Madison Gilbert

AP Biology

Handest

29 September 2016

Enzyme Lab Write-Up

Hypothesis:​ If heated water is added to the substrate and enzyme test tubes before they are

combined, then peroxidase (the enzyme) will become denatured, and the guaiacol compound will

express less color in the resulting test tube.

Procedure:

1. Gather two test tubes, and label one “substrate” and the other “enzyme”. Add 8 mL of

distilled water, 0.5 mL of .1% hydrogen peroxide, and 0.25 mL of guaiacol to the

“substrate” test tube. Cover and invert two times.

2. Add 7 mL of distilled water and 1.7 mL of peroxidase to the “enzyme” test tube. Cover

and invert two times.

3. Combine the “substrate” and “enzyme” test tubes into a completely new test tube. Cover

and invert two times.

4. Time the reaction for five minutes, and record the color change based off the 1-10 ranged

color chart.

5. Repeat steps 1-4, but add water that has been warmed on a hot plate (specifically at a

temperature of 172.6*F) in place of the distilled water. Be sure to control for the other

variables, by not changing any of the aforementioned measurements. Slight changes in

measurement could completely alter the results of the lab.

Data:

Time Passed (in seconds) Color Change

10 2

20 4

30 5

40 7

50 8

60 9

70 10

80 10

90 10
Table 1. Portrays the color change (substrates broken down) based off the reaction time.

Fig 1. Visually demonstrates and depicts the data points and results from the base lab.
 Time Passed (in seconds) Color Change

10 6

20 10

30 10

40 10

50 10

60 10

70 10

80 10

90 10
Table 2. Portrays the results from the second procedure, where hot water was substituted in for
the distilled water.

Fig 2. Demonstrated the data points from Table 2 when distilled water was replaced by heated
water.
Fig 3. Demonstrates the difference between both procedures, and how a variable can alter the
effects of peroxidase.

Data Analysis:

According to my graph, the baseline results were slightly lower than the variable results.

Shown in Figure 3, the baseline experiment was expressed by the maroon line; it’s color change

increased at a comparably linear rate, until maxing out at 10 (a complete color shift) during the

last few seconds of the experiment. The aqua line represents the variable experiment, in which

we substituted distilled water for heated water (172.6*F); the reaction rate appeared faster in this

lab because the color change increases slightly, and then quickly maxes out at 20 seconds.

According to this graph, we can conclude that the variable experiment simply had a faster

reaction rate than the baseline test. Specifically, the variable test reached a max color shift

approximately 50 seconds before the baseline experiment does.

Conclusion:
 The data from the variable lab did not prove our hypothesis because, instead of there

being a lack of color change due to enzyme denaturation, there was an increased amount of color

change occurring at a faster pace. We hypothesized that the peroxidase would be denatured, but

in reality the enzyme peroxidase functions optimally at higher temperatures. Different enzymes

prefer different operating temperatures, so an enzyme will become denatured when it is exposed

to a temperature that it is not accustomed to, or not genetically able to function in.

The independent variable in my group’s variable experiment was the temperature of the

heated water (172.6*F). The dependent variable was the amount of color change based off the

1-10 ranged color chart. The control variables include the amount of peroxidase, the amount of

guaiacol, the amount of .1% hydrogen peroxide, and the amount of water (because only the

temperature of the water changed). Also the type of test tubes, syringe, droppers, and test tube

rack would be considered control variables because they did not change throughout the course of

the baseline and variable labs.

As with any other student scientists, we considered sources of error that could have been

evident during the course of the experiment. One major source of error would be the fact that I

accidentally spilled 1 mL from our combined test tube when I was inverting it during our

variable lab (sorry!). Due to this unfortunate mishap, we can no longer be certain of the amounts

of substances we so carefully measured and added to the test tubes. Another factor that we could

not measure was the amount of heat loss that occurred when we transferred the “substrate” and

“enzyme” test tubes into the combined test tube.

During the celery demonstration, we witnessed bubbles as the product of the enzymic

reaction taking place. During our peroxidase lab, we did not experience these bubbles because
the guaiacol compound reacted with the oxygen so quickly that it would have been impossible to

visibly see it bubble. Also, instead of creating bubbles as a product of the reaction, the guaiacol

turned the oxygen product into a visibly brown color.

If an enzymic reaction begins to level off, and nothing is denaturing or inhibiting the

enzyme, then we can assume that the concentration of substrates has decreased since the enzyme

was introduced. This makes sense because in any given situation, there are only so many

substrates. Once the enzymes have created as many products as possible, given the total amount

of substrates, then the reaction will level off because there are no longer any substrates available,

only products. If the enzymes don’t have anything to react with, than obviously no reaction will

occur.

If an active preparation of peroxidase is exposed to the trypsin enzyme, then it may be

inactive when it is re-assayed. Peroxidase, and other enzymes, can be described as proteins. By

understanding the word “proteolytic” we can comprehend the effects of trypsin on peroxidase.

The first cluster, “proteo-”, alludes to proteins, while the second cluster, “-lytic”, alludes to

breaking down, which leads to an understanding of trypsin. Trypsin, based off its proteolytic

connection, works to break down the proteins that it comes into contact with. So, once trypsin is

introduced to peroxidase, it will begin the reaction of breaking it down. Therefore, the

peroxidase will be completely inactive when it is re-assayed.

The idea of an increased substrate concentration can have similar end results as when

there is a decreased substrate concentration. When there is too much of a substrate, in this case

hydrogen peroxide, then it will act as an inhibitor to itself. Specifically, the peroxide will operate

as a competitive inhibitor, and will make it difficult for any substrates to react with an enzyme
because they are all essentially blocking each other from the active site. Imagine a time when

you were stuck in an elevator, and more and more people began coming on after every floor.

Eventually the concentration of people will become so dense that it’s impossible to move; the

same concept can be applied to enzymes completely surrounded by substrates.

Hydrogen peroxide has been proven to be a cell damaging agent. However, hydrogen

peroxide is produced naturally within cells during normal cell metabolism. The production of

hydrogen peroxide becomes negative when it is produced in excessive amounts. Oxidative stress

and disease are possible results of over producing hydrogen peroxide. Hydrogen peroxide also

plays a beneficial role in cells as a signaling molecule in lymphocytes; based off the studies of

beneficial hydrogen peroxide, we can conclude that it is only damaging in excessive amounts

(Nindl, 2004). Catalase is an enzyme that works in both animal and plant cells to break down the

harmful hydrogen peroxide substance. Catalase ensures that the amount of hydrogen peroxide

never reaches a point where it would become detrimental to the cell. Peroxidase works in much

the same way (Blue, 2016). By making sure that hydrogen peroxide never reaches a harmful

point, but is still present in the cell at lower levels (in order to receive the beneficial aspects),

catalase and peroxidase (and other enzymes who operate in the same way) play a large role in the

survival of organisms.
 Works Cited

Blue, Marie Luise. "Characteristics of a Catalase Enzyme." ​Our Everyday Life​. Our Everyday

Life, 2016. Web. 29 Sept. 2016.

Nindl, G., N. R. Peterson, E. F. Hughes, L. R. Waite, and M. T. Johnson. "Effect of Hydrogen

Peroxide on Proliferation, Apoptosis and Interleukin-2 Production of Jurkat T Cells."

PubMed.org​. USA.gov, 12 Mar. 2004. Web. 29 Sept. 2016.