Biology please see attached filwe to complete this lab. the directions are in the first document, which helps you fill out the second document which is a w

Biology please see attached filwe to complete this lab. the directions are in the first document, which helps you fill out the second document which is a w

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Biology please see attached filwe to complete this lab. the directions are in the first document, which helps you fill out the second document which is a worksheet, please include the graphs, for full rating. BIOLOGY 211 LA-4 Enzymes Page 1 of 5

LEARNING OBJECTIVES

· Understand how an assay works.

· Compare the rate of peroxidase activity in varying conditions.

· Create a graph of the results and draw conclusions to support or reject your hypothesis

BACKGROUND INFORMATION

(Background adapted from Biology 160 Lab Manual at Seattle Central College)

A wide variety of chemical reactions must occur all the time inside living cells in order to keep them functioning properly. In very general terms, a chemical reaction can be described as:

reactants à products example 1: A + B à C example 2: E à F + G + H

In example 1, two different reactant molecules join together to form a single product. In example 2, one reactant breaks down into three separate products. Chemical reactions do not occur instantaneously, however. Without “help,” most of the chemical reactions that occur inside cells would not proceed rapidly enough to maintain life. Enzymes are proteins (produced by cells) that catalyze chemical reactions. This means that they speed up the rate at which reactions occur, without being used up or transformed into something else in the process.

An enzyme capable of catalyzing the reaction in example 1 above might do its job by attaching to substances A and B, and holding them in position so that a bond can easily form between them. Without an enzyme, the reaction would not occur until a molecule of A and molecule of B just happened to bump into each other at precisely the right orientation for the bond to form. In example 2, the enzyme’s job might be to hold molecule E in such a way that the bonds attaching its monomers become stressed, and are therefore more likely to break.

Notice that in both of the situations described above, the enzyme must physically hold the reactant molecule(s) in order to work. Because enzymes are proteins, hydrogen and sometimes ionic bonds contribute to their specific and often quite elaborate shapes. In order for an enzyme to catalyze a reaction, it must fit together precisely with the reactant(s), like a lock and key. For enzymes the reactants that physically touch the enzyme are referred to as the “substrate”. The particular part of the enzyme where the substrate fits in is called the active site. Because their shapes are so specific, most enzymes are capable of catalyzing only one or a few specific reactions. We can now describe a chemical reaction catalyzed by an enzyme more specifically than we did above.

substrate(s) + enzyme à substrate-enzyme complex à product(s) + enzyme

The substrate-enzyme complex exists only briefly before the substrate(s) is changed into the product(s), which separate from the enzyme. The enzyme is now available to join with new substrate molecules, and repeat the process. A single enzyme molecule may catalyze thousands of reactions each second!

However, enzymes can be inactivated by factors which either cause changes in their shapes, or block their active sites. If the substrate and enzyme no longer fit together, the reaction will not proceed rapidly. The hydrogen bonds that contribute to the unique shape of each different protein are relatively weak, and they can be disrupted. Some enzymes function well over a broad range of temperature and pH values, however, many enzymes can only hold their proper shapes under specific temperature or pH conditions. The figure below shows how enzyme activity might increase with increasing temperature. Recall that as temperature rises, molecule vibration becomes faster. As molecules vibrate faster, there is a greater likelihood that substrate and enzyme molecules will encounter each other and a reaction will occur. However, note that the reaction rate abruptly decreases after a certain temperature. At this temperature, the enzyme likely denatured.

Chemical inhibitors are substances other than the substrate, enzyme, or product that can stop or slow the rate of a reaction. Competitive inhibitors are similar in shape to the substrate, and so can fit into the enzyme’s active site and physically block the formation of the enzyme-substrate complex. Noncompetitive inhibitors interact with some part of the enzyme other than the active site, and by doing so, they cause the protein’s shape to change enough that the substrate is no longer able to fit into the active site.

In this lab you will be observing the setup and results from experiments using turnip hydrogen peroxidase.

Hydrogen peroxide is a byproduct of cellular respiration (and lignin production and defense mechanisms in plants) that is toxic. The enzyme hydrogen peroxidase converts hydrogen peroxide to water and oxygen, which are not toxic. In humans, peroxidase (also called catalase) is produced and secreted from glandular organs. Many parts of plants produce peroxidase. The general reaction for peroxidase is given below:

2H2O2 à 2H2O + O2

It is often difficult to distinguish by appearance the reactants and the products, so just by looking, you may not be able to tell whether a reaction occurred. For this reason, colorimetric (color-producing) assays are often used to determine whether a reaction occurred. In a colorimetric assay, either the reactant or the product interacts with a molecule that changes color. Recall that iodine turned dark purple in the presence of starch. Iodine can be used as an assay for the presence of starch.

For this reaction, a molecule called guaiacol is provided in the reaction. Guaiacol interacts with the oxygen produced by the reaction to produce a yellow to brown product called tetraguaiacol. Therefore, you can tell how much product is produced by how deep the yellow to brown color is. This can be quantified using a spectrophotometer. Tetraguaiacol absorbs wavelengths best at 500nm. The more tetraguaiacol in solution, the greater the measurement of absorbance will be.

Notes about experimental design

The experiments demonstrated in the video do not necessarily follow the elements of experimental design. Consider the following points when designing your own experiment:

Controls: Especially for a colorimetric assay, you need to make sure you have a negative and a positive control, so that you know what the results look like when there is no activity/ reaction and what the results look like when there is activity/ reaction. The negative control gives you something to compare your results. If your results are different than the control, you can say a reaction happened. The positive control shows you what a positive result looks like. If your positive control looks different than the negative control, you can be assured that your assay is working.

Replication: It is always possible that something happened in one of your tubes. Maybe you forgot to add something or your sample was not representative of general results. Replication allows you to determine if your sample was representative. 2 replicates would be a bare minimum for any experiment.

INSTRUCTIONS FOR COMPLETING THE LAB

You will observe a chemical reaction in 5 different temperatures and analyze the results.

EXPERIMENT 1 (observe, do not attempt at home)

1. Collect the solutions needed in 4 beakers as shown in this video: https://youtu.be/xb0DnRc_CRQ

2. You will set up 2 tubes for each reaction. The first tube (1a, etc) will contain the substrate, H2O2, and the 2nd tube (1b, etc) will contain the peroxidase extract. Prepare five pairs of substrate and enzyme tubes by adding each solution as shown in table below.

3. Label each set of 2 tubes with a temperature. 5 temperatures will be tested, 4 °C (ice bath), 23° C (room temperature), 32 °C warm water bath, 65 °C hot water bath and 100 °C (boiling water) Label an additional tube as control.

4. Put each pair of tubes in the corresponding temperature and leave for 10 minutes.

5. Mix the substrate (a) and enzyme (b) solutions together by first pouring each substrate tube contents into an enzyme tube and then pouring the enzyme tube contents back into the substrate tube. You can observe this step and the next step in this video: https://youtu.be/xATgd4jrzro

6. Use a spectrophotometer to measure the absorbance of the solution at 500nm in each tube after 2 minutes.

If you are still unsure about how the lab is set up, you can additionally watch this video: https://youtu.be/f_ywfz0n0y8

Tubes 1a&1b, 2a&2b, etc. will be combined, so that the enzyme and substrate are found in the same tube.

tube

Temperature (C)

buffer (pH5), mL

H2O2, mL

Peroxidase extract, mL

Guaiacol,, mL

TOTAL, mL

1a

0

2

0

1

3

1b

4

0

1

0

5

2a

23°

0

2

0

1

3

2b

23°

4

0

1

0

5

3a

32°

0

2

0

1

3

3b

32°

4

0

1

0

5

4a

65°

0

2

0

1

3

4b

65°

4

0

1

0

5

5a

100°

0

2

0

1

3

5b

100°

4

0

1

0

5

6a

23°

2

0

0

1

3

6b

23°

4

0

1

0

5

Results for Experiment 1:

For results, the color will be quantified with a spectrophotometer at 2 minutes. The figure below shows a sample range of absorbance at 500 nm from 0-0.4. Results for the experiment itself are in the table.

https://apcentral.collegeboard.org/pdf/bio-lab13-enzymeactivity.pdf

Temperature

4

23

32

65

100

23

Absorbance (500nm)

0.1

0.25

0.4

0.1

0

0

Fill in page 1 of the worksheet. You will need to construct a graph for each experiment. Use Excel if you are able. Other options are Google Sheets or draw neatly by hand and take a picture. You will need to decide in each case whether a column graph or line graph is more appropriate. Remember that graphs need a descriptive title and labeled axes. I recommend not including the negative control on your graph. If making a line graph in Excel, use scatter plot with line instead of line graph. Paste your graphs into the worksheet and use them to answer the questions.

EXPERIMENT 2

This experiment will be similar, but instead of each tube being in a different temperature, it will have a different pH buffer. Note that only pH buffer 5 was used for all tubes in the previous experiment.

1. Label each set of two tubes with a pH. (3, 5, 7, and 9) Label an additional pair as control.

2. Prepare five pairs of substrate and enzyme tubes, as shown in the table below.

3. Working quickly, pour a substrate tube and a pH tube together and back into original tube.

4. Place each tube in a tube rack, as they are mixed.

5. Use a spectrophotometer to measure the absorbance of the solution at 500nm in each tube after 2 minutes.

tube

buffer pH value

Buffer, mL

H2O2, mL

Extract, mL

Guiacol,mL

TOTAL,mL

1a

3

0

2

0

1

3

1b

3

4

0

1

0

5

2a

5

0

2

0

1

3

2b

5

4

0

1

0

5

3a

7

0

2

0

1

3

3b

7

4

0

1

0

5

4a

9

0

2

0

1

3

4b

9

4

0

1

0

5

5a

5

2

0

0

1

3

5b

5

4

0

1

0

5

Results for Experiment 2:

pH

3

5

7

9

5

Color at 2 minutes

0.1

0.4

0.25

0

0

Fill in page 2 of the worksheet. Construct a graph to answer the questions at the bottom of the page.

Experiment Proposal

Now it is time to formulate a hypothesis and design an experiment. You can type in your proposal on the last page of the worksheet. Your proposal should include the following.

Introduction

State your question and hypothesis. Identify the independent and dependent variables. Make a prediction.

Materials & Methods

Begin with a list of the supplies and solutions you would need. Which variables would need to be controlled? Next, write a numbered list of steps you would take to conduct the experiment and measure the results. Be specific and thorough enough that someone else could repeat what you did without having to ask you for clarification. You may include a diagram (drawn by hand or computer-generated) to supplement your explanation, if appropriate.

Results

You will not have actual results, but you should include a data table which you could use to record your results. You can put your predicted results in the table. You should also indicate the title and axis labels for the graph you would construct.

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