Notes

Observing and Measuring Proteins (enzymes) at Work

Introduction:
Like the shape of a gear, determines its function or the shape of a ball determines which sport it is used in, shape can determine what function many things have. Proteins serve various functions within our bodies and makes us who we are:

Some proteins are structural like hair and your finger nails,
Some proteins are the colors of your eyes, hair, and skin (structural),
Some proteins gave you male or female characteristics (hormones)
Some proteins, like enzymes, control the rate of chemical reactions that occur in your cells.

Whatever the type of protein, its shape determines its function. If the shape changes, the protein won’t continue to work correctly. pH and temperature can change the shape of enzymes, therefore effecting their function. Enzymes are proteins with very specific functions. For instance, venom from snakes contains 90-95% protein. A rattlesnake’s venom contains the enzyme phospholipase.

What do you think phospholipase breaks down?
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Why would it be important for snakes to have venom that contains enzymes? What about them physically requires this tool?
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In order to obtain energy and building blocks from food, the digestive system must break down proteins, fats and carbohydrates. In this process, specific enzymes catalyze hydrolysis (splitting) reactions in which foods are broken down into monomers (single molecules).

Enzymes act to maintain homeostatic (optimum levels for the reaction) levels of chemicals in your body, digest food, and even remove toxic waste from your cells (also considered a form of digestion).

In order to obtain energy and building blocks from food, the digestive system must break down proteins, fats, and carbohydrates. In this process, specific enzymes catalyze hydrolysis (splitting with water) reactions in which foods are broken down into monomers.


Based on the picture below, how do enzymes do this?
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Enzymes are highly selective biological catalysts, meaning that each enzyme only speeds up a reaction by lowering the amount of activation energy needed for the reaction to occur (in other words, they make the reaction easier to happen).

The molecules that an enzyme works with are called substrates. The substrates bind to a region on the enzyme called the active site. Often called the lock-and-key model, the active site of an enzyme is precisely shaped to hold specific substrates. The chemical reactions result in a new product or molecule that then separates from the enzyme. The enzyme is them reused to catalyze other reactions; Enzymes are the ultimate recyclers, they can be reused!















Referring to the figure above and the background information, what is the role of enzymes in a reaction?
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How do your cells get rid of toxic waste materials?

Every cell in your body is naturally creating waste that is potentially toxic. If the cell doesn’t get rid of it or change it into something that is not toxic the cell may be damaged and die. Your cells generally don’t die as a result because they are always converting the toxic material into less toxic material. Enzymes are generally the tool used to breakdown these toxic substances.

In this lab you will observe how catalase (an enzyme in many types of cells) breaks down hydrogen peroxide (produced by and toxic to cells) into water and oxygen gas.

That means that 2 molecules of hydrogen peroxide (H2O2) break down into 2 molecules of water and 1 molecule of oxygen gas (O2). Use the space below to write out a balanced chemical equation for the chemical reaction described above. If you are confused make sure to ask your teacher for help.





Reactants Products












Part 1: Cellular Enzymes and Hydrogen Peroxide

Purpose:

The purpose of this lab is to observe how cells use enzymes to break down toxic cellular material (hydrogen peroxide) into harmless water and oxygen gas.

Materials:
Goggles
1-50 mL beakers with tissue samples (liver)
3% Hydrogen Peroxide solution
Thermometer
Glass stirring rod
Stopwatch
1 test tubes and rack


SAFETY CONSIDERATIONS – The chemicals you are working with are real. They should be considered dangerous and should be treated according to the ETHS safety contract that you signed. YOU MUST WEAR SAFETY GOGGLES ON YOUR EYES AT ALL TIMES AT YOUR LAB STATION

Procedure:
Pre-lab preparation: You will need to label test tubes and get your stop watch ready. These reactions will occur quickly. All qualitative and quantitative observations should be recorded in the data table provided (last page).


Test 1 – Establish a control. READ ALL OF TEST 1 BEFORE STARTING
How does catalase act under unaltered conditions? In order to keep yourself organized, consider checking off each step as it is completed. All collected data should be recorded in the data table.

Place 2 mL of the 3% hydrogen peroxide into one clean test tube labeled “liver.” Insert thermometer and record the starting temperature of the hydrogen peroxide. Leave the thermometer in place.

Prepare your stop watch and begin the count as soon as the 1 mL of the liver solution is added into the test tube labeled “liver.” Create a data table to record the temperature every 5 seconds for the duration of the reaction. If necessary, continue the data table until the reaction is complete.




Data Table 1

Time (secs)
Temperature:


0


5


15


20


25


30


35



Starting Temperature________
Ending Temperature_________
Percent increase/decrease* in temperature as a result of the reaction __________

*To find percent change of temperature:

new temp – old temp
old temp

Observe the reaction and record in the data table.
What gas is being released? ____________
Where is that gas coming from? _________. Refer to the chemical equation in the introduction.
Assign an appropriate “Reaction Rate” ________for the reaction. (ie; how immediate was the reaction? Reference chart above)
Did the test tube get warmer or cooler? _____________.
Why do you think that happened? ______________














Part 2: Catalase in cells.

Purpose:

Determine what types of tissue contains catalase, answering: Do all cells have catalase?

Materials:
Water bath, 40 C
Goggles
2 50 mL beakers (tissue samples – catalase source
homogenates – liver and potato)
8 test tubes and rack
HCl (acid) in dropper bottle
10 mL graduated cylinder
Thermometer
NaOH (base) in dropper bottle
Ice Bath 0 C
pH papers
Glass stirring rod
Test tube tongs
3% Hydrogen Peroxide solution
Hot Plate (water baths)
100 C
Stopwatch

Test 1: The types of tissues that contain catalase

Check off each step as it is completed and record all data in the data table.

Label 2 clean test tubes: liver tissue and potato.
Place 2 mL of 3% hydrogen peroxide into each of the labeled test tubes.
Prepare your stop watch and time from first bubble to last bubble as start time and end time respectively. Complete each tissue sample before proceeding to the next.
Add 1 mL of the liquid liver tissue to the labeled test tube and record the reaction rate ________, starting temperature ________ and ending temperatures _________, and qualitative observations.
Record start time ___________ and end time ___________
Add 1mL of the liquid potato to the labeled test tube and record the reaction rate _________ , starting temperature ________ and ending temperatures _________, and qualitative observations.
Record start time ________ and end time _________.

Catalase Reaction Data
Test No.
Catalase Source
Temp
Start
Temp
End
Reaction Time
(Seconds)
Reaction
Rate
(0-3)
Observations: Qualitative/Quantitative
2
Liver











2
Potato











*Reaction time is the amount of time from when you added the hydrogen peroxide to when the bubbles stopped.

Test 2 – The effect of temperature on catalase

Purpose: Determine the effect of temperature on enzymes, answering: will the catalase enzyme continue to work at high and low temperatures?

**** Before you do anything else****
Label one test tube with your name/period/frozen liver.
Place 2 mL of liver homogenate in tube and place in freezer until needed for test 2.


Hypothesize: What happens to enzymes at high temperatures? Will the catalase enzyme continue to work at high and low temperature extremes?
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Check off each step as it is completed and record your data in the data table.


Label three test tubes (100 ◦C Liver, 65 ◦C, and 0 ◦C Liver)
Put 2 mL of liver tissue into each of the labeled test tubes.

100◦C HOT
Place one test tube labeled “100 ◦C Liver” in a boiling water bath for 5 minutes.
Remove the test tube from the hot water bath using test tube holders and place in the rack. Insert the thermometer and allow the test tube to cool to room temperature (about 24 ◦C). CAUTION - HOT GLASS and LIQUID!
Add 1 mL of hydrogen peroxide to the test tube and record the reaction rate _________ and ending temperature _________. Did the reaction occur?________
This data should be transferred to the test 3 data (boiled) on the data table.

40 ◦C Warm
Place one test tube labeled “40 ◦C Liver” into the 40 ◦C water bath for 5 minutes.
Remove test tube from warm water bath using test tube holders and place in the rack. Insert the thermometer and allow the test tube to cool to room temperature (about 24 ◦C). This will be warm so please be careful.
Add 1 mL of hydrogen peroxide to the test tube and record the reaction rate _________ and ending temperature __________. Did the reaction occur? _______
This data should be transferred to the test 3 data (warm) on the data table.



0 ◦C COLD
Place one test tube labeled “0 ◦C Liver” into the freezer until the temperature reads 0 ◦C and the homogenate is slushy/solid. THIS SHOULD HAVE BEEN DONE BEFORE THE HOT TEST!!!
Remove test tube from freezer and place in the rack.
With the thermometer in the test tube, add 2 mL of hydrogen peroxide to the test tube and record the reaction rate _________ and ending temperature _______.
Transfer this data to the test 3 data (frozen) on the data table


Catalase Reaction Data
Test No.
Catalase Source
Temp
Start
Temp
End
pH
Reaction Time
(Seconds)*
Reaction
Rate
(0-3)
Observations: Qualitative/Quantitative
3
Warm Liver












3
Boiled Liver












3
Frozen Liver












*Reaction time is the amount of time from when you added the hydrogen peroxide to when the bubbles stopped.

Test 3 – The Effect of pH on catalase

Purpose: Determine the effect that pH has on an enzyme, answering: will enzymes continue to work when the pH is changed?

Hypothesize: What happens to enzymes at different pH levels? Will the experiment continue to work at high and low pH extremes?
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Check off each step as it is completed.

Label three clean test tubes H2O, HCl, and NaOH.
Add 2 mL of hydrogen peroxide to each of the 3 clean, labeled test tubes.
Add 3 drops of H2O to the H2O test tube.
Using a glass stirring rod, remove a drop of the solution and place this on the pH test strip, record the pH of the above test tube. ______
Add 2 mL of liquid liver to the H2O test tube and record the reaction rate _______ and the reaction time (sec) ______ and ending temperature ____.
Add 3 drops of HCl (hydrochloric acid) to the labeled HCl test tube.
Caution HCl is a dangerous acid. Use a glass stirring rod to remove a drop of the solution and place on the pH test paper. Using pH paper record the pH level of test tube 1. ______
Add 2 mL of liquid liver to the HCl test tube and record the reaction rate_______ and the reaction time (sec) ______ and ending temperature ______.

Add 3 drops of NaOH to the labeled NaOH test tube.
Caution NaOH is a dangerous base. Use a glass stirring rod to remove a drop of the solution and place on the pH test paper. Using pH paper record the pH level of the above test tube. _______
Add 2 mL of liquid liver to the NaOH test tube and record the reaction rate _______ and the reaction time (sec) ______ and ending temperature ______.


Catalase Reaction Data
Test No.
Catalase Source
Temp
Start
Temp
End
pH
Reaction Time
(Seconds)*
Reaction
Rate
(0-3)
Observations: Qualitative/Quantitative
4
Acidic Liver














4
Basic Liver














*Reaction time is the amount of time from when you added the hydrogen peroxide to when the bubbles stopped.



Results:

Add all of your collected data to this sheet so that you can compare your data to your control.

Catalase Reaction Data
Test No.
Catalase Source
Temp
Start
Temp
End
pH
Reaction Time
(Seconds)
Reaction
Rate
(0-3)
Observations: Qualitative/Quantitative
1
Liver












2
Liver












2
Potato












3
Liver












3
Boiled Liver












3
Warm Liver












3
Frozen Liver












4
Liver












4
Acidic Liver












4
Basic Liver












*Reaction time is the amount of time from when you added the hydrogen peroxide to when the bubbles stopped.

Graphs:

Using the data collected above construct graphs for “Temperature and Catalase Reaction Rate” and “pH and Catalase Reaction Rate” on google sheets. Make sure to include all appropriate labels and titles.










Data Analysis and Conclusion:

Review: Purpose of this lab

Observe how cells use enzymes to break down toxic cellular material (hydrogen peroxide) into harmless water and oxygen gas.
Observe how acidic and basic pH levels can affect how well the enzymes work.
Determine the optimal (best) pH level for the enzyme, catalase.
Observe how temperature can affect how well the enzymes work.
Determine the optimal (best) temperature for the enzyme, catalase.

pH Review:

After completing this lab, review pH and the pH scale. Remember that the pH scale is a measure of how many H+ and -OH There are in a solution. Acids have a higher concentration of H+, thus the stronger the acid the lower it is on the pH scale (smaller the number), and the more H+ ions are available to chemically react. Bases have a higher concentration of -OH ions, thus the stronger the base the higher it is on the pH scale (larger the number), and the more -OH ions are available to chemically react. The pH scale measures pH levels between 0 (acidic) and 14 (basic) with 7 being neutral. The pH of your stomach is about 2, therefore the enzymes that digest food in your stomach work best at that pH level.

Conclusion:

Use the questions below to organize and construct your thoughtful analysis and conclusion of the enzyme lab. These questions are designed to guide your thought process as you write your analysis.

Conclusion: Use your background information (and your unit packet) and your data to write a conclusion, using Claim, Evidence, and Reasoning format.

Within this conclusion you should state a claim (could be refuting or accepting your hypothesis, or answering the original question), use your evidence (so that is your background information and what you know from your unit packet), and reason (so how you argue that your claim is correct).










Organize your thoughts in the table below.

Claim: A statement or conclusion that answers the original question/problem.










Evidence: Scientific data that supports the claim. The data needs to be appropriate and sufficient to support the claim.





























Reasoning: A justification that connects the evidence to the claim. It shows why the data counts as evidence by using appropriate and sufficient scientific principles.


Within your conclusion you should be using the following questions as a guide for writing the conclusion section of this lab report. This DOES NOT MEAN that you answer all of these questions in this order, but that these questions are meant to make you think about what your results actually mean. Most of these questions will be answered within your conclusion.

What are enzymes?
Describe the relationship between catalase and hydrogen peroxide. Indicate which is the enzyme, which is the substrate and what occurs during the reaction. It may be helpful to write the equation.
What is the optimum pH of catalase?
What do high temperatures do to enzymes?
Why did the reaction not proceed at 100º C ?
Why did the reaction proceed slowly at 0º C?
What is your body temperature in Celsius?
What effect does temperature have on the activity level of an enzyme? (Hint: Look at what happened in test tubes 1 and 3). Why do you think it might be bad to have a high fever?
How does temperature and pH affect the reaction rate of catalase? Based on your results does there appear to be an optimal (best) pH range for catalase? Propose a way to refine your experiment to find the exact or OPTIMAL pH and temperature of catalase.
What factors could have influenced the reaction time and rating of the chemical reaction between the liver and the hydrogen peroxide? Give two factors. Explain each.
Summarize the graph below, discussing the role of enzymes and activation energy.