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RUNNING HEAD: COLOR VS SUBSTRATE

Color Change due to Amount of Substrate

Rylee Nepple

Harrison High School

February 10, 2020

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RUNNING HEAD: COLOR VS SUBSTRATE
Purpose: The purpose of this lab was to determine whether or not the amount of substrate present

in the reaction had an effect on the rate of reaction.

Hypothesis Procedure 1: As time passes, the reaction will continue to occur, causing the solution

to turn darker in color.

Hypothesis Procedure 2: A neutral pH will result in the most significant color change.

Hypothesis Procedure 3: The more substrate there was present in the solution, the quicker the

color change would occur and the darker the color would turn.

Null Hypothesis: The amount of substrate or pH present or the length of the reaction will have no

effect on the rate of reaction or color change.

Variables: Ind- amount of substrate

Dep- rate of reaction/color change

Constants- Total volume in beaker, amount of guaiacol

Control- No substrate present

Procedure 1: Determining a Baseline

1. Using two test tubes, mark one “substrate and the other “enzyme”. To substrate tube, add

7mL of distilled water, 0.3 mL of 0.1 percent hydrogen peroxide, and 0.2 mL guaiacol for

a total volume of 7.5 mL. Cover test tube with Parafilm and gently mix.

2. To enzyme tube, add 6.0 mL of water and 1.5 mL of peroxidase for a total volume of 7.5

mL. Cover the test tube with Parafilm and gently mix.

3. Combine the contents of the two test tubes into another clean test tube, cover with

Parafilm, invert twice to mix, and place in test tube rack. Immediately begin timing the

reaction.
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RUNNING HEAD: COLOR VS SUBSTRATE
4. Observe the color change for the next 5 minutes. Rotate the tube before each reading.

Record the observed color each minute for the 5 minutes.

5. Use the color chart (A chart showing images of the differing change in color from clear to

brown, ranging from 1, clear, to 9, the darkest brown) to help you quantify change in

color over time.

Procedure 2: Determining the Effect of pH on Enzymatic Activity

1. Using clean test tubes, make 3 tubes of substrate following step 1 from Procedure 1 and 3

tubes of enzyme

 For each enzyme tube, add 6.0 mL of a specific pH solution ( pH= 4, 7, or 10),

and 1.5 mL of peroxidase for a total volume of 7.5 mL

Cover each tube with Parafilm and gently mix

2. Combine the substrate and enzyme tube of each pair, cover with Parafilm, gently mix,

and place in test tube rack. Immediately begin timing the reactions.

3. Record the observed color each minute for 1-5 minutes.

4. Use the color chart to quantify color change and record results.

Procedure 3: Determining the Effect of Substrate on Enzymatic Activity

1. Prepare 6 test tubes of substrate with varying amounts of hydrogen peroxide and H2O to

keep total volume of beaker consistent. Amount of guaiacol remains the same for all

tubes.

 Tube 1: 7.3 mL H2O, 0mL hydrogen peroxide, 0.2 Ml guaiacol

 Tube 2: 7.0 mL H2O, 0.3 mL hydrogen peroxide, 0.2 mL guaiacol

 Tube 3: 6.8 mL H2O, 0.5 mL hydrogen peroxide, 0.2 mL guaiacol

 Tube 4: 6.3 mL H2O, 1.0 mL hydrogen peroxide, 0.2 mL guaiacol

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RUNNING HEAD: COLOR VS SUBSTRATE
 Tube 5: 5.8 mL H2O, 1.5 mL hydrogen peroxide, 0.2 mL guaiacol

 Tube 6: 5.3 mL H2O, 2.0 mL hydrogen peroxide, 0.2 mL guaiacol

Cover each tube with Parafilm and gently mix.

2. Prepare 6 test tubes of enzyme, following step 2 in Procedure 1.

3. Combine the substrate and enzyme tubes for all six pairs, cover with Parafilm, gently

mix, and place in test tube rack. Immediately begin timing reaction.

4. Record color at each 1 minute interval for a total of 5 minutes for each tube.

5. Use the color chart to quantify the color change.

Data:

Procedure 1- Baseline

Time (minutes) Number based of Color Chart

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

Baseline
10
9
8
Color Chart Reaidng

7
6
5 Baseline
4
3
2
1
0
0 1 2 3 4 5
Time (minutes)
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RUNNING HEAD: COLOR VS SUBSTRATE
Procedure 2- Determining the Effect of pH on Enzymatic Activity Based on Color Chart

Time (minutes) pH= 4 pH= 7 pH= 10

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

Time vs Color Change

10
9
8
Color Chart Reading

7
6 pH= 4
5 pH= 7
4 pH=10

3
2
1
0
0 1 2 3 4 5
Time (minutes)

Procedure 3- Color Change of Varying Amount of Substrate Based on Color Chart

Time Tube 1 Tube 2 Tube 3 Tube 4 Tube 5 Tube 6

(minutes)
0 0 1 1 1 1 1
1 0 3 4 3 5 3
2 0 5 7 6 8 7
3 0 7 8 6 9 7
4 0 8 9 8 10 8
5 0 9 9 8 10 8
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RUNNING HEAD: COLOR VS SUBSTRATE

Time vs Color Change

12

10

Tube 1
Color Chart Reading

8
Tube 2
Tube 3
6
Tube 4
Tube 5
4 Tube 6

0
0 1 2 3 4 5
Time (minutes)

Conclusions: From the gathered evidence, it can be concluded that to higher amount of substrate

will yield a faster reaction rate with more significant color change, until a certain point. Overall,

as the amount of substrate was increased, the reaction time increased as well, resulting in a

darker color, excluding test tube 6, which had the most substrate. This is because there is only so

much enzyme to speed up the reaction, so once a certain amount of substrate (in this case, 1.5

mL) is surpassed, the reaction will no longer increase in speed. This evidence supports the

hypothesis that the more substrate there is, the faster the color will change.

Discussion of Theory: In this experiment, we demonstrated the theory that adding more substrate

will result in a faster color change, or rate of reaction. This theory was supported by the data.

The reaction is sped up when more substrate is added because there is more substrate available to

react with the catalytic enzyme. In low concentrations of substrate, there is extra enzyme that is

not being used to speed up the reaction. However, as you add more substrate, more of the

enzyme is used up, causing the reaction to occur at a faster rate. This is true until you reach a
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certain concentration of substrate; in this case anything over 1.5 mL. This is true because at this

point you have equal amounts of enzyme (peroxidase) and substrate (hydrogen peroxide). This is

the most effective the reaction can be, because there is no more enzyme to speed up the reaction

of the excess substrate. The color change is caused by the guaiacol, which turns brown in the

presence of oxygen. The darker the brown, the more oxygen there was present. This helps us

measure the rate of the reaction, as oxygen is a product of the reaction.

Experimental Sources of Error: A possible source of error appears to be test tube 4. This test is

the only one that did not follow the pattern of increased rate of reaction when there was an

increase in the substrate concentration. This is possibly due to inaccurate measurement when

adding substrate or enzyme to the tube, or by reading the color incorrectly in comparison with

the color chart provided. This makes the overall correlation weaker.

Questions:

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

The limiting factor was the substrate because there were only 0.3 mL of substrate but

there were 1.5 mL of enzyme.

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?

The four factors that vary are temperature, pH, enzyme concentration, and substrate

concentration. I think all of these factors could affect enzyme activity. As seen in the lab,

the variation in pH changes the results, with lower and neutral pH yielding a faster

reaction rate. The concentration of the substrate also affects the results, as those with a

higher concentration result in a faster reaction time. The concentration of the enzyme

would produce results similar to that of the concentration of the substrate. The
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temperature will also have an effect because molecules are more active at high

temperatures, meaning they have a higher likelihood of bumping into another molecule,

causing them to react.

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

springs?

These specific types of bacteria have adapted their enzymes to be able to survive in the

extreme temperatures of hot springs, allowing the bacteria to survive.

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.

The enzyme evolved in similar ways many times throughout life as it was forced to adapt

in different conditions repeatedly to be able to survive.

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.

The best soil conditions would be neutral pH, high temperature, and equal concentrations

of enzyme and substrate. A neutral pH resulted in the highest rate of reaction, so the

beans would grow the fastest in this condition. At high temperatures, the molecules are

more likely to collide and react. However, the temperature should not be too high, as it

can cause the enzyme’s hydrogen bonds to break, resulting in denaturing. Equal amounts

of substrate and enzyme will cause the reaction to happen the most efficiently, as there is

no limiting factor, allowing the beans to with maximum productivity.