Enzymes & Experimental Design

Enzymes & Experimental Design

Objectives:

To continue to practice and apply hypothesis testing.
To continue to practice experimental design.
To gain a better understanding of enzymes and some conditions (temperature, pH, and enzyme and substrate concentration) that affect enzyme activity and the rate of an enzyme-catalyzed reaction.
To learn the concepts behind the workings of the spectrophotometer.
To learn to use the spectrophotometer.
To understand these terms: enzyme, enzyme activity, active site, substrate, enzyme-substrate complex, product, denature, variable, control.

Now that we have spent some time considering the steps scientists go through in “doing science”, we’ll try our hands at formulating our own hypotheses, and designing experiments to test them. Unlike other labs you have done, this will not be a “cookbook” exercise. You will be writing your own protocol. To do this effectively, you must read this lab thoroughly, and complete the prelab assignments, before coming to class!

The subject of our inquiry will be enzymes and the various factors that influence their activity. Without enzymes, most biochemical reactions would take place at a rate far too slow to keep pace with the metabolic needs and other life functions of organisms. Enzymes are catalysts that speed up chemical reactions but are not themselves consumed or changed by the reactions.

The cell's biological catalysts are proteins. These enzymes have a very complex three-dimensional structure consisting of one or more polypeptide chains folded to form an active siteaspecial area into which the substrate (material to be acted on by the enzyme) will fit.

Changes in temperature, alterations in pH, the addition of certain ions or molecules, and the presence of inhibitors all may affect the structure of an enzyme's active site and thus the ability of the enzyme to catalyze the reaction (“enzyme activity”), and hence the rate of the reaction in which it participates. The rate of an enzymatic reaction can also be affected by the relative concentrations of enzyme and substrate in the reaction mixture.

During this exercise you will study the activity of the enzyme catecholase contained in some fruits and vegetables. Peeled potatoes and bruised fruits turn brown when exposed to air because catecholase facilitates a reaction between catechol and oxygen. In the presence of oxygen, the compound catechol is oxidized by the removal of two hydrogen atoms. Catechol is thus converted to benzoquinone, and oxygen is reduced by the addition of two hydrogen atoms to form water. Benzoquinone molecules then link together to form long branched chains. These chains are the structural backbones of the red and brown melanoid pigments that cause darkening. Keep in mind that whenever you use potato juice in the following experiments, you are using an enzyme preparation (in which the enzyme is catecholase).

MATERIALS (per group)

12-18 test tubes / 12 pieces of Parafilm / 2 pipets
250 ml beaker / 1 test tube rack

MATERIALS (to share)

pH 7 phosphate buffer / 50 °C water bath / extra pipets
catechol, with pipets / ice / wax pencils
potato extract, with pipets / spectrophotometer / Kimwipes ®
HCl and NaOH solutions / thermometers / pH Indicator Paper

preparation

Before lab, read over this lab and material in the book on enzymes. Generate a list of variables, or factors that might affect the rate at which an enzyme can catalyze the conversion of substrate to product. Think about how you might test the effects of some of these variables. In the space provided on the sheet at the end of the lab, select one of the variables you identified, and outline the protocol you would use to test its effects. (You may want to coordinate with your lab group in selecting a variable, but eachstudent needs to write out their own protocol before coming to lab.) Think carefully about how many conditions you might want to test in your experiment, and what type of controls you might need to use to validate your results. Be sure to consult the sample protocol below as you plan your experiment.

General procedures

1.Work in groups (the size of the groups will be determined by the size of the class and by the amount of equipment available). Your instructor will consult with each group to approve their protocol before you begin your experiment!

Sample protocol- Use this protocol as a guide in writing your own!

Identify the variable you wish to explore, and establish the conditions under which you will test the activity level of our enzyme, catecholase. For instance, if you choose to explore the effects of temperature, you’ll need to decide on the exact temperatures you wish to test, and set-up the appropriate ice baths or warm water baths. You should test your enzyme under three conditions. For example, your group may choose to test your enzyme at 4 °C, 24 °C, and 50 °C. Be aware that you may need to adapt your experiment to fit the time and supplies available in the lab.

Label three tubes with a wax pencil so that you can distinguish one treatment from the next. Prepare the tubes, each containing 3 ml of pH 7 phosphate buffer, and 0.5 ml of potato juice (our enzyme!), and 0.5 ml of water (use a different pipette for the water and the potato juice). These will be the blanks for the three conditions you decide to test.

Fill three additional test tubes with 3 ml of pH 7 phosphate buffer, and 0.5 ml of potato juice. Label these tubes such that you can distinguish them from your blanks. These are your “experimental” tubes.

You will have a bottle of catechol, the substrate. When you are ready to begin your experiment, you can add catechol directly from the bottle using a new pipette for measuring.

With your experimental conditions established, add 0.5 ml of catechol to each of your experimental tubes. (Water has been used to replace this in your blanks.) Cover each tube with Parafilm and invert it several times to mix the contents.

6. Allow the reaction to proceed for 6 minutes. In the meantime, prepare to take a reading on the Spectronic 20 (“Spec 20” for short). Youshould be sure to turn on your Spec 20 before your experiment as it will need 10-15 minutes to warm up! Be sure the wavelength is set to 420. Adjust the Spectronic 20 to total absorbance, or zero transmission using the zeroing knob (on/off) on the left. Turn it until the needle aligns on top of the line, just to the left of “2” on the absorbance scale. This is called “zeroing” the Spec. (See Figure 1.)

7. After 6 minutes, use the blank for your first treatment to adjust the machine to 0% absorbance using the blanking knob. This is called “blanking” the Spec 20. Wipe condensation off the tube before reading. Then read the absorbance of your experimental tube and record your reading in Table 1.
8. Repeat for the other 2 experimental tubes. Remember to use appropriate blanks at each condition you test. / Figure 1: Baush & Lomb, Spectronic 20

zeroing knob (L)blanking Knob (R)
Sample chamber above zeroing knob

Clean-up

When done, please empty all tubes in the sink, rinse them out, place them in your test tube rack, and put the rack on the table at the front. Throw away your pipets, pieces of Parafilm, and any papers. Wipe your desk off.

DATA analysis

Graph the data in Table 1 (use graph paper). Be sure to label the x- and y-axes appropriately. Think carefully about what type of graph might be most appropriate for the data you have generated.

Fill in the rest of Table 2.

Based on the data, did you support or reject your hypothesis?
Explain your reasoning for your conclusion: why did you choose support or reject?.

Identify the optimal condition for the enzyme activity.
Be prepared to present your results to the class.
Lab 4- Enzyme Activity: To Hand In

Name: ______

This and the following page.

A graph of the data attached to these two pages.

Answers to Questions

Pre-Lab

1. Identify three factors you expect to affect the activity of the enzyme, catechcolase. For each of the factors you identify, describe why you expect the enzyme to be affected. Note that these are all possible hypotheses you might test in your experiment!

2. Select one of the factors you have identified above and, in the space below, write out the protocol for an experiment you might conduct to test your hypothesis. Use the example provided in the lab handout as a template for your protocol. Be precise as these are the directions you will be following in lab!

Data Analysis

Table 1. Effect of ______on Enzyme Activity after 6 minutes

Absorbance (420 nm)
Condition 1: ______/ Condition 2:
______/ Condition 3:
______
Control (blank)
Treatment

Table 2

Hypothesis:
Prediction:
Interpretation (circle one): support reject
Reasoning for choosing "support" or "reject" (if supported, why; if rejected, why):

What is the optimal condition for this enzyme reaction? Why did you choose it as the optimal condition?

additional questions-

Answer these questions on a separate page.

1. What would pH do to enzyme activity, and why?

2. a. If an enzyme were isolated from an invertebrate organism, such as a clam, that lived in seawater that averages14 °C, what would you predict would be the optimal temperature for that enzyme, and why?

b. If an enzyme were isolated from a mammal, such as a seal, that lived in seawater that averages 14 °C, what temperature would be optimal for the enzyme? Why?

3. Define enzyme denaturation in terms of protein structure.

4. What would a graph of the relationship between substrate concentration and rate of reaction look like, and why? What is a substrate?

5. How could you calculate the rate of reaction for your enzyme?

6. How would you expect increasing enzyme concentration to affect the rate of an enzyme-catalyzed reaction in the presence of unlimited substrate?