Lab – Factors That Affect Enzyme Activity



Lab – The Effect of Temperature on Enzyme Activity

Background

Some fruits contain enzymes capable of hydrolyzing proteins. If you are hoping to tenderize a steak, the enzymes found in papaya can be helpful. However, not all enzymes found in fruit can be helpful all the time. The enzyme Actinidin found in fresh kiwi fruit is capable of hydrolyzing proteins found in dairy products. As the milk proteins are digested acids are released and the food ends up having a very unpleasant bitter taste.

Pineapple contains a digestive enzyme called bromelain which is capable of breaking down the proteins such as gelatin – the main ingredient in Jell-O. Gelatin is a processed version of the protein collagen, a simple protein that makes up one-third of all proteins in the human body. The main source of the collagen that is used in Jell-O comes from hooves, bones, connective tissue found on cows, horses and pigs. Along with collagen, Jell-O consists of water, food coloring, sugar, and artificial flavours. Collagen is found in all living animals. This protein is what gives body parts strength, flexibility, and protection. There are five major categories of collagen that range from the fibres in your eyes to the structure of placentas. To harvest the collagen needed for gelatin the Jell-O Corporation turns to natural sources found in cows, horses, and pigs. The animals' body parts which were previously mentioned are ground up to expose the proteins within. After they are ground up the bio matter is then treated with a strong acid or base, which breaks down the cellular structures of the collagen to release the proteins from connective tissue. Once the proteins become separated from the tissues the bio-mass is then discarded. Then, the mixture created from the released proteins (collagen proteins, which are the basis of Jell-O) and the strong acid or base is boiled.

Bromelain is used in many meat tenderizers for this purpose (cooking ham with pineapple makes it tender). If you have ever read the directions on a Jell-O package, then you will have seen that it warns you not to put pineapple chunks into the Jell-O. This is because Jell-O is a protein mesh with trapped pockets of liquid and the bromelain cuts the protein chains and keeps the gelatin from gelling properly.

Pineapple plants produce this enzyme as a self-defence mechanism. The sap of the pineapple plant contains much higher concentrations of bromelain and can cause severe pain if eaten.

Purpose:

Today we are going to use fresh, frozen, and canned pineapple to see how changes in temperature can affect bromelain’s ability to digest the protein gelatin.

Materials:

• 4 test tubes

• Hot JELL-O

• Fresh pineapple

• Canned pineapple

• Frozen pineapple

• Knife

• Sharpie

• Beaker with ice water bath

• Weigh Boat

• Electronic Balance

Procedure

1. Using your knowledge of how enzymes function, record a hypothesis that predicts the results of this lab investigation for EACH of your test conditions.

2. Label 4 test tubes with the numbers 1-4.

3. Prepare an ice water bath in a beaker.

4. Select pieces of fresh, canned and frozen pineapple that are approximately equal sizes. The pieces should be just small enough to slide into your test tubes.

5. Using the electronic scale, weigh your smallest piece and record its mass below. Weigh and trim the other pieces as necessary so that each piece has the same mass measured to one decimal place. ** Do this quickly so your frozen pineapple doesn’t melt too much!

Type of Pineapple Mass

_________________ - ____________ g

_________________ - ____________ g

_________________ - ____________ g

6. Gently mash each piece of pineapple in the weigh boat to increase the surface area where bromelain can react with the gelatin molecules in the Jell-O.

7. Place your pineapple pieces and any juices that are in the weigh boat into their corresponding test tubes:

• Test tube #1 – Control (Jell-O only, no pineapple)

• Test tube #2 – Fresh Pineapple

• Test tube #3 – Frozen Pineapple

• Test tube #4 – Canned Pineapple

8. Using a graduated cylinder add 20 mL of liquid Jell-O to each test tube.

9. Gently mix the contents of the tubes by rolling them upright between the palms of your

hands.

10. Place all four test tubes into an ice water bath in your beaker.

11. While you are waiting for the Jell-O to set, complete the data table as part of your results

section for this lab.

12. Every 2 minutes check to see if the Jell-O is setting in the tubes. When test tube #1 (control) has set 100% the experiment is over. Record your observations in your table.

Time (mins) |0 |2 |4 |6 |8 |10 |12 |14 |16 |18 |20 | |Test Tube #1

Control | | | | | | | | | | | | |Test Tube #2

Fresh | | | | | | | | | | | | |Test Tube #3

Frozen | | | | | | | | | | | | |Test Tube #4

Canned | | | | | | | | | | | | |Results:

Table 1. Percent solidification of Jell-O over time

Formal Lab Write-Up Assignment Total /26 marks

Once you have completed your experiment write up a formal lab report. Follow instructions carefully!! Writing a proper lab report is an important skill to learn.

Your lab report must be typed, double-spaced and include:

1 marks Your name, date, lab partners, title etc.

1 mark Purpose - Why are we doing this lab?

2 marks Hypothesis - Make an educated guess about the results of this lab. Make sure to include hypotheses for each test condition. Do not attempt to provide reasons for your hypotheses, simply state them i.e. "If _____[I do this] _____, then

_____[this]_____ will happen."

1 mark Materials – List out the materials used in this lab.

1 mark Procedure - (Simply refer to the procedure in this lab – DO NOT write it out)

7 marks Results -

(2 marks) 1) Data Table - proper format all relevant information included

Mass of pineapple pieces and any other observations made

(5 marks) 2) Graph - title, X and Y axis labels, legend, 4 lines, appropriate scale

If you do not have excel – use this link to create a graph:

3) Data sheet that you made any other observations or records of data. Will be stapled to back of report.

10 marks Discussion - (paragraph form, double spaced – DO NOT simply answer the questions, discuss what you have learned and show me that you can apply your knowledge)

Things to include in your discussion:

(1 mark) Discuss why we used a control in this experiment.

(4 marks) Summarize your observations for each test condition in the lab. Use the information in your notes about how temperature affects enzyme activity to explain what you observed for each condition. ** This is most effectively done if you write a paragraph that explains each test condition.

(5 marks) Were your results for each condition what you had hypothesized? Why or why not? Explain. Consider this as you interpret your results: Do you think our experimental set-up had any influence on the results for any of the test conditions?

3 marks Conclusion

(2 marks) Was there evidence to support or reject each of your hypotheses? Explain while summarizing what you have learned about the effects of temperature on enzyme activity.

(1 mark) Provide an example of how you could apply this newly learned knowledge in the future.

The Effect of Temperature on Enzyme Activity

Supplementary Questions /10 marks

1. Why did test tube #1 only contain JELL-O e.g. explain the importance of a control? 1 mark

2. The process of canning involves exposing the pineapple to high temperatures for extended periods of time. How does this process change bromelain? 1 mark

3. Check out the following website:



Look around this website to help you answer the following question.

List and describe the steps involved in scientific method. 4 marks

4. You hypothesize that the hormone thyroxin will increase the heart rate of mice. Use the following materials; design a controlled experiment to test this hypothesis. The design will require that you have a purpose, hypothesis, materials, and procedure (how you would carry it out). Please do not go any further. We do not need to expose mice to any unnecessary testing.

For this experiment you acquire the following materials:

▪ A population of 30 mice

▪ 2 Identical cages to house the mice

▪ Food with thyroxin and food without thyroxin

▪ Heart rate monitors 4 marks

(answer by typing up on the computer and printing it out. Please remember to double space.)

The Effect of pH on Enzyme Activity

Background

Extremes in pH levels, like temperature, cause alterations in the structure, and thus the function, of enzyme molecules. Also, varying concentrations of pH levels in solution affect the activity of the enzyme. In groups you will test the effect of different concentrations of an acid (HCl) or a base (NaOH) on the activity of the enzyme Bromelin.

MATERIALS PER GROUP:

(Group A will need 1 ml HCl, Group B will need 1 ml NaOH)

• 21 test tubes

• 10 ml graduated cylinder

• 10 - 5 ml pipettes

• test tube rack

• water

• 30 ml frozen pineapple juice concentrate, thawed

• 1 pkg Knox gelatin, prepared according to instructions on the package.

PROCEDURE:

1. GROUP A: Label 10 test tubes A-1, A-2, A-3, etc. up to A-10.

GROUP B: Label 10 test tubes B-1, B-2, B-3, etc. up to B-10.

2. Carefully measure 3 ml pineapple juice into each of the labeled test tubes.

3. Place 9 ml water into the 10 empty test tubes. Label one of these tubes as CONTROL and set it aside.

4. Make a serial dilution in the 9 test tubes by following the dilution table drawn below.

Group A use HCl, Group B use NaOH. *** Use a different pipette for each dilution.

5. Transfer 3 ml of diluted pH concentrations into each test tube of pineapple juice, using the dilution table below as a guide. Record the concentration of solution placed in each test tube. (Example: A-1 = 1:1 dil, A-2 = 1:10 dil, A-3 = 1:100 dil, etc.) The solution concentrations become weaker as the test tube numbers progress from 1 - 9.

6. Transfer 3 ml of water from CONTROL tube into tube A-10 (or B-10).

7. Add 10 ml gelatin to each test tube, A-1 to A-10 (or B-1 to B-10). Shake well being careful not to let the acid or base come into contact with skin.

8. Refrigerate test tubes overnight.

9. On day 2, check each test tube for liquidity of the contents. Record observations.

ANALYSIS:

1. At what concentration of acid or base was the first signs of liquidity observed?

2. What conclusion can be derived as to the acid/base concentration at which bromelin's molecular structure is altered?

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