no. UP2261). The thermometer was allowed to equilibrate with
the temperature of this solution for 1 minute. Once the temperature became steady, 4 mL of activated yeast solution was added.
The temperature of the solution was recorded immediately upon
addition of yeast and then every 10 seconds for a total of 100 seconds
Temperature: To test the effect of temperature on the activity of
the enzyme, 4 mL of the yeast solution was heated in a dry heat
block at 100°C for 10 minutes; after heating, it was placed on ice
for 4 minutes to rapidly cool, then finally equilibrated to room temperature for 5 minutes. This treated yeast mixture was then used in
place of the 4 mL of activated yeast solution in the baseline procedure described above.
pH: To investigate the effect of pH on the activity of the
enzyme, the pH of 4 mL of the yeast suspension was raised to
pH 12.0 by the dropwise addition of 1 M NaOH. The pH was
monitored using pH test strips. This mixture was equilibrated at
room temperature for 2 minutes and then used in place of the
4 mL of activated yeast solution in the baseline procedure
2× Substrate: To investigate the effect of changing substrate
concentration on the activity of the enzyme, 1 mL of 3% H2O2
was substituted for the 0.5 mL of 3% H2O2 + 0.5 mL water. This
substitution doubles the concentration of substrate in the test
tube. The digital thermometer was allowed to equilibrate with
the temperature of this solution by placing the thermometer in
the solution for 1 minute. Once the temperature became steady,
4 mL of activated yeast solution was added as before, and the
temperature change was recorded as described above.
0.5× Enzyme: To investigate the effect of changing enzyme concentration on the activity of the enzyme, 2 mL of activated yeast solution
was added to 2 mL of water. The substitution of 4 mL of activated yeast
solution with 2 mL of yeast solution and 2 mL of water effectively dilutes
the enzyme in the reaction to half of the original concentration. This
mixture was used in place of the 4 mL of activated yeast solution in
the baseline procedure described above.
The 3°F increase in temperature in the baseline experiments confirmed that the experimental setup was efficient in capturing the
indirect enzyme activity. Plotting the change in temperature as a
function of time for the baseline procedure exhibits a classic
enzyme activity graph (Figure 1). The reaction is quite rapid. Temperature increases 3°F within the first 20 seconds, and the reaction
reaches completion less than 1 minute after addition of substrate.
Results of all experiments are listed in Table 2.
Alterations in optimum temperature and pH of the enzyme
substantially affect the progression of an enzyme-catalyzed reaction.
Heating the enzyme to 100°C (Figure 2) or increasing the enzyme
Table 2. Temperature (°F) change indicating enzyme activity.
Time (sec) Baseline Temperature pH 2× Substrate 0.5× Enzyme
0 72.5 72.3 72.0 72.5 72.3
10 75.5 72.3 72.5 77.1 74.0
20 75.5 72.3 72.5 77.9 74.7
30 76.0 72.3 72.5 78.0 74.9
40 76.1 72.3 72.5 78.0 75.0
50 76.1 72.3 72.5 78.3 75.0
60 76.2 72.3 72.5 78.2 75.0
70 76.2 72.3 72.5 78.2 75.0
80 76.2 72.3 72.5 78.2 75.0
90 76.2 72.3 72.5 78.1 75.0
100 76.2 72.3 72.5 78.1 75.0
Figure 1. Temperature increase over time as a result of the
exothermic reaction of yeast peroxidase. A suspension of yeast
activated with sucrose was mixed with H2O2, and temperature
increase was measured at 10-second intervals for 100 seconds.