ENERGY OF ACTIVATION AND RELATIVE RATES
FST 605 - Laboratory
Date Performed: January 13, 2004
Report Due: January 20, 2004
Reagents:
Sucrose - 100 mM in . 05 M acetate buffer
at pH 4. 8.
Invertase Enzyme Solution
3, 5-Dinitrosalicylic Acid Reagent - l g 3,
5-dinitrosalicylic acid ( DNSA) + 30 g sodium potassium tartrate
+ 20 ml of 2N NaOH to 100 ml with water.
Procedures:
The effects of temperature on enzyme
activity. A number of water baths ranging from 4°C to 40°C will
be found in the room. Each group is to perform analysis at RT and
at two additional temperatures. These temperatures will be
assigned by theinstructor . For each temperature, place a tube
containing 20 ml of substrate solution and another containing 1.0
ml of enzyme solution in the water bath and allow them to
equilibrate for 5 minutes. Place 2 ml of DNSA solution in each of
5 test tubes. Place 0.2 ml of enzyme solution in the tube
containing the substrate and mix well by inverting the tube
twice. Remove 1.0 ml of this solution and add it to a tube
containing 2 .0 ml of DNSA. This sample will be counted as the 0
time sample. Remove additional samples at 2, 4, 6 and 8 minutes.
Place the tubes in a boiling water bath for exactly 5 minutes.
Cool the tubes for 5 minutes, then record the absorbance of the
solution at 540 nm.
Results
Present all of your data. Plot the reaction
rate data obtained for the three temperatures you performed. Data
obtained by other groups at other temperatures will be given to
you by the instructor. Use this information to obtain an energy
of activation for the inversion of sucrose by the enzyme.
Questions
1.What is the Ea you obtained for the
reaction? How does this compare to literature values?
2. The reported Ea for the acid is 25.2
Kcal/mole. What would be the relative rates of these two
reactions at 50°C?
3. At what temperature does the rate of the
inversion of sucrose by acid equal the rate obtained by the
enzyme at 37°C?
4. Given the information obtained in
Question 1 if you measured the rate of invertase at 30°C but
later found that your thermometer was not properly calibrated and
the reaction was actually measured at 33°C, how large would your
error be?
5. An enzyme catalyzed reaction was found
to be 3 times faster at 37°C than at 12°C. What was the energy
of activation of the enzyme catalyzed reaction? What can you say
about DG of the reaction?