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Learning Objectives:
After completing this lab you should be able to:
Test hypotheses regarding the effect of temperature and pH on enzyme activity
Explain substrate specificity
Generate graphs of enzymatic rates as a way of presenting data
Calculate averages and standard deviations
Interpret data to draw conclusions
The following items will be needed for this lab:
Microsoft Excel (or other spreadsheet software of your choice)
Left to their own devices, chemical reactions in biological systems would occur very slowly if at all. This prospect presents a clear disadvantage to living organisms trying to carry out the processes necessary for survival. It would thus be advantageous to have a way of making these reactions occur in a more timely fashion. Enzymes are primarily protein molecules that act as catalysts for biochemical reactions. A catalyst is a substance that speeds or otherwise facilitates a chemical reaction by lowering the activation energy, the amount of energy that must be put into the system in order for the reactants to form products. In the process of the reaction the enzyme is not itself altered and may thus participate in further reactions leading to the same products. Catalysis can be described as follows: substrate (the molecule undergoing a reaction) binds to the enzyme (the catalyst) forming an intermediate complex; in this intermediate complex the substrate is altered and then released from the enzyme as product; the enzyme, unchanged in the reaction, can then participate in another reaction with another substrate molecule. This can also be summarized by the following equation:
Enzyme + Substrate D Enzyme-Substrate Complex D Products + Enzyme.
As part of today’s lab you will investigate the action of the enzyme lactase on lactose (the sugar found in milk). Many people are lactose intolerant- that is, they don’t produce sufficient quantities of the enzyme lactase needed to break down lactose. Fortunately, lactase can be taken as a dietary supplement in pill form to help with the digestion of lactose. Lactase, like most other enzymes, has a very specific three- dimensional conformation and reacts only with specific substrates (e.g., lactose). Lactose is a disaccharide composed of galactose and glucose linked by a covalent bond. The enzyme lactase separates the galactose and glucose by inserting a water molecule between the two sugars. This type of reaction is very common and is an example of hydrolysis. The breakdown of lactose by lactase can be summarized as follows:
lactose + lactase + H2O D lactose-lactase complex D galactose + glucose + lactase
Enzymatic activity is affected by a number of parameters (e.g., pH and temperature) and part of today’s exercise will involve examining the effect that some of these parameters have on the relative rate at which the enzyme lactase catalyzes the breakdown of lactose into galactose and glucose. We will assay for the production of glucose as a measurement of lactase activity under various conditions. Portable glucose meters were used to measure glucose levels and we will analyze those data as part of this lab.
Part 1- Enzyme Specificity: Lactase
The enzyme lactase breaks down lactose into galactose and glucose. Sucrose (common table sugar) is a disaccharide composed of fructose and glucose linked by a covalent bond.
? Make a prediction: Imagine mixing lactase and milk in a tube and letting it sit for 30 minutes at room temperature. Do you predict glucose levels in the tube to be high, moderate, or low after 30 minutes? Explain your reasoning.
? Make a prediction: Imagine mixing water and milk in a tube and letting it sit for 30 minutes at room temperature. Do you predict glucose levels in the tube to be high, moderate, or low after 30 minutes? Explain your reasoning.
? Make a prediction: Lactose and sucrose are both disaccharides with similar (but not identical) structures. Imagine mixing lactase and sucrose in a tube and letting it sit for 30 minutes at room temperature. Do you predict glucose levels in the tube to be high, moderate, or low after 30 minutes? Explain your reasoning.
Let’s test your predictions using data from experiments with lactase dietary supplement and glucose test strips. Experiments were conducted as described below.
Lactase enzyme solution was prepared by dissolving one lactase tablet in 200 ml of tap water.
Three different treatments were prepared as described below.Tube 1- add 1ml milk (and get ready to add 0.5 ml lactase – but don’t add it yet!).
Tube 2- add 1ml milk and 0.5 ml water.
Tube 3- add 1ml of 5% sucrose (and get ready to add 0.5 ml lactase – but don’t add it yet!).
When ready to begin timing, go ahead and add 0.5 ml lactase to tubes 1 and 3. Gently swirl the tubes to mix the contents of each.
Use the glucose meter to immediately test for the presence of glucose in all three tubes and record your results for time “zero” in the table below.
Test glucose levels again after 30 minutes.
Results from ten different groups performing the experiment above are presented below. Table 1. Results of lactase specificity.
Solutions
Glucose Levels (mg/dL)
0 min
30 min
Tube 1: milk + lactase
0
423
0
543
1
244
2
540
0
465
5
552
3
550
1
478
0
504
0
489
Tube 2: milk + water
2
3
3
0
0
0
0
2
0
1
1
0
2
4
2
2
0
0
4
0
Tube 3: sucrose + lactase
2
2
0
3
0
1
4
0
1
0
0
3
3
1
2
0
0
1
0
0
Group Data:
For this activity, you will be asked to produce bar graphs based on the group data above showing the mean (average) glucose levels at 30 min for each treatment along with the standard deviation (which is a formal way of showing how spread out the numbers are). For example, a bar graph for one of the treatments might look something like:
Click here for a tutorial on how to make a bar graph in Excel.
The standard deviation is often important to know and has a bearing on many statistical tests. For example, what is the average of the following three numbers: 5, 5, 5? You should recognize that the average is 5. The average of the following three numbers is also 5: 1, 1, 13. However, there is more variation in the second set of numbers than in the first set!
If you look into the formula for calculating standard deviation, it may seem overwhelming at first sight. However, it is actually very easy!
Calculate the mean (average) of the numbers in the results. For example, you might calculate the average glucose level for part 1, spot 1 (10 min) based on the class results.
Then, for each individual number (individual glucose reading), subtract the mean and square the result.
Then, calculate the mean of those squared differences.
Then, take the square root of that mean. That’s it—you have now calculated the standard deviation!
Example:
Let’s say the class results for spot 1 glucose levels at 10 minutes are as follows: 5, 2, 7, 7, 4, 8.
Calculate the mean- the mean is 5.5.
Subtract the mean from each individual number and square the result- you get the following: 0.25, 12.25, 2.25, 2.25, 2.25, 6.25.
The mean of those squared differences is: 4.25
The square root of 4.25 is: 2.061552813 (and that is the standard deviation for this set of numbers!). If you take a look at the sample graph above, you’ll see that the mean is 5.5 and the standard deviation is roughly 2.
In reality, people rarely calculate means and standard deviations by hand anymore. Instead, we rely on computers to do it for us! Although this exercise may seem tedious, it is very common to see standard deviation error bars in graphs of results—you’ll now have a better appreciation for what those mean!
Use the space below to calculate the mean and standard deviations for each of the three treatments in Part 1-Lactase Specificity (based on the group data for the 30 min readings).
On the following page, make a single bar chart showing the mean and standard deviations for all three treatments. You are encouraged to use Microsoft Excel (or other spreadsheet software of your choice) to create your graphs.
CALCULATIONS:
Graph the results (averages + Standard Deviation) for each of the three solutions using proper formatting. You may do this in Excel (or any other program you are used to), the drawing function on your computer, or simply hand draw your graph and insert a picture.
Graph paper below can be used if you prefer to print it out and hand draw your graph.
Explain these results. Do they match your predictions? Why or why not? What can you conclude about the specificity of lactase? What was the purpose of tube 2?
Part 2- Effect of pH on Enzyme Activity
Lactase, like most other enzymes, is a protein and its catalytic properties can be influenced by factors such as pH and temperature. Many enzymes become denatured when placed in extreme conditions (e.g., too acidic or too hot). When enzymes become denatured, their specific three-dimensional conformation is disrupted and they lose their ability to catalyze reactions. This portion of today’s exercise will examine the effect of pH on lactase activity.
Make a prediction: The optimal pH for lactase activity has been reported to be around 6.0. What do you predict will happen to lactase activity when placed in solutions with pH values below and above 6.0? Explain your reasoning.
Let’s test your predictions using data from experiments with lactase dietary supplement, glucose test strips,
buffers at various pH values. Experiments were conducted as described below.
Prepare your lactase enzyme solutions by dissolving one lactase tablet in 200 ml of buffer at each of the following pH values: 2, 6, 10. Stir until dissolved.
Obtain a spot plate, milk, and glucose test strips.
You will prepare three different treatments, each with a different pH. To save time, you are encouraged to get all reactions going as soon as possible. However, you will have to test for glucose at a specific time interval so be prepared to start timing once you prepare the three treatments.
Add 1 ml milk to each of 3 spots.
Then, add 0.5 ml pH 2 lactase to spot 1; 0.5 ml pH 6 lactase to spot 2, and 0.5 ml pH 10 lactase to spot 3. Be sure you remember the contents of each spot.
Immediately test for the presence of glucose in all three spots and record your results for time “zero” in the table below.
Test glucose levels again after 10 minutes.
Results from ten different groups performing the experiment above are presented on the next page.
Table 2. Effect of pH on Lactase Activity
Solutions
Glucose Levels (mg/dL)
0 min
30 min
pH 2
0
323
0
443
1
244
2
440
4
465
5
452
3
450
1
378
0
404
3
389
pH 6
2
412
3
509
3
245
0
523
0
487
1
534
2
579
2
418
4
565
4
489
pH 10
2
123
0
243
2
144
4
240
1
265
5
252
3
250
2
178
0
204
0
189
Graph the results (averages + Standard Deviation) for each of the three solutions using proper formatting. You will likely create a bar graph for your results. You may do this in Excel (or any other program you are used to), the drawing function on your computer, or simply hand draw your graph and insert a picture.
Graph paper below can be used if you prefer to print it out and hand draw your graph.
1. Explain these results. Do they match your predictions? Why or why not? What can you conclude about the effect of pH on lactase activity?
2. Does it make sense for the enzyme to work at low pH values? Consider this in the context that the Lactaid brand of lactase must be able to work in peoples’ stomachs.
Part 3- Effect of Temperature on Enzyme Activity
As mentioned before, temperature can also have an effect on the three-dimensional conformation of enzyme molecules and, as a result, can influence their activity. This portion of today’s lab will examine the effect of temperature on lactase activity.
Make a prediction: The optimal temperature for lactase activity has been reported to be around 37o C. What do you predict will happen to lactase activity when placed in temperatures below and above 37oC? Explain your reasoning.
Let’s test your prediction using data from experiments with lactase dietary supplements at various temperatures and glucose test strips. Experiments were conducted as described below.
Use the lactase solution at different temperatures for this activity.
Obtain a spot plate, milk, and glucose test strips.
You will prepare three different treatments, each at a different temperature. You will also include results from Part 1, spot 1 above (room temperature) for comparison. To save time, you are encouraged to get all reactions going as soon as possible. However, you will have to test for glucose at specific time intervals so be prepared to start timing once you prepare the three treatments.
Spot 1- add 1ml milk and 0.5 ml lactase that has been kept in the refrigerator (4o C). Record the time, test for glucose as time zero, and place back in the refrigerator.
Spot 2- add 1 ml milk and 0.5 ml lactase that has been kept in the incubator (37o C). Record the time, test for glucose as time zero, and place back in the incubator.
Spot 3- add 1 ml milk and 0.5 ml lactase that has been boiled (100o C). Record the time, test for glucose as time zero. Note: this treatment can simply be left to sit at room temperature. If the enzyme was denatured by boiling, then the effect of boiling will not be reversed.
Test each treatment for glucose levels again at the end of 10 minutes and record below.
Results from ten different groups performing the experiment above are presented on the next page.
Table 3. Effect of Temperature on Lactase Activity
Solutions
Glucose Levels (mg/dL)
0 min
30 min
4o C (refrigerator)
0
123
4
143
1
144
2
140
4
165
5
152
3
150
1
178
2
104
3
189
22o C
(room temp)- From Part1, Tube 1
0
423
0
543
1
244
2
540
0
465
5
552
3
550
1
478
0
504
0
489
37o C
(incubator)
2
623
5
643
2
644
4
640
1
665
5
652
3
650
2
678
5
704
0
689
100o C
(boiled)
3
5
2
4
2
2
4
4
3
4
1
1
0
2
5
5
2
2
3
4
Graph the results (averages + Standard Deviation) for each of the four solutions using proper formatting. You will likely create a bar graph for your results. You may do this in Excel (or any other program you are used to), the drawing function on your computer, or simply hand draw your graph and insert a picture.
Graph paper below can be used if you prefer to print it out and hand draw your graph.
1. Explain these results. Do they match your predictions? Why or why not? What can you conclude about the effect of temperature on lactase activity? 2. Consider the results with the boiled lactase. Explain why they are so dramatically different from the other results. Concluding Questions:
1.Explain how a molecule that mimics the substrate for a certain enzyme might affect the rate at which the normal reaction will occur. 2. Beano is a commercially available product that contains alpha galactosidase and aids in the digestion of specific glycolipids and glycoproteins found in many beans. Would you expect Beano to be effective at breaking down lactose? Explain (be sure to include a discussion of the role of the active site during enzymatic reactions).
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