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Laboratory 5:
The Properties of Enzymes

By: Lexi Grubbs

Honors Biology Period 3
11/27/15

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Introduction:
There is so much about organisms that the world doesnt know about, the enzyme being
one of the prime examples. Enzymes, a substance made up of proteins, are used to hurry the rate
of chemical reaction in organisms. Humans wouldnt be able to continue living, or even exist in
general, without this substance. Theyre specific, meaning that the specific type of enzymes only
react to their certain reactants (Saul, 2012).
Obviously, we know some facts about the enzyme. However, do we really know what
enzymes are? Do we truly know all of their properties and functions? The answer is probably
not. With this lab, though, we will being learning more and more.
Every enzyme has a specific pH. This pH is specific to the enzyme, making sure that
enzyme only reacts to its precise reactants. Changing the pH could affect the shape, the
properties and their shape/ charge, and will not allow the enzyme to go through catalysis.
Catalysis is when the chemical reaction further accelerates (BC CUNY, n/a).
In this experiment, a Spectronic 20 Spectrophotometer will used. This device measures
and compares a light beams intensity both before and after it passes through the solvent. We will
only be focusing on one of the two readings that the machine measure, which is the absorbance,
also known as the log of transmittance. It gather the information by reading the range of
wavelengths, which the user must select and set themselves (NMSU, 2006).
How does one know if the hydrogen peroxide has been broken down? What is the
reaction it has with peroxidase? The answer is Guaiacol. Guaiacol, the indicator, will react with
the solvents. If oxygen is present, then the solvent will turn from its clear color to a brown color.
You will know that all of the reactant has been used up once the solution changes color. On a

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graph, it will show a plateau were the reactants line will no longer increase or decrease. The
Spectronic 20 Spectrophotometer records the color change (Williams, 2015).
Absorption tell us that there are enzymes present in the reaction happening within the
Spectronic 20 Spectrophotometer. On a graph, the line would be escalating upwards, meaning
that enzymes are present. These enzymes can be reused over and over again throughout the
project or life if need be. If there is hardly any movement upwards of the slope on the graph, then
the enzymes have denatured, meaning that they have died and will not return. They are not, nor
will they ever be, able to be reused.
In conclusion, the purpose for this experiment is to discover the rate of reaction between
H202 (hydrogen peroxide) and peroxidase and also find out the optimum pH of the peroxidase,
which means the one that works the best under a strict set of conditions. If this is the case, then I
hypothesize that the optimum pH is eight. I say this because I dont want to pick the control
group due to the fact that we are basing our findings off of this group, and eight isnt too acidic;
its directly following the control, which has a pH of seven.
Materials:
1.
2.
3.
4.
5.

Distilled Water
Turnip Extract
Five Cuvettes of the same brand or type.
Kimwipes
A Spectronic 20 Spectrophotometer

Procedure:
1. Set up and power you Spec 20 using the activation knob.
2. Allow the Spec 20 to hit up for at least 15 minutes.
3. If you want to change the wave length, do so now by using the control knob.

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4. Change the he left side of the scale to read infinity absorbance by turning the activation
knob. NOTE: the chamber should be empty and covered while doing this step.
5. While using two of the same type of cuvettes (tubes), always hold the tube at the tops by
the opening. Holding on the sides may cause a false reading.
6. Fill the cuvette to the half way point with either distilled water or the solvent being used.
Remove any moisture or fingerprints from the outside of the cuvette with napkins of
Kimwipes. This cuvette is the reference tube, the tube that you will base your findings on.
7. Use the light control knob, which is placed on the right side, make sure that the machine
is set at zero absorbance. The absorbance will removed from the solution.
8. Place the tube into the chamber and close lid.
9. The sample solution should now fill the new cuvette, be wiped down like in Step 6, and
replace the reference tube in the chamber. Record data directly from the scale on the
right.

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Absorbance Rates in Peroxidaise at Different Rates

2.5
2
1.5

Absorbance

1
0.5
0

Time (in Seconds)

control

pH 9

pH:8

pH: 5

pH: 3

Results:

This graph depicts all five pHss absorbencies and how they progress over the course of
the time. The pH of the control is at 7, while the others are listed as such in the graph itself. The
horizontal axis is representative of the time in which data was recorded, which spanned from the
very entry of the cuvette into the chamber to eleven minutes. The vertical axis is representative
of the absorbance, which can go anywhere from zero to two-and-a-half.
Discussion:
My hypothesis was wrong. The enzyme with the pH of eight did not plateau at all during
the time given. Both the pH of his five and the pH if three also did not reach a full reaction. The
pH of three hardly reacted at all, meaning that the enzymes are denatured. They have died from
the solvents being mixed.

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The reference cuvette, the cuvette with the pH of seven inside, reacted the quickest and
did reach the maximum absorbance at 1.999. The pH of nine comes in second because it didnt
react quite as fast but did, in fact, plateau. The optimum pH, besides the control group, is the pH
of nine. If the control was not the reference, however, then the control group would be the
appropriate optimum pH for peroxidase.
As for sources of error that couldve been corrected, there is always a possibility of
fingerprints or moisture still being present on the sides of the cuvette, thus creating a false
reading. Also, on the Spectronic 20 Spectrophotometer, the absorbance often changed itself on
the scale. Sometimes it would decrease and increase without any actual movement of the knob.
The machine could have been giving of false readings. This occurred prior to the cuvette being
added, so there is no real way to know if it affected the test. Finally, human error is always
inevitable. Somebody couldve mixed the solvents wrong, wrote down the wrong data at the
wrong times, and so and so forth. Theres so much that couldve gone wrong, and some of those
events actually happened throughout this experiment.
Following this experiment, I now feel inspired to continue on learning about peroxidase.
Are there any conditions that would change the findings discovered? For example, would
changing the environment of the experiment affect the results that were found here? What would
happen if you mix peroxidase with a compound other than hydrogen peroxide and then redid the
experiment? Also, if you did this and changed the environment, would that also change? Having
said this, I hope to one day be able to try out all of these ideas to further quench my thirst for the
knowledge of enzymes and peroxidase that I so long for.

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References:
Saul, Leif. "Enzyme Characteristics." Dr. Saul's Biology in Motion. Leif Saul, 2012. Web.
http://www.biologyinmotion.com/minilec/wrench.html
Williams, Carly. Laboratory 5: Properties of Enzymes. Lab handout. Cardinal Wuerl North
Catholic High School. Cranberry Township. 2015. Print
"Spectronic 20." New Mexico State University. NMSU Board of Regents, 2006. Web. 27 Nov.
2015.
http://web.nmsu.edu/~kburke/Instrumentation/Spectronic_20.html
"The Effect of PH on Enzyme Activity." Brooklyn College City University of New York.
Brooklyn College City University of New York. Web. 27 Nov. 2015.
http://academic.brooklyn.cuny.edu/biology/bio4fv/page/ph_and_.htm