Students Design IDH Experiment

LAB #3

Environmental Parameters of Enzyme Activity

Focused Reading: "Molecular Structure..." pp 132-133. Stop at bottom of page 133.
"Enzyme activity..." pp134-136. Srop @ "Allosteric enzymes..."
"Enzymes are sensitive..." pp 138-139. Stop @ "Summary..."
Bring a calculator to lab.

Goals for this Lab:

In this week's lab you will determine the effects of environmental perturbations of our standard assay conditions. You will use what you learned last week and apply that information to this week's lab.

I. Introduction

Last week you:
1. Learned how to perform isocitrate dehydrogenase (IDH) assays.
2. Examined the relationship between activity and amount of enzyme in an assay.
3. Examined the relationship between activity and substrate concentration.
4. Learned how to present experimental data in graphic form.
5. Chose one of the following experiments (Options A F) to complete.
6. Designed an experimental protocol for that experiment.

II. Methods and Materials

You will use the same general methods that you used in Lab #2. All equipment, solutions and supplies required to carry out your experiments have been prepared and ready for your use. You may wish to review your protocol again and assign specific tasks before you start your experiments.

Option A: The Effects of pH on IDH Activity

Hypothesis: pH of the assay buffer will have no effect on IDH activity.

To test this hypothesis, you will need to follow a protocol that holds all conditions constant except the pH of the assay buffer.

Table 4: The effects of varying pH of the assay buffer.

Wells	pH	Buffer	NADP+	Isocitrate	IDH
A 1-3
A 4-6
B 1-3
B 4-6
C 1-3
C 4	Blank

Table: Data from varying pH of the assay buffer.

Time (min)	pH	pH	pH	pH	pH
0
0.5
1
1.5
2
2.5
3

Considerations - Experiment on pH

Does IDH activity vary when the pH of the assay mixture varies, or are levels of activity the same regardless of pH? Do you need to test activity other pH values? Explain how the pH of the assay mixture might affect activity of an enzyme.

Option B: Does IDH Have a Metal Ion Requirement?

Hypothesis: IDH activity does not require a divalent metal in the assay solution.

To test this hypothesis, you will need to follow a protocol that holds all conditions constant except the presence or absence of divalent metal ions in the assay solution.

Table 5: Does IDH Have a Metal Ion Requirement?

Wells	Buffer **	Metal, ul	NADP+	Isocitrate	IDH
D 1-3		None
D 4-6		EDTA, 10
E 1-3		Mg+2, 10
E 4-6		Mn+2, 10
F 1-3		Cu+2, 10
F 4-6		Zn+2, 10
G 1		Blank

**You will want to use a buffer that does not contain any added Mg+2

Table 5a: Data from cofactor experiment.

Time	None	EDTA	Mg+2	Mn+2	Cu+2	Zn+2
0
0.5
1
1.5
2
2.5
3

Considerations - Experiment on metal ions

Does IDH require a divalent metal ion for activity? Does additional Mg2+ added to the standard assay buffer increase activity? What does this observation mean? Does the addition of Mn2+ added to the standard assay buffer increase activity? What does this observation mean? How do you explain the effects of Cu2+ and Zn2+ on IDH activity?

Option C: The Effects of Temperature on IDH Stability

Hypothesis: Exposure to 37° C will have no effect on IDH stability.

To test this hypothesis, you will need to follow a protocol that holds all conditions constant except temperature. You will incubate samples of IDH at 37°C for 0, 2, 4, 6, 8, 10 min prior to assaying activity. Old all samples on ice until incubations are complete and assay at the same time.

Table 6: Does exposure to 37°C affect IDH stability?

Wells	Min, 37°C	Buffer	NADP+	Isocitrate	IDH
D 1-3
D 4-6
E 1-3
E 4-6
F 1-3
F 4-6
G 1	Blank

Table 6a: Data from termperature experiment.

Time, min	Min	Min	Min	Min	Min	Min
0
0.5
1
1.5
2
2.5
3

Considerations - Experiment on temperature pre-treatment

Is IDH stable at 37°C? How can your results be explained? How would you design this experiment to test your hypothesis further?

Option D: Does enzyme activity vary with concentration of NADP+?

Problem: What is the relationship between the rate of a reaction and the amount of coenzyme in the assay solution when the amount of enzyme is held constant? Before you start this experiment, develop an hypothesis and sketch a graph predicting the relationship of activity vs substrate concentration.

Table 7: The effects of varying the concentration of NADP+.

Wells	Buffer	NADP+	Isocitrate	IDH
D 1-3
D 4-6
E 1-3
E 4-6
F 1-3
F 4		0

*The concentration of these NADP+ solutions will be provided by the Instructor. The second number refers to the volume [µl] to be used. NOTE: In these assays we will initiate reactions with isocitrate. Do not add isocitrate until your plate is in the plate reader.

Table 7a: Data from varying the concentration of NADP+.

Time, min	mM	mM	mM	mM	mM
0
0.5
1
1.5
2
2.5
3

Considerations - Experiment varying NADP+ concentration

Organize the data from your experiment. Determine the mean activity for each concentration of NADP+. Construct a graph that compares activity as a function of NADP+ concentration. Do your data support your hypothesis? Is the relationship between activity and concentration of NADP+ linear? Explain this relationship, referring to your figures as necessary.

Option E: The Effects of NaCl on IDH Activity

Hypothesis: Sodium chloride will have no effect on IDH activity.

To test this hypothesis, you will need to follow a protocol that holds all conditions constant except concentration of NaCl in the assay solution.

Table 8: The effects of NaCl on IDH activity.

Wells	Buffer	5M NaCl	[NaCl} M	NADP+	Isocitrate	IDH
D 1-3
D 4-6
E 1-3
E 4-6
F 1-3
F 4-6
G 1			Blank

Table 8a: Data from the effects of NaCl on IDH activity.

Time (min)	Conc. 1	Conc. 2	Conc. 3	Conc. 4	Conc. 5	Conc. 6
0
0.5
1
1.5
2
2.5
3

Considerations - Experiment on salt concentrations

Did you observe differences in activity between the treatments? What was the relationship between the concentration of NaCl and activity? Explain how salt might affect enzyme activity.

Option F: Does IDH activity vary among different organisms or tissues?

This is an "open ended" experiment; you may design a single, additional experiment, comparable to the ones listed above, or expand these topics into a research project of wider magnitude.

1. You may chose to survey IDH activity:
i. In a wide variety of related species [think salad bar].
ii. In different tissues of a single species [think chicken giblets].
2. Homogenize your samples in cold Assay Buffer, using a kitchen blender.
3. Filter the homogenate through two layers of cheesecloth into a small beaker that is on ice.
4. Transfer 1 ml samples to 1.5 ml microfuge tubes, spin for 5 minutes.
5. Transfer the supernatant to clean 1.5 ml microfuge tubes, on ice.
6. Use standard conditions to assay IDH activity.

Before You Leave Lab

1. Be certain that you have collected all of the data you need to make your
experiment complete.
2. Be certain that each member of the group fully understands what was done
and has a copy of all of your data.
3. Schedule a meeting of your group [why not now?] to analyze your results
and prepare slides for your group's oral presentation.

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