/min) and can be used to calculate initial velocity (v0). To convert the
slope to Δ[substrate]/time, you will use a special extinction coefficient (see below).
To obtain the catalytic activity values, you can divide Δ[substrate]/time by the concentration of enzyme in the experiment.
Just be sure to have the units of enzyme concentration be the same as the units of substrate concentration so that the units
cancel out.
Please bring a memory stick where the measured data can be stored! The data can be shared later for each lab
member via email or everyone can save the data to their own memory stick.
2.
Purpose of the lab
Your lab assignment is to run the reaction (DHF + DHFR + NADPH → THF + DHFR + NADP+) and monitor the
disappearance of the substrate (DHF) and cofactor (NADPH). This data will allow you to determine the catalytic activity
of the enzyme you purified. You will also do the experiment with different enzyme (DHFR) concentrations to optimize
the experiment. DHFR concentration is the variable, all other parameters are kept constant.
Your assignment is to examine the effect of DHFR (enzyme) concentration on the time dependence of the DHFR
catalyzed reaction. You will also use the data to determine the catalytic activity of DHFR.
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3.
Agenda for the Day
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4.
You must have worked through Math Moment problems before coming to class. Upon entry, each student shows
their work to instructor to receive points.
Instructor presentation on Measuring catalytic activity
In class, groups review Math Moment Problems to prepare for experimental design.
Conduct the experiment in groups. Each student individually records data in their personal laboratory notebook.
Clean up
Background
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Useful information can be found in Chapter 8B. Please, read this section.
The enzyme that catalyzes the reaction is DHFR (dihydrofolate reductase) (~18,600 Da). DHFR catalyzes the
conversion of substrate DHF (dihydrofolate) to product THF (tetrahydrofolate).
NADPH (nicotinamide adenine dinucleotide phosphate) is the cofactor required for the reaction. NADPH donates
a hydride (H-) to DHF. NADPH is converted to NADP+ in the reaction. The reaction thus has two starting
materials (DHF and NADPH) and two products (THF and NADP+).
Detecting the reaction: DHF (substrate) and NADPH (cofactor) absorb more light at 340 nm than THF (product)
and NADP+. Today, the plate reader will be set so that it takes many absorbance measurements as the reaction
proceeds at specific time intervals. This is the “kinetic setting”. The plate reader will only give the time values
and the absorbance values, you will use Excel to graph and analyze your data.
The initial velocity for the DHFR reaction will be determined by measuring the rate of enzyme-dependent
decrease in absorbance at 340 nm using the extinction coefficient of 13.2 mM-1 cm-1 and Beer’s law. This means
that the values you obtain for the ΔAbs/time (using a 0.5 cm pathlength in the microtiter plate well) will be
divided by this extinction coefficient and pathlength (0.5 cm) as stated in Beer’s law. Note what the units are
when you do this division when extinction coefficient is expressed in units of mM-1*cm-1 à
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The assay will be conducted in a microtiterplate. The pathlength will be 0.5 cm. Different wells will be identical
experiments except that each one will have a different enzyme concentration.
Provide your kcat value in units of 1/seconds. Note that the instrument give the data in minutes and you will need
to do a conversion.
Concentrations we will use in the assay (final in microtiterplate well) are NADPH (100 µM) and DHF (100 µM).
A Mastermix is provided that contains buffer, NADPH, and DHF at appropriate concentrations.
DHF is a suspension and you must extensively mix Mastermix before each time you add it to another well.
DHF is light sensitive and must be protected from light as much as possible during the experiment (use aluminum
foil for this purpose).
Math Moment
1. What three molecules will be needed in the microtiterplate well, in addition to the buffer, to start the DHFR catalyzed
reaction?
2. What are the products of the reaction catalyzed by DHFR?
3. What wavelength must the plate reader be set to in order to monitor the reaction? Do the starting materials DHF and
NADPH or the products THF and NADP+ absorb more light at this wavelength?
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4. What molecules are present in the beginning of the reaction (time = 0 seconds) in the microtiterplate well?
5. Which molecules are being consumed during the reaction? Which molecules are being made during the reaction?
6. You will be provided a Mastermix solution containing DHF at 118 µM and NAPDH also at 118 µM in buffer. If you
mix 170 µL of mastermix with 30 µL of enzyme solution in a microtiterplate well, what is the final concentration of DHF
in the well? What is the final concentration of NADPH in the well?
7. Do you expect to see increase or decrease in absorbance at 340 nm as the reaction proceeds? Why?
8. What is the extinction coefficient you will use to convert the enzyme-dependent decrease in absorbance at 340 nm to
change in substrate/cofactor concentration? Include units in your answer. See background section.
9. We want to measure kcat, which is equal to Vmax/[E]. Explain why in our experiment the concentration of the
substrate and the cofactor must be much greater than then concentration of the enzyme (saturating concentrations). Think
about the shape of the Michaelis Menten curve.
10. What will happen to the absorbance vs. time slope if you double the amount of enzyme in the assay?
11. If the initial, linear portion of the line has a slope (Δabsorbance/Δtime x cm) of 0.02/second, use the extinction
coefficient above to convert this to a slope that gives the Δ[DHF]/Δtime. Remember to include units in your answer.
Note: you can use Beer’s law in the following way:
Δ(absorbance) = ε * 0.5 cm * Δ[DHF]
Note: Solve for Δ[DHF]!
12. If the enzyme concentration in the assay in the question above was 30 nM, what is the catalytic activity? Note: you
will want to divide the Δ[DHF]/Δtime value by the enzyme concentration (see the equation in the introduction). The units
of the [DHF] and [DHFR] will cancel out as long as you make sure that they are in the same units. You can do an unit
conversion.
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13. Let’s say that you are provided a Mastermix of NADPH and DHF at appropriate concentrations to get the correct
final concentrations where enzyme is saturated with NADPH and DHF. You will add 30 µM of enzyme solution to the
well for a total concentration of 200 µL. You want to make 8 solutions with different concentrations of enzyme. You
want each one to be a 2-fold dilution of the previous one. Please, write into the table how you will make the solutions.
Name of
D1
D2
D3
D4
D5
D6
D7
D8
100 µL of DHFR stock
50 µL of D1
Volume of buffer to add (µL)
0
50
Total volume (µL)
100
100
100
100
100
100
100
100
Final Dilution Factor
1-fold (no dilution)
2-fold dilution
14. See the possible data below. On the graph, label the data 1-6 from the experiment with the HIGHEST enzyme
concentration (data 1) to the lowest enzyme concentration (data 6). Remember that the slope of the initial decrease is the
initial velocity. Think about what happens to the initial velocity as you increase enzyme concentration. Also, label the 3
sets of data that would be best for determining the kcat. When you analyze you data experimental data for this module, do
it for all the data sets that have a good initial slope and then take the average. Remember that you will want to measure the
initial velocity (the initial decrease). Once the reaction runs out of substrate, there is no longer decrease and you see a
horizontal line (plateau). Provide your answer in units of 1/second (note that x-axis here is in minutes). You will need to
do the same conversion for your experimental data.
0.6
Absorbance at 340 nm
0.5
0.4
0.3
0.2
0.1
0
0:00:00
0:01:26
0:02:53
0:04:19 0:05:46 0:07:12
Time in minutes
0:08:38
0:10:05
0:11:31
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15. Use the data shown in filled circles in the figure in the question above to determine the kcat for the experiment in
units of 1/second. Use the extinction coefficient provided in the Background Section. Assume that the enzyme
concentration in this experiment was 2 nM.
16. Below in the graph, draw a reaction curve that you would like to see (Absorbance vs. time). Now add a curve for what
the data would look like if you use way too much enzyme (use thicker line). Think about how, in practice, it will take you
a few moments to mix your solutions in a well, then press the button to start measuring, so your time 0 on the graph is
actually a little bit after the reaction was initiated. Hint: is it possible that the data looks like a straight line. Where would
the line be? Finally, add a line for what the data would look like if you had way too little enzyme (dotted line).
Plot Absorbance (concentration of substrate) vs. time. Label your axes.
6.
Supplies Provided
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Ice buckets and ice
Micropipetters and tips
Multichannel pipettor
96-well microtiter plates
Eppendorf tubes
plate shaker
UV-VIS plate reader
Buffer (40 mM HEPES at pH 6.8)
Mastermix solution containing DHF and NAPDH both at 118 µM in 40 mM Hepes pH 6.8 buffer.
Enzyme (DHFR)R solution (the purified enzyme from Module 6). Note: it will be necessary to convert the
enzyme concentration into units of µM.
Aluminum foil
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7.
Experimental Protocol
1. Label 8 tubes D1 – D8.
2. Make serial dilutions of the DHFR enzyme stock in small Eppendorf tubes as shown in the table below. Mix each
dilution vigorously before making the next dilution. For example, mix D2 vigorously before making D3. Fill in the final
concentrations in the table below (they are based on stock concentration of enzyme).
Dilution
D1
D2
D3
D4
D5
D6
D7
D8
100 µL of DHFR stock
50 µL of D1
50 µL of D2
50 µL of D3
50 µL of D4
50 µL of D5
50 µL of D6
50 µL of D7
Volume of buffer to add (µL)
0
50
50
50
50
50
50
50
Total volume (µL)
100
100
100
100
100
100
100
100
Final concentration
3. Mix the MASTERMIX (provided) vigorously. You must mix MASTERMIX each time before you pipet from it. Place
170 uL of MASTERMIX in wells A1 – A9 in a microtiter plate. Keep plate covered with foil (DHF is sensitive to light).
4. Add 30 µL of buffer to well A9. This is your “no enzyme” control. Since there is no enzyme in well A9, do you
expect to see any change in Absorbance over time?
5.
Add 50 µL solution D8 to well B8. (Note, not the experiment well A8 but next to it!)
Add 50 µL solution D7 to well B7.
Add 50 µL solution D6 to well B6.
Add 50 µL solution D5 to well B5.
Add 50 µL solution D4 to well B4.
Add 50 µL solution D3 to well B3.
Add 50 µL solution D2 to well B2.
Add 50 µL solution D1 to well B1.
6. You must do the next steps (up to when you hit read on the plate reader) as quickly as you can because the reaction will
start when you add enzyme (DHFR). So go to the instrument, make sure all settings are ready to go. Then place plate on
the tray. Only then do the following. Use a multichannel pipettor to transfer 30 µL of solutions from wells B1-B8 to
wells A1-A8.
7. Read the plate (10 minutes).
8. Look at your data. Be prepared to repeat the experiment if necessary.
8.
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Common Mistakes and Some Advice
Remember good pipetting technique. Use the correct pipettor (P20, P200, or P1000) for the volume you
measuring. Please, handle pipettors gently.
In solution, DHF is a suspension. If the suspension is left sitting for a while the solid and liquid phases separate.
Therefore, before you use the mastermix, mix it by pipetting it up and down.
DHF is sensitive to light, keep mastermix covered with foil when not using it.
MAKE SURE TO LABEL TUBES AND RECORD THE WELLS.
Be careful with pipetting. If too forcefully pipetted, reagents may splash into other wells.
Think in which order to fill the wells.
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9.
Vocabulary
Catalytic activity, substrate, cofactor, reaction rate, Kcat
10.
Safety
You must wear safety glasses when conducting the experiment. You must never eat or drink in the laboratory. You will
need to wait in line to use the plate reader. Please, be patient when waiting for your turn to use the plate shaker and plate
reader. Be gentle with the plate reader and plate shaker, these are delicate instruments. Any observed violations of these
rules will result in lower final grade and/or removal from the lab. These safety items are solely the responsibility of the
student.
11.
Clean up
For clean-up, return the remaining original solution to the instructor. Discard dilutions in the sink and eppendorf tubes in
the regular trash. Do not throw anything in the biohazard waster. Wash 96-well plates and leave them at the sink to dry.
Mark the well that you used with a marker. Return pipettors in the correct boxes, the last person puts the boxes in the
cabinet in the back of the lab. Dry your ice buckets and place them back in the cabinet. Place all other items where you
got them from. Make sure they are clean. Leave your bench ready for the next class to start working.
12.
Homework
Data from modules 7 and 8 will combined into a formal lab report (one per group). When analyzing data, you should be
able to use a few of your slopes (a few wells) to calculate kcat. The ones that finished too quickly or too slowly will not
be useful. Remember to use the initial slope (initial linear decrease) to obtain your slope (Δabsorbance/Δtime). Then
convert it to Δ[DHF]/Δtime. Finally, divide by [E] to determine kcat.
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