Unit 2: Labs 2-3 Enzyme Lab – Make Up Module

Enzyme Lab Handout (covers 2 weeks of Online Make Up labs) –  Includes areas for filling out your tables and answering questions.

Make sure you have Excel or access to Google Sheets.  As an NCSU student, you can download Microsoft Office 365 from NC State’s OIT software site for free!

Read along in your Enzyme Handout as you work with the information in this webpage.  Fill in and answer questions accordingly.  Your Enzyme Handout will be collected and graded later.

Enzyme Proteries
Effect of Inhibitors

How Cells Work – Enzymes

Chemical Reactions and Activation Energy

Graph of Activation Energy

Catalase

Catalase structure
Catalase

Animation – Enzyme Function and the difference between competitive and non-competitive inhibitors.

Activity 1: Qualitative Study of Catalase

2H2O2 + CATALASE —-> 2H2O + O2 + CATALASE

Catalase is prepared by blending a potato in cold water, then filtered by straining the mixture through 4 layers of cheesecloth.  The catalase solution is then kept cold on ice.  Why would filtering and maintaining a cold temperature be important for the catalase solution?

Procedure A Testing the Activity of Catalase

Follow steps 1-3 and complete Table 1 using the results in the pictures below.  The picture on the left shows the appearance of catalase and boiled catalase.  What would account for the difference in appearance of the boiled catalase?  Where did the “chunks” come from?  The picture on the right shows the reaction in tubes 1, 2, 3, and 4 after 5 minutes of the catalase reaction.

Procedure B Testing the Activity of Catalase Under Six Different Environmental Conditions

You will be assigned to work with ONE of the six environmental conditions (Groups) below.  Propose a plausible hypothesis for your assigned experiment.  The same basic reaction as tube #3 in Procedure A is followed, but treated with an assigned environmental condition.  Answer all questions and fill in the Table for your assigned treatment in your lab manual.   

Your assigned Group for Procedure B will be the same Group assignments for Procedure D (further below). 

Group I: Effect of Substrate Concentration on Catalase
Group II: Effect of Hydroxylamine and Subs. Concentration
Group III: Effect of Salt Concentration on Catalase
Group IV: Effect of the Metal Copper Sulfate
Group V: Effect of pH*
Group VI: Effect of Temperature
pH Test strip reading

Activity 2: Quantitative Study of Catalase

2H2O2 + CATALASE + GUAIACOL (COLORLESS) —-> 2H2O + CATALASE + TETRAGUAIACOL (ORANGE-BROWN)

We can study the same reaction as above in a more detailed manner by fine tuning the experiment.  Instead of learning about the relative activity of catalase, observing more or less reaction, we can introduce a sensitive color indicator to detect the presence of one of the reaction products.  The color indicator guaiacol will change from clear to orange-brown as it binds to the O2 produced in this reaction forming tetraguaiacol.  This color change can be measured using a spectrophotometer.  These data can then be graphed and rates determined for the baseline reaction of catalase, but also for the various treatments.

Procedure C Using a Spectrophotometer to Monitor Baseline Catalase Activity

Carefully read the Procedure C background.  This will help you fill in and answer the questions and Table 9 in within the Procedure C directions in your lab handout.  We will use a Genesys spectrophotometers and square disposable cuvettes.  If you have not used a spectrophotometer before, view the videos below.  It is very important to use an appropriate blank for each treatment that is run.  The blank will contain all the same components as the treatment tube, minus the enzyme.  This way we will know that any change in color is resulting from the activity of the enzyme itself.

How spectrophotometers work – transmittance and absorbance

How to use a Genesys 10 Spectrophotometer

Sample Data for Procedure C

Use this data to create a graph monitoring the baseline catalase reaction.  Be sure to label the graph correctly with all appropriate graphing components outlined by your TA.

Determining  Baseline data –  Be sure to label the graph correctly with all appropriate graphing components outlined by your TA and indicated in the Resources –> Appendix D: Graphing Using Excel.

  • Add a trendline and the equation of the trendline to your graph.
Spectrophotometer and cuvettes
Procedure C, Blank, initial and final (t=5 minutes)
Spectrophotometer with blank and t=5 minute cuvettes

Week 2 of Enzymes – Wait and do this part Enzyme’s Week 2 (May 30)

Procedure D Using a Spectrophotometer to Monitor the Activity of Catalase Under Different Environmental Factors

Since you have already learned about the effect of ONE environmental condition on catalase, select the data set for the SAME Group below that you already studied in Procedure B.  In each of these data sets, the baseline reaction is run, but then students designed 2-3 additional experiments to further test their hypothesis of effects of their assigned treatment on catalase activity.  While the same basic procedure was followed for these additional experiments, the blank may have changed.  Why might the same blank not always be the most appropriate as you change the treatment?  What does the blank tube constitute?  Refer to Hints at the back of the lab handout.

The provided data includes new baseline data (Procedure C) and experimental treatment data (Procedure D) collected by different student groups.

 

Group I: Substrate Concentration data Group II: Hydroxylamine and Subs. Concentration data
Group III: Salt Concentration data Group IV: Copper Sulfate data
Group V: pH data Group VI: Temperature data

 

Graphing Resources:

Graphing with Excel – Appendix D listed on the Resources page (Appendix Resources for BIO 181 and 183).  This lists the 5 key criteria for a proper graph (p. 204) and also shows how to add a trendline and equation for the best fit line (p. 203).  These are both important skills needed for the Enzyme lab assignment and later lab units and Unit assignments in BIO 183 labs.

Understanding and Representing Data – Appendix E listed on the Resources page (Appendix Resources for BIO 181 and 183).

Take Home Assignment: Enzyme Lab

This assignment will help you review the key components of writing an Introduction and Results section, an finding pertinent references for your research. It is worth 30 points and due at the start of lab Mar. 6-8.

Click on this box to download the assignment details.

Help for Writing the Different Sections of Lab Reports

*Tips for Writing a Review Paper*(opens in new window)  –  scroll down to see Choosing a Topic, What to Consider, and Structure of Your Review Paper. 

Finding and Citing References in your Lab Report