Laboratory 5: Properties of Enzymes

Alex Dorsch
11/27/15
Period 4

Introduction:
An enzyme is a protein that speeds up the rate of chemical reactions by acting as a
catalyst. Since enzymes are a type of protein, they are made from long chains of amino acids.
Enzymes help our bodies perform important functions such as digestion. They are also located in
an organisms liver, with the purpose of turning hydrogen peroxide into water and oxygen. The
pancreas also produces enzymes to break down macromolecules. Enzymes work with molecules
known as substrates, which bind to a region of the enzyme known as the active site. Some
characteristics of enzymes include that they are reusable. Since they are unchanged by a
chemical reaction, they can be reused. Enzymes are specific in their choice of reactants, known
as substrates, and in the reactions they catalyze. By weakening the bonds in substrates, enzymes
lower the activation energy. (Biggs et al., 2012)
There are three things that can affect enzyme activity. Some cofactors, which are
inorganic substrates, are needed to help the substrate attach to the active site of the enzyme (Kerr,
2015). For example, in order for hemoglobin to pick up oxygen, it must have iron. Unlike
cofactors, coenzymes are organic. Their purpose is to help enzymes function. Next are
competitive and noncompetitive inhibitors. Competitive inhibitors are chemicals that are similar
to an enzymes normal substrate and compete with it for the active site. Noncompetitive
inhibitors do not enter the active site, but they do bind to another part of the enzyme, forcing the
enzyme to change shape and alter the active site. These inhibitors can denature the enzyme,
which is irreversible. If an enzymes structure changes, it is no longer able to act as a catalyst.
Extreme temperatures are the most dangerous out of the three factors. High temperatures can
result in the enzyme being denatured by destroying its bonds and shape (Adam, 2015). Low
temperatures cause the enzyme to decrease its rate of interaction with substrates and slow down

(alevelnotes.com). Most enzymes work best in environments with a pH around six to eight,
which is close to neutral. Extreme pH levels can damage the substrate or its bonds, which would
limit the ability of the substrate to react with the enzyme. This would cause a further decrease in
activity because, as when we tested the substance with a pH of three, the enzymes absorbance
leveled off because it literally fell apart (Adam-Day, 2015).
In this experiment we will be testing an enzyme found in horseradish, turnips, and
potatoes known as peroxidase. In order to do so, the extract containing peroxidase is mixed with
water, hydrogen peroxide, and Guaiacol. If Peroxidase is present, the hydrogen peroxide will
break down to water and oxygen. The oxygen will then react with the Guaiacol and produce a
brown coloration. Guaiacol will be used to help determine the rate of reaction, how fast
hydrogen peroxide turns into water and oxygen, based on how dark the color gets. As the
solution of enzyme and substrate turns darker brown, the amount of absorbance increases
because more product is being produced. The machine we will be using is called a Spec 20, short
for spectrophotometer. It measures the intensity of a light beam before and after it passes through
a sample and compares the two results. If we look at the hydrogen peroxide reaction with the
presence of peroxidase in five different pH environments, then we will see that the optimum pH
for peroxidase is seven.

Materials:

Spectronic 20 spectrophotometer
Peroxidase
Guaiacol
Hydrogen Peroxide
Cuvettes

Tissue
Turnip Extract
Distilled Water
Pipette
Graduated Cylinders
Different pH Solutions

Procedures:
1. Adjust Spec 20 so wave length is 460
2. Adjust the Spec 20 to zero absorbance
3. Take tube filled with distilled water and turnip extract and put it in the Spec 20 as a
reference tube. This is the control group.
4. Pour tubes labeled enzyme and substrate into the tube labeled #1
5. Place finger over the end of the tube and shake to mix the substances
6. Place tube in the Spec 20 for 10 minutes
7. Record absorbances at set times
8. Take tube #1 and place it on the blue rack
9. Place the control group back in the Spec 20
10. Pour substrate, enzyme, and pH 9 into the same tube
11. Place finger over the end of the tube and shake to mix the substances
12. Place tube in the Spec 20 for 10 minutes
13. Record absorbances at set times
14. Place tube back on the blue rack

Results:

Figure 1

Figure 2

Figure 1 is showing the basic reaction as a pH of seven, which is absorbance versus time.
It accelerates quickly with a small level off at 90, before accelerating again. It finally levels off
for good at 300 seconds. In Figure 2 we are comparing the different pH effects of three, five,
seven, eight, and nine. The pH of three instantly levels off with no acceleration. The pH of five
does not level off and accelerates slowly. The pH of seven shows the fastest acceleration, and
levels off at 300 seconds. The pH of eight does not level off and accelerates quickly. The pH of
nine accelerates slowly, but not as slow as the pH of five.

Discussion:
By looking at Figure 2 we can determine that the enzyme with the pH of three was the
slowest because it does not reach maximum absorbance. The enzyme mixed with a pH of five,
eight, and nine tell us it was a non-enzyme-catalyzed reaction. The enzyme mixed with the pH of
seven demonstrates an enzyme-catalyzed reaction. The pH seven was the optimum pH for
peroxidase because it accelerated much quicker than the other pH samples we tested. There were
several errors that could have been made when performing this experiment. We might have
recorded a wrong number at the wrong time. Also, the liquids might not have been shaken
enough to mix them. Any of these errors would have affected our results.

References:
Adam-Day, S. (2015). Factors affecting Enzyme Activity. Alevelnotes.com
Biggs et al. (2012). Biology. Columbus, OH: McGraw-Hill Companies Inc.

Kerr,

S.

(2015).

How

Education.seattlepi.com

Do

Cofactors

&

Coenzymes

Affect

Enzyme

Activity?