Enzyme Pre-Lab
By Adam Wanetik, Spencer Olesky
And also Daniel Broadie
What are Enzymes?
• Enzymes are proteins, each with a unique
physical structure
• They are “catalysts” which means that they
speed up reactions
• The shape of each enzyme fits the shape of
the reacting molecule for which the enzyme
serves as a catalyst
Substrate Complex
• In an enzyme catalyzed reaction, the reacting
molecules are called the substrate. Substrate
molecules combine with the enzymes forming
a enzyme substrate complex
• In this chemical reaction the products are
formed and the enzyme is released, and it is
unchanged from its original structure
Catechol and Benzoquinone
• Catechol is a clear organic compound which
occurs naturally in trace amounts
• Catechol can be found in foods such as apples,
bananas and potatoes
• Catechol oxidase is the enzyme which
catalyzes the color in catechol to turn in into
benzoquinone
• Benzoquinone (or benzo brown) is what is the
brown color that increases as your food ages
Catechol and Benzoquinone
Form Fits Function
• Much like DNA and RNA, the form of the
enzyme determines it’s function
• For enzymes to function correctly they also
need a cofactor/coenzymes
• Many vitamins and minerals serve as
coenzymes
• Substances that interfere with coenzymes or
cofactors will also inhibit the enzyme
The Lab
• In this lab, we will look at the inhibition of
enzyme activity by specific chemicals called
inhibitors
• The inhibitors that we will use is called
phenylthiourea (PTU)
• Catechol oxidase’s cofactor is copper
• PTU can combine with the copper in catechol
oxidase to inhibit its enzymatic activity
The Lab
• Inhibitors affect enzymes in one of two ways:
Competitive inhibition takes place when two
molecules structurally similar compete for an
active site on the molecule
• In the experiment we will do, we will find out
if PTU is a competitive or non-competitive
inhibitor
The Lab
• We will measure the production of
benzoquinone in the presence and absence of
the enzyme
• We will test this on potatoes both in the
presence of the enzyme preincubated with
PTU, and preincubated with various
concentrations of lemon juice
Materials
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2 potatoes, peeled and cut into chunks
1 Blender
3 Liters cold distilled water
1 1000-ml beaker
1 Large funnel
Several layers of cheesecloth
Materials
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Colorimeter
Colorimeter cuvettes
Distilled water
Pipettes
Stock solutions of
1% Catechol (Poison!)
Enzyme Catechol Oxidase (potato extract)
Enzyme Preincubated in Phenylthiourea (PTU) (Poison!)
Enzyme Preincubated in pure Lemon Juice (pH = 6.5)
Enzyme Preincubated in 1:100 Lemon Juice (pH = 6.5)
Enzyme Preincubated in 1:100000 Lemon Juice (pH = 6.5)
Procedure
• Peel and chunk the potatoes. Put the peeled,
chunked potatoes in a blender. Add 700ml of
cold distilled water and blend at high speed
for 2 minutes.
• Line a large funnel with several layers of
cheesecloth and place the funnel in a 1000 ml
beaker, which has been placed in a container
of ice. Filter the potato juice through the
beaker
Procedure
• The filtrate is the potato juice-catechol
oxidase extract. It is called "catechol oxidase”
throughout the exercises.
• Potatoes also contain catechol, so you will
need to keep the catechol oxidase extract on
ice at all times to retard any natural chemical
reaction that might occur.
Procedure
• Next, we test with different concentrations of
the lemon juice (different pH solutions) on the
rate of production of Benzoquinone
• Make serial dilutions by taking 10 ml of lemon
juice to a graduated cylinder and bring to
100ml. Then take 10 ml of that solution and
bring it to 10 ml etc.
Preparation of Lemon Juice
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Concentration
Pure
1:10
1:100
1:1000
1:10000
1:100000
pH
2.5
3.2
4
4.8
5.6
6.54
Table 1. Experimental Setup
ACHTUNG!
• Catechol, hydroquinone, and phenylthiourea are
poisons and catechol, in concentration, is a
mutagen. Use caution handling these chemicals.
Avoid contact with these solutions. If a spill
occurs, immediately wipe up the spill with dry
paper towels and then use disinfectant solution
on the spill site. Dispose of all towels in a
designated contamination location. Be sure to
notify your instructor of the spill and your cleanup procedures. Your personal safety and the
safety of other students is most important.
Preparation of Solutions
Preparation of 1% Catechol
Add 10 grams of Catechol to beaker and bring to 1 liter
Preparation of 1% PTU
Add 1 gram of PTU to beaker and bring to 100ml
Preparation of Lemon Juice Concentrations
In this part of the lab we will test the effect of various concentrations
of lemon juice (different pH solutions) on the rate of production of
Benzoquinone.
Lemon Juice is commonly used to preserve fruit. This is because
Lemon Juice contains a 5-6% Citric acid solution which will denature
the catechol oxidase, thus slowing the oxidation of Catechol to
Benzoquinone.
Enzyme Concentration Determination
• Determine what concentration of Potato extract
will catalyze the reaction at such a rate such that
the reaction reactions completion in around 5
minutes.
• Add 4ml of Catechol to 1 ml of enzyme (so
technically no longer a 1% Catechol solution)
• Put in Colorimeter and watch reaction proceed.
• Make serial dilutions of Enzyme until a “good”
rate of reaction is achieved.
Standardizing the Colorimeter
• Insert water filled cuvette into colorimeter.
• Push and hold green button. Colorimeter is
now calibrated.
• Make sure you colorimeter is set to BLUE
Absorption.
• Hit run just as you add the final solution to
your cuvette and hit stop just as you reach 10
minutes.
Standardizing the Colorimeter
• Repeat for each run and it will make for a
beautiful graph.
• Make sure that the cuvette is DRY and free of
finger prints
Measuring Production of
Benzoquinone
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Put Catechol into Cuvette
Put water into Cuvette
Put Enzyme into Cuvette
Put Cap on Cuvette, invert 2 times to mix, and
place into colorimeter.
• Take colorimeter readings every 15 seconds.
Measuring Production of
Benzoquinone
• Remove cuvette from colorimeter every
minute, invert twice and immediately return
cuvette to colorimeter.
• Take readings for 10 minutes.
• (note: in the interest of time, skip the 2
control conditions, the teacher will do those)
Data Analysis
• Graph your raw data
• Calculate your maximum rate for each
condition.