Name ______________________________________
Enzymes Lab
Before you begin, save this Lab Report Template on your computer as LastNameAPBIOEnzymes
Go to Biology Labs Online site (http://www.biologylabsonline.com) and log onto the Enzymes
Lab.
Read all the instructions below BEFORE you start!
Read the Background Information on enzymes so that you understand the terms used in this
online lab as well as how enzymes function. Then you will be ready to experiment with the effects
of temperature, pH, and substrate concentration on enzyme function. You will be completing
Assignments 1, 2, and 3 for this lab.
To start the Enzyme Lab, scroll down on the left to find the START LAB link. Click and wait for the Enzyme
Lab to open. You will see a lab bench. You can click on the various items to see their names and functions.
You will also see links to the experiment area, a data plot area, a notebook, a calculator, a glossary, a help
function, as well as links to the introduction and the assignments. Please spend time getting comfortable
with the lab interface before you start!
Experiment 1: Temperature Optimum for Invertase Activity
a.
b.
c.
d.
e.
f.
g.
h.
i.
j.
k.
Set the water bath at 30oC, the substrate concentration at 90 mM sucrose, leave the pH at 7.0, and
do not add any inhibitors.
Click Add Enzyme & Go: A plot of product concentration vs time will appear. The enzyme kinetic
data is plotted as data points in solid black circles.
Move the red line to get a line-of-best-fit. Try to align the red line so that you have an equal number
of data points above and below the line. Use your best judgment!
Record the value of the slope in the data chart below.
The record data button only functions when you have properly determined the slope.
Click Clear Experiment, make sure the pH is 7.0 and the sucrose concentration is 90 mM. (These
should not change, but it is a good habit to check!)
Increase temperature to 35oC, Add Enzyme and Go, determine the slope of the line (rate), and
record.
Repeat at 5o increments until you reach 85o C.
Click on Plot Data and you will see a data chart.
The Assignments area of the Enzyme Lab provides details on how to use the data plotting function
and what the abbreviations mean. Refer to this as necessary.
You will not be able to cut and paste this data chart accurately into this lab report, so make sure
that you have recorded the rate data (slope) in the data chart below. You will be able to copy, cut,
and paste the graphs!
Title your graph: Experiment 1—Temperature Optimum
The default plot is Vo vs [S] so you need to change this to Vo vs Temp.
Shift-Click to highlight the rows you want to include in the plot. Make sure you have highlighted all
the temperature rows!
Click Plot Selected Data and then move the arrow/ vertical line to find the temperature maximum.
Click Export Graph and then copy and paste it below your data chart!
AP Biology
Johns Hopkins Center for Talented Youth
Fall 2006/2007
Temperature
(oC)
30
35
40
45
50
55
60
65
70
75
80
85
Rate
(mM/min)
Paste your Vo vs Temperature Plot here:
Experiment 1 Analysis:
1.
What is the optimal temperature for invertase activity? Is this what you expected? Would invertase
isolated from any two organisms (for example, yeast invertase vs. invertase from the small intestine
of humans) show the same temperature optimum? Why or why not? Explain your answers.
2.
Explain why temperatures lower or higher than the optimum cause decreases in invertase activity.
What is happening to the enzyme to produce these decreases in activity?
3.
Carefully examine the curve for VO vs. Temperature. Is the slope of the line on both sides of the
curve the same or different? If the slope of the line to the left of maximum velocity is different from
the slope of the line to the right of maximum velocity, explain why this is. What is responsible for
these differences in enzyme kinetics?
4. If you were to carry out these temperature experiments at a higher or lower [S], what effect would
[S] have on the temperature optimum for invertase? How would you test this?
Experiment 1 Conclusion: The temperature optimum for invertase is _________________.
AP Biology
Johns Hopkins Center for Talented Youth
Fall 2006/2007
Experiment 2: pH Optimum for Invertase
a.
b.
c.
d.
e.
f.
g.
h.
Set the water bath at the temperature optimum you determined in Experiment 1, the substrate
concentration at 90 mM sucrose, leave the pH at 3.0, and do not add any inhibitors.
Click Add Enzyme & Go: Find the line-of-best-fit as you did in Experiment 1.
Record the value of the slope in the data chart below.
Click Clear Experiment and repeat using the pH values in the data chart below.
Click on Plot Data to see a data chart.
Title your graph: Experiment 2—pH Optimum
The default plot is Vo vs [S] so you need to change this to Vo vs pH
Shift-Click to highlight all the rows for the pH experiment
If you have run this experiment right after the temperature experiment, all the data is listed. You
need to scroll down to where you set the optimum temperature and adjusted the pH values. If you
get an error message, check to see that you have highlighted only the rows where you changed
pH.
Change the plot to # 2, click Plot Selected Data, and then move the arrow/ vertical line to find the
pH maximum.
Click Export Graph and then copy and paste it below your data chart!
Rate
(mM/min)
pH
3.0
3.4
4.0
4.4
5.0
5.4
6.0
6.4
7.0
7.4
8.0
8.4
9.0
9.4
10.0
Paste your Vo vs pH Plot here:
Experiment 2 Analysis
1.
What is the optimal pH for invertase activity? Why and how do pH changes affect invertase
activity?
2.
If the slope of the line to the left of maximum velocity is different from the slope of the line to the
right of maximum velocity, explain why this is. What is responsible for these differences in enzyme
kinetics?
3.
If you were to carry out these pH experiments at a higher or lower temperature, what effect would
this have on the pH optimum for invertase? How would you test this?
Experiment 2 Conclusion: The pH optimum for invertase is ________________.
AP Biology
Johns Hopkins Center for Talented Youth
Fall 2006/2007
Experiment 3: The Effect of Substrate Concentration on Invertase Activity
By now you are familiar with the operation of the Enzyme Lab, so the instructions will be
simpler:
a.
b.
c.
d.
e.
f.
g.
h.
i.
Set the water bath at the temperature optimum you determined in Experiment 1 and the pH at the
optimum you determined in Experiment 2.
Start with an initial substrate concentration [S] of 0.00 mM
Click Add Enzyme & Go: Find the line-of-best-fit as you did in Experiments 1 and 2.
Record the value of the slope in the data chart below.
Click Clear Experiment and repeat using the[S] values in the data chart below.
Click on Plot Data to see a data chart.
Title your graph: Experiment 3—Substrate Concentration.
And set plot type to Vo vs [S]
Shift-Click to highlight all the rows for the [S] experiment
If you have run this experiment right after the temperature and pH experiments, all the data is
listed. You need to scroll down to where you set the optimum temperature and pH and adjusted [S]
Change the plot to # 3, click Plot Selected Data. You will have to move the vertical and horizontal
arrows/lines line to find Vmax and Km
Click Export Graph and then copy and paste it below your data chart!
Note: You do NOT have to set yp Lineweaver-Burk or Eadie-Hofstee Plots, as suggested in the
online instructions.
Sucrose Concentration [S]
mM
2.5
5.0
20.0
40.0
60.0
80.0
100.0
120.0
140.0
160.0
180.0
200.0
220.0
240.0
260.0
280.0
320.0
340.0
360.0
380.0
400.0
Rate
(mM/min)
Paste your Vo vs pH Plot here:
AP Biology
Johns Hopkins Center for Talented Youth
Fall 2006/2007
Experiment 3 Analysis
1. What was the relationship between [S] and invertase activity? Was this relationship what you
expected? Why or why not? Explain your answers.
2. What do the values for Vmax and Km indicate?
3.
What do you think would happen if you carried out an experiment with concentrations of
sucrose greater than 400 mM? How would you test this?
Experiment 3 Conclusion:
The maximum velocity for Invertase activity at ____ o and pH ___ is _______
Assignments 4 and 5 are NOT required, but you may perform them if you want to!
AP Biology
Johns Hopkins Center for Talented Youth
Fall 2006/2007