Enzymatic Activity Levels vs Temperature

2/4/2020-2/5/2020

Purpose: The purpose of this lab it to see how different variables, such as temperature substrate

concentration, and enzyme concentration, affect the activity levels of an enzyme.

Hypothesis (procedure a): If the enzyme and substrate react with each other, the tube will

produce oxygen and begin to turn brown because of the guaiacol (oxygen indicator).

 Null Hypothesis: If the enzyme and substrate are mixed together, then there will be no

reaction.

Hypothesis (procedure b): If the pH is closer to neutral, then the rate of the reaction will

happen faster than the reaction of pH 4 and 10 because pH 7 is closest to body pH, where the

reactions happen.

 Null Hypothesis: If the pH differs between each tube, then it will not have an effect on

the rate of the reaction.

Hypothesis (created procedure): If the temperature of the tube is within the optimal range for

an enzyme, then the reaction will happen quicker because of the higher number of particle

collisions between the enzymes and substrates.

 Null Hypothesis: If the temperature is changed, it will have no effect on enzymatic

activity.

Procedure part A:
 1) Obtain 2 test tubes. Label one substrate and one enzyme. To the substrate tube, at 7 mL

of distilled water, .3 mL of 1% hydrogen peroxide, and .2 mL of guaiacol. Cover tube

with Parafilm and gently mix.

2) To enzyme tube, add 6 mL of distilled water and 1.5 mL of peroxidase. Cover with

Parafilm and mix gently.

3) Combine contents of the two tubes in another test tube and immediately cover with

Parafilm. Invert twice to mix and begin timing the reaction.

4) Observe the reaction over the next 5 minutes (0, 1, 2, 3, 4, 5) and record a color activity

level (1-10) compared to the given chart.

5) Graph the change in color over time

Procedure part B:

1) Obtain 6 test tubes and label 3 substrate and 3 enzyme. Add the same amounts of

substances to the substrate tubes as before. To the enzyme tubes, add a different pH

buffer solution to each (4, 7, 10). Cover the 6 tubes with Parafilm and gently mix.

2) Combine substrate and enzyme tubes (total of 3 tubes now), cover with Parafilm, and

invert twice to mix. Immediately begin timing the reaction.

3) Record color change using the same chart as procedure 1, and for minutes 0, 1, 2, 3, 4, 5.

4) Graph data as color intensity versus pH

Created Procedure:

1. Put safety goggles on to protect eyes from chemicals

2. Obtain 8 test tubes (4 for enzyme and 4 for substrate) and a test tube rack
3. Set a 350mL beaker of water (300 mL water inside it) onto a hot plate and set it to heat

level 6. Obtain a bowl of ice and another beaker with the same amount of water inside it

for later use.

4. Into the substrate tube, add 7 mL of distilled water, .3 mL of 0.1% hydrogen peroxide,

and .2 mL of guaiacol (total of 7.5 mL inside tube)

5. Into the enzyme tube, add 6 mL of distilled water and 1.5 mL of peroxidase (total of 7.5

mL inside tube)

6. Cover all 8 tubes with Parafilm and gently mix.

7. Measure temperature of beaker on hot plate and add ice to the beaker of plain water. The

beaker on the hot plate should measure 55 degrees Celsius. The ice beaker should

measure 9 degrees Celsius.

8. An enzyme activity level chart should be at the table. Use that for comparison.

9. Once they have been mixed, combine the tubes of substrate and enzyme, cover with

Parafilm, invert twice to mix, and place in the 55 degree beaker. Record the level of

enzymatic activity after each minute for 5 minutes.

10. Repeat the process with the test tube and place it into the beaker with ice in it. Record

enzymatic activity level after each minute for 5 minutes.

11. After the tube is taken out of the hot beaker, turn the temperature up to 8 on the hot plate

and wait for the water to boil. Combine the enzyme and substrate tube, cover with

Parafilm, invert twice, and place inside the boiling water. Record activity for five minutes

as before.
 12. Remove the tube from the ice water and place the beaker into the ice bowl. Let the

temperature drop to 2 degrees Celsius and then place a combined test tube in for 5

minutes and record the activity levels.

13. Be sure to clean the tables and dispose of all trash in the proper receptacles.

Data:

Time (minutes) Color Level (1-10)

0 1
1 5
2 6
3 8
4 8
5 9

Time pH Color Time pH Color Time pH Color

(Minutes) (constant) (1- (minutes) (constant) (1- (minutes) (constant) (1-

10) 10) 10)

0 4 0 0 7 0 0 10 0
1 4 6 1 7 7 1 10 0
2 4 8 2 7 8 2 10 0
3 4 9 3 7 9 3 10 0
4 4 9 4 7 10 4 10 0
5 4 10 5 7 10 5 10 1

Time (minutes) Temperature (degrees Color (1-10)

Celsius) (constant)
0 55 0
1 55 6
2 55 8
3 55 9
4 55 10
5 55 10
Time (minutes) Temperature (degrees Color (1-10)

Celsius) (constant)
0 100 3
1 100 7
2 100 8
3 100 9
4 100 10
5 100 10

Time (minutes) Temperature (degrees Color (1-10)

Celsius) (constant)
0 9 0
1 9 2
2 9 3
3 9 5
4 9 5
5 9 6

Time (minutes) Temperature (degrees Color (1-10)

Celsius) (constant)
0 2 0
1 2 1
2 2 3
3 2 3
4 2 4
5 2 5

Calculations and Graphs:

*series 1= pH 4, series 2= pH 7, series 3= pH 10
*series 1 = 55 degrees Celsius, series 2 = 100 degrees Celsius, series 3 = 9 degrees Celsius,

series 4 = 2 degrees Celsius

Conclusion:

The hypothesis was proven because the optimal temperature range is between 35 and 55 degrees

Celsius, and the temperature that worked the best was 55 degrees Celsius.

Discussion of Theory:

Peroxidase reacts with hydrogen peroxide to produce oxygen. Guaiacol in an indicator of

oxygen, which is a product of the reaction between peroxidase and hydrogen peroxide. The

speed of the reaction depends on temperature, pH and concentrations of enzymes/substrates. The

substrate must also fit within the active site on the enzyme. When hydrogen peroxide binds with
peroxidase, oxygen is produced. The guaiacol will turn a shade of brown depending on the stage

of the reaction, and can be compared using an analysis chart given in the lab materials.

Experimental Sources of Error:

One thing that could affect the data is having inaccurate measurements. This could cause the data

to be skewed because the lab would have two independent variables and not just one. The error

could also occur because of the color chart. Because the reaction kept going, the colors were hard

to compare and obtain a solid reading of the color.

Questions:

1. Was the limiting factor of your baseline reaction the enzyme or the substrate?

The limiting factor was the substrate because that can be used up, but enzymes are reusable. The

enzymes will continue to create a reaction until the substrate runs out.

2. What are four factors that vary in the environment in which organisms live? Which of

these factors do you think could affect enzyme activity?

Four factors that affect the environment is pH, temperature, substrate concentration (amount of

food), and enzyme concentration within the organism. I believe all of these can affect the activity

of an enzyme because they can speed up a reaction, slow down a reaction, or denature the

enzyme or protein.

3. How do some organisms, like archaebacteria, survive in extreme environments like hot

springs?
Archaebacteria have evolved over the years in order to survive in these extreme conditions. They

have a high level of fatty acids inside them and very compact proteins to help prevent the

denaturing of the enzymes within them.

4. Some enzymes are commonly found in all living domains, like E. coli, corn, fungi, and

sheep. Did this enzyme evolve numerous times in numerous organisms or just once early

in the history of life? Explain your answer.

In order for an evolution to be favored, it needs to be better than the current model. The enzymes

that can survive in multiple environments most likely evolved once at the beginning to save

energy and become a favored trait. This would ensure the survival of the enzymes in different

areas of life and growth.

5. Consider a farmer growing soybeans, what would be the best soil conditions for

maximum productivity? Be sure to explain your reasoning for your answer. Hint: you're

going to need to provide optimal conditions for a variety of factors.

Soybeans grow the best when the soil around them has warmed to 60 degrees Fahrenheit, and

when the air around them had warmed to 60-70 degrees Fahrenheit. This is because the soybean

plant is not frost proof and will easily freeze. They grow best in a soil pH of 6.3-6.5 because it

maximizes nutrient productivity. The water soybeans prefer is close to neutral, but if it is slightly

below 7, it will not be extremely detrimental to the soybeans.