THE ENZYME KINETIC ACTIVITY OF

CHYMOTRPYSIN: TEMPERATURE AND pH EFFECTS

Enzyme activity is influenced by temperature and pH of the reaction conditions. The purpose of
this experiment is to explore both temperature and pH effects on the activity of the enzyme
called chymotrypsin. A series of enzymatic reactions will be constructed in the following
manner:

1. Place 2.0 mL of pH 5.0, 6.0, 7.0, 8.0, 9.0 and 10.0 buffer respectively into separate
reaction cuvettes (13x100 test tubes).

2. Pipette 0.25 mL of stock chymotrypsin enzyme solution into each of the cuvettes.

3. Mix each of the tubes thoroughly by covering with Parafilm and inverting several times.

4. Beginning with the pH 5.0 buffer, take the cuvette and calibrate your SpectroVis Plus.
Your blank is the buffer and enzyme without substrate.

5. Select “configure spectrophotometer” (second icon to the left of the collect button) and
click on absorbance versus time. Set the wavelength as close as possible to 410nm.

6. Select “data collection” and change the duration time to 5 minutes and collect
measurements every 30 seconds.

7. You are now ready to start collecting your absorbance data. To start the enzymatic
reaction, take the cuvette out of the instrument and pipette 0.25 mL of substrate known
as N-glutaryl-L phenylalanine-p-nitroanilide (GPNA) solution into the cuvette, place a
small piece of Parafilm over the opening of the tube and MIX the contents. AS FAST AS
POSSIBLE, PLACE THE CUVETTE INTO THE CUVETTE SLOT AND CLICK ON THE
GREEN “COLLECT” BUTTON.

8. The SpectroVis Plus software will tabulate the absorbance data and automatically stop
after the 5-minute reaction time. ***Write down a few data points for each enzyme
condition in case the columns get mixed up later. ***Label the top of the column in
Loggerpro to be sure the columns are clear. The latest run in Loggerpro is always in
column A and B.

9. After completing the data collection for the pH 5.0 buffer sample, repeat the same
process using the pH 6.0 tube. NOTE: After you click on the green “COLLECT” button
the second time, you will be prompted to “store latest run” before the next absorbance
readings will be recorded. It is important that you move through this step as quickly as
possible. Repeat until all the tubes including the pH 10 buffer have been evaluated.

Now that you have completed kinetic studies for each of the 6-different buffered enzyme
reactions, line-up six (6) new, dry cuvettes in a test tube rack to test the effect of temperature on
enzyme kinetics.

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 10. Add 2.0 mL of pH 7.0 buffer to each of the 6 cuvettes (13x100 test tubes).

11. Add 0.25 mL of chymotrypsin enzyme to each of the cuvettes.

12. Taking each one of the six tubes at a time, sit one of them in a 0°C water bath for 10
min. or longer, the next one into a 10°C water bath, another into a 20°C water bath, the
other into a 30°C water bath, the other into a 40°C water bath, and the last into a 50°C
bath. Be sure to cover your tubes with Parafilm so that condensation on the lid to the
water baths does not drip into your reaction tube.

13. After 10 minutes or longer remove one of the tubes to start your experimental
measurements of temperature effects on enzyme activity.

14. TAKE ONE TUBE AT A TIME FROM THE WATER BATHS AFTER AT LEAST 10
MINUTES OF INCUBATION, DRY THE OUTSIDE OF THE TUBE. Calibrate the
SpectroVis Plus using the reaction cuvette containing pH 7.0 buffer and enzyme without
substrate as the blank, just as you did in the previous section.

15. Add 0.25 mL of GPNA substrate as fast as possible, mix the tube contents, and
measure the absorbance value over a 5.0-minute period as done in the earlier section.

16. Repeat this for each of the five (5) remaining tubes.

17. Cleanup: Reaction liquid pour in hazardous waste. Regular glass test tubes dispose of
in glass waste.

DATA ANALYSIS:

1. Plot the absorbance versus time for each reaction tube (you can combine tubes 1 to 6,
effect of pH, on one graph and tubes 7-12, effect of temperature, on a second graph).

2. Determine the slope of each line (linear regression of the trend line for each reaction).
The slope is equal to the initial velocity (ΔA/min) for each reaction tube.

3. Convert the change in absorbance per minute (ΔA/min) into change in GPNA
concentration per minute (e.g. μM/min) using Beer’s law and a molar extinction
coefficient of 8200 M-1cm-1 for GPNA.

4. Plot the change in concentration per minute (μM/min) which equals the velocity, versus
the pH for each of the buffer effects observed in each of the six reactions.

5. Plot the change in concentration per minute (μM/min) which equals the velocity, versus
the temperature for each of the temperature effects observed at pH 7.0 for the second
set of reactions.

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Some information for the data analysis:

1. Stock solution of the substrates GPNA and SPNA is at 1.5 x 10-2 M in DMF

2. Stock solution of the enzyme chymotrypsin was made by dissolving 0.25 g of

chymotrypsin in 100 mL of 1 x 10-3 M HCl. Molecular weight of chymotrypsin is
approximately 25,000.

Structure of GPNA