Enzyme Activity Lab 13

8/10/2019 Enzyme Activity Lab 13

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Enzyme Activity Lab 13

AP Biology

(Peroxidase + Hydrogen Peroxide Complex Peroxidase + Water + Oxygen)

2H2O2 2H2O + O2(gas)


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Learning Objectives

The student is able to design a plan forcollecting data to show that all biological systemsare affected by complex biotic and abioticinteractions (2D1 & SP 4.2, SP 7.2).

The student is able to use models to predict andjustify that changes in the subcomponents of abiological polymer affect the functionality of themolecule (4A1

& SP 6.1, SP 6.4). The student is able to analyze data to identifyhow molecular interactions affect structure andfunction (4B1 & SP 5.1).


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Basic background information

Basic protein structure

The concept of induced fit

The role of enzymes That structure, function, and environment

are all required for maximal function of

enzymatic reactions


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2H2O2 2H2O + O2(gas)

Peroxidase is an enzyme that breaks

down peroxides, such as hydrogen

peroxide, and is produced by most cells in

their peroxisomes. Peroxide is a toxic

byproduct of aerobic metabolism. Various

factors abiotic and biotic could have

a major influence on the efficiency of thisreaction.


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Label carefully

Gather all materials

Plan your procedure and know what you are

doing before you start. Do a mock run

There is timing involved, so be prepared to

start the timer as soon as you mix the

materials.

Make data charts ahead of time so you have

a place to put your results.


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So, what is this lab about?

Turnip peroxidase is the enzyme that

catalyzes the reaction that breaks down

hydrogen peroxide into water and oxygen.


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guaiacol

We can see the reaction because we are

adding guaiacol, an indicator of oxygen due to a

color change that occurs in its presence. The

more oxygen the deeper the brown the colorbecomes. The compound guaiacol has a high

affinity for oxygen, and in solution,it binds

instantly with oxygen to form tetraguaiacol,

which is brownish in color. The greater theamount of oxygen produced, the darker brown

the solution will become.


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We can qualitatively or quantitativelymeasure the color change after allowing the

reaction to occur. A color palette is prepared by placing

different amounts of enzyme and substratemixture with distilled water so that the finalpercent of the solutions varies by 10% ineach of the 11 test tubes prepared.

This will provide a way to view the different

colors that can be seen for the differentamounts of oxygen released at maximumproduction. It will be used for comparison forthe other reactions.


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Baseline is a universal term for most chemical

reactions. In this investigation, the term

is used to establish a standard for a reaction. Thus,when manipulating components of a reaction (in

this case, substrate or enzyme) you have a

reference point to help understand what occurred

in the reaction. The baseline may vary withdifferent scenarios pertinent to the design of the

experiment, such as altering the environment in

which the reaction occurs. In this scenario, different

conditions can be compared, and the effects ofchanging an environmental variable (e.g., pH)

can be determined.


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Color palette


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Rate can have more than one applicabledefinition because this lab has two major optionsof approach, i.e., using a color palette and/or aspectrophotometer to measure percent of lightabsorbance. When using a color palette tocompare the change in a reaction, you can inferincrease, decrease, or no change in the rate; thisinference is usually called the relative rate of thereaction.

.


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Part 1 Baseline materials+ labeling

1. E= enzyme the cold, turnip enzyme

label a 2.5 ml syringe to use to measure this.2. P= product (oxygen) which is shown when

guaiacolreacts with it and turns brown

label a 2.5ml syringe to use to measure this

3. NB= buffer pH7 neutral buffer

label a 10ml syringe to use to measure this

4. S= substrate, Hydrogen peroxide

label a 2.5ml syringe to use to measure this

5. test tube- label SPNB substrate, product, neutral buffer

6. Test tube- label ENB enzyme, neutral buffer


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A SPNB

2ml Substrate hydrogen

peroxide

1ml P = guaiacol

1ml NB neutral Buffer pH 7

B ENB


1ml E= Enzyme turnip peroxidase

Cover with parafilm and mix . Use a disposable pipette

to transfer tube A to tube B. Cover and mix.

Immediately observe by comparing to the color palette and begin timing!

Observe every minute for 5 minutes.

Calculate the rate for the baseline. Color change/% oxygen over time

Time

minutes 0 1 2 3 4 5Scale/

number


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A SPNB

2ml Substrate hydrogen

peroxide

1ml P = guaiacol


B ENB3ml NB neutral Buffer pH 7

1ml E Enzyme turnip peroxidase

Time

minutes 0 1 2 3 4 5Scale/

number

Immediately observe by comparing to the color palette and begin timing!

Observe every minute for 5 minutes.

Calculate the rate for the baseline. Color change/% oxygen over time


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2 ml S= hydrogen peroxide

1 ml P=product indicator

guaiacol

Tubes 1,2,4,9,11,12

1 ml NB neutral buffer

Tubes 3,5,6,7,8,10

1 ml E=turnip peroxidase solution

3 ml buffer of the correct pH for the tube. For

example tube 3 use pH 3, tube 5 pH of 5 etc!

Make a data

chart!


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2 ml S= hydrogen peroxide

1 ml P=product indicator

guaiacol

Tubes 1,2,4,9,11,12

1 ml NB neutral buffer

Tubes 3,5,6,7,8,10

1 ml E=turnip peroxidase solution

3 ml buffer of the correct pH for the tube. For

example tube 3 use pH 3, tube 5 pH of 5 etc!

Be ready!

Mix tube 1 with tube 3.

Observe at time zero and

every minute for 5 minutes!


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rate

Calculate the rate for each tube.

How much oxygen produced in 5 minutes?

Based on the readings over the 5 minutetime period compared to the color change

or % of oxygen.

% change over time= rate


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graph

Rate for each pH


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Assessment Questions

#3. If you omitted the enzyme?

If you omitted the substrate?

If you omitted the indicator? Based on your answer to #4 develop a

specific question to test for part 3 of the lab.

Your group must submit an experimentalplan for approval.


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Part 3

Complete your experiment planning sheet

Record your data.

Data analysis What conclusion can be drawn from your

groups data?

Create a presentation to share yourfindings

Documents

Enzyme Activity Lab 13