The document describes two experiments on how temperature and pH affect the activity of the enzyme turnip peroxidase. The temperature experiment found that activity peaked at 32°C and decreased at higher and lower temperatures, supporting that temperature affects reaction rate. The pH experiment determined that activity was highest at pH 5 and lower at more acidic or basic pH levels, demonstrating pH is a determining factor. A proposed experiment on salt concentration hypothesizes that reactivity will decrease with increasing salt concentration.

1. BIOLOGY 211LA-4 EnzymesPage 2 of 2
Effect of temperature on the activity of turnip peroxidase
(If you are struggling with any of the questions, I recommend
thoroughly reading through the first two pages of Enzyme Lab
introduction.)
Question: How does temperature affect the absorption of an
enzyme?
Null hypothesis (avoid using the word “will” in your
hypothesis): Enzyme activity is not affected by temperature.
(If you need help with hypothesis or independent and dependent
variables, I suggest reviewing background information from
Diffusion Lab.)
In this experiment, what are the independent and dependent
variables?
independent: temperature of the solution
dependent: reaction rate (light absorption)
What is the name of the enzyme in this enzyme-mediated
reaction?Peroxidase
What is the substrate molecule in this enzyme-mediated
reaction?________Hydrogen peroxide________
What is/are the product(s) produced in this enzyme-mediated
reaction?H2O & O2
Which tube served as a negative control?
______6____________________
How do you know it is a negative control?

2. 6 is missing substrate in both 6a and 6b.
Paste in your graph below:
Do the data support the null hypothesis? Explain, using the
data (numbers) you obtained to support your conclusion.
The data does not support the null hypothesis because the
temperature was a determining factor of light absorption. When
the temperature was 32 °C, the light absorption peaked at 200
nm (0.4*500 nm), and decreased at a steady incline after that,
dropping to 50 nm at 65 °C, and denaturing at 100 °C. This
shows that there was a peak temperature that aided in enzyme
absorbance. In addition, the light absorption before this peak
temperature was low when it was a colder temperature, reading
at about 50 nm at 4 °C. This shows that when it is too cold or
too hot, the light absorption is affected, proving that
temperature is a determining factor of enzyme reaction rate,
which is measured through light absorption.
Of those tested, what is the optimum temperature for turnip
peroxidase? ______32 °C_________
Why is activity lower at other temperatures? The activity at
very high temperatures is affected because the enzyme is
denatured due to the breaking of bonds which cannot be
repaired, which alters the shape of the protein. Enzymes have a
desired temperature range and when it is too cold, the shape
tightens, and this makes it harder to perform proper functions.

3. When it is too hot, the shape of the enzyme loosens. Thi s
affects how easily the enzymes perform their function and will
slow down the process. At lower temperatures, the enzyme
reaction rate is also decreased, and less light is shown to be
absorbed. This is because there is less kinetic energy when the
temperature is colder, and when the particles collide there will
be less activation energy, or reaction rate.
Effect of pH on the activity of turnip peroxidase
Question: When the pH is very acidic or alkaline will this
negatively affect the productivity of the enzymes?
Null hypothesis (avoid using the word “will” in your
hypothesis): pH is not a determining factor of enzyme
functionality.
In this experiment, what are the independent and dependent
variables?
independent: pH
dependent: reaction rate (color of the solution)
Which tube served as a negative control? ______tube
5____________________
Which tube served as a positive control? (which would you to
predict to have optimal pH for turnip peroxidase)
_________2_________________
Paste in your graph below:
*Note: The top of the graph shows pH 9 at 0, it is not left blank.

4. Do the data support the null hypothesis? Explain, using the
data (numbers) you obtained to substantiate your conclusion.
The data does not support the null hypothesis since there was no
reaction at very low and at higher pH levels. Since the color
determined whether a reaction occurred or not, we can see that
at pH 3, there was some reaction, but much lower than the
others. This shows that the reaction was a lot weaker since it
was 0.1 and therefore it was affected by pH, but still possible.
We can see that the peak pH level is next for this reaction,
measured at pH 5 as 0.4 which proves that there is an optimum
pH level, hence pH is a determining factor. The next best
recorded pH level was 7, where the color of the reaction was
0.25, which is better than the reaction at pH 3, showing that the
reaction is best in an acidic environment more towards the
neutral spectrum (7 pH). Finally, we see an alkaline solution,
pH 9, which has no color, showing that there was no reaction.
This proves that a basic solution is not viable for a reaction
with turnip peroxidase. Thus, pH is a determining factor for
enzyme reactions and whether or not they function properly.
Of those tested, what is the optimum pH for turnip peroxidase?

5. _____pH 5__________
Why is activity lower at other pH values?
Activity is lower at other pH values because enzymes have an
ideal pH level to function properly. A protein may denature by
pH sticking to polar amino acids. In addition, the enzymes will
slow down and eventually stop in non-ideal pH values.
EXPERIMENT PROPOSAL
Question: How does salt concentration affect the absorbance of
an enzyme?
independent: Salt
dependent: reaction rate (color of the solution)
Null hypothesis: Reactivity of enzymes does not decrease as
salt concentration increases
Materials:
· Test tubes
· Micropipette
· Spectrophotometer
· pH buffer
· Turnip peroxidase (enzyme)
· Guaiacol (indicator)
· Hydrogen Peroxide (substrate)
· NaCl solution (0%, 5%, 10%, 15%)
Methods: Make a control group with everything but the NaCl.
Then, add 0.5 mL of NaCl (different percentages) in each tube,
plus the same solution that is in the control tube. Mix and put
the tubes by the spectrophotometer at 500 nm and record
absorbance after 2 minutes.
Sodium chloride percentage

6. 0%
5%
10%
15%
20%
Color at 2 minutes
0.4
0.25
0.20
0.15
0.1
Graph:
Results: The NaCl caused a decrease in enzymatic absorption.
The absorbance peak was at 0% and was lowest at 20% NaCl,
and as the sodium chloride decreases as more sodium is added
this shows that NaCl hinders the reaction rate of the peroxidase.
This is shown from a decrease in the color, which measures the
enzymatic reactivity.
How Temperature Affects the Absorption of Light Which
Measures Reaction Rate
Absorbance42332651000.10.250.40.10
Temperature (°C)
Absorbance (500 nm)
How pH Affects the Color of the

7. Solution
to Determine Whether a Reaction Occured
Series 135790.10.40.250
pH level
Color at 2 Minutes
How Sodium Chloride Concentration Affects the Color of the