This is a three week experiment for 290 points. 3 prelabs 1 presentation one formal report This is the most demanding experiment of the semester - YOU MUST COME PREPARED AND ORGANIZED OR YOU WILL NOT FINISH.  NO EXTRA TIME WILL BE ALLOTTED!    DOUBLE ALL ASSAY AMOUNTS OF MATERIALS IN THIS LABORATORY.

For week 1 prepare the procedure for preparation of the wheat germ extract and the effects of time, concentration, temperature and pH as well as the appropriate background information. You should also complete prelab questions 1-4 for week 1.

*The procedure given for this experiment is not detailed. THE PROCEDURE YOU WILL TURN IN MUST BE!
*You must include any calculations, prepare data tables and decide the step by step plan for the laboratory.
In other words you must fill in the detail. There will be 3 prelab grades for this experiment (90 points!)

For the second week you should complete the determination of the Michaelis Constant (K_m) for p-Nitrophenyl Phosphate Hydrolysis by Acid Phosphatase and catalytic inhibition of the enzyme, and  the prelab questions 5-8.

For the final portion of this experiment, week 3, you will be required to develop your own procedure! (These questions should help guide you towards factors you need to consider in your experiment development. For week 3 you should prepare the inhibitor assays and question 9.  The inhibitors and there concentrations will be given during week two.

IMPORTANT NOTE: You will find that you will be most successful in lab if you prepare a procedure/data table for each assay. I will grade prelabs for success.  Each column heading should be a component of the mixture and a column for totals and for absorption data.  The rows should be your test tube numbers (or vice versa).  Each cell should list the appropriate volume of each component .  The total volumes should be consistent.  This will allow you to keep track of the significant number of test tubes and assays you will be juggling.

Reading - For week 1 and 2

Laboratory text (Robyt and White): Review 2.8 and 2.9, 3.2 - Study chapter 9 This is a fabulous supplement to your Class work Read it

Link to a kinetics site

Prelab Questions (completed in notebook prior to class):

Below are questions that you would need to answer prior to carrying out the phosphatase inhibitor experiment that you have designed for the third week of lab.
1. a) What will be your source of the acid phosphatase?
b) How is the enzyme extracted andsolubilized?
2. a) Why is it necessary to dilute the enzyme crude extract?
b) Explain how you would prepare 1.0 ml of a 1/10 dilution of the crude extract. Be specific in your answer and be sure to show any calculations used.
3. What buffer will you use for the assays? (Be specific)
4. What will the reaction volume be (before adding KOH)?
5. What factors must be considered when determining the reaction time for the assays?
6. What will you use as a blank to zero the spectrometer? Be specific for each assay
7. What control(s) must be done? Why are these necessary? Be specific for each assay
8. What is(are) the variable(s) in your experimental design?
9. What concentration(s) of inhibitor are you planning to use? What factors did you consider in determining this? (discussion of this will occur in week 2)
10. List any references you utilized in designing this experiment.

** - Once you have answered these questions see the data provided at the end of the experimental procedure.

1. Kinetics and the theory of enzyme action
2. Initial velocity and its determination
3. Enzyme reactions as a function of pH, temperature and concentration
4. Enzyme activity
5. Michaelis Menton Kinetics
6. Enzyme Inhibition - competitive, noncompetitive, mixed and uncompetitive
7. Allosteric Enzymes
8. Enzyme specificity

1. Prepare a standard solution of wheat germ phosphatase
2. Prepare a standard assay
3. Examine the effects of the variables time, concentration, temperature and pH on enzyme activity
4. Explore the kinetics of the reaction and determine Michaelis Constant (K_m) for p-Nitrophenyl Phosphate Hydrolysis by Acid Phosphatase
5. Determination of the Catalytic Inhibition of the Enzyme

In this experiment, you will develop the optimal parameters for the measurement of a phosphate ester hydrolyzing enzyme activity. The particular phosphatase enzyme we will be studying is present in wheat germ. The enzyme is termed acid phosphatase because it has a relatively low pH optimum. The enzyme is capable of hydrolyzing a number of phosphate ester substrates, including an artificial substrate p-nitrophenyl phosphate. Upon hydrolysis of p-nitrophenyl phosphate, p-nitrophenol and inorganic phosphate are produced:

the product of this reaction, p-nitrophenol, absorbs visible light at alkaline pH values. Because the substrate has no absorption in the visible region of the spectrum, the enzymatic activity can be followed by following the rate of p-nitrophenol formation. This can be measured spectrophotometricly by absorbance at l_max. The Beer-Lamber law. A=Ebc,  can be used to calculate the concentration of product in each assay. The literature value for the extinction coefficient (aka molar absorptivity) of p-nitrophenol is 18.8 x 10⁶ cm²/mole at the 405 nm.¹ In this experiment the enyme assay for wheat germ acid phosphatase activity will be optimized with respect to time, amount of wheat germ extract present (a[protein]), temperature, pH and, [p-nitrophenylphosphate]. If time permits, the effect of inhibitors on the phosphatase will also be studied. For these initial characterizations of the optimum parameters for the assay of the enzyme, we will utilize a crude extract from wheat germ.

¹The units for this extinction coefficient come from ml/molcm which is equivalent to  cm³/mol cm or cm²/mol. The path length of the spec 20 is 1 cm.

Preparation of a Wheat Germ Crude Extract

On each day that you do an experiment to optimize the enzyme assay, you will prepare a fresh extract of wheat germ proteins by the following procedure:

1. All solutions utilized in the preparation of the crude extract should be ice cold, and the crude extract must be kept on ice or in the refrigerator at all times.

2. Weigh about 0.5 g of wheat germ and place it in a cold mortar and pestle. Add 2.5 ml of 20 mM sodium acetate, pH 5.7, containing 2 mM MgCl₂, and grind  until the wheat germ is well ground.

3. Using a Pasteur pipette, place the entire quantity of the extract (homogenate) into a 1.5 ml polypropylene microcentrifuge tube, let sit on ice for 10 min, then centrifuge for 2 min in the microcentrifuge, spinning at the high setting. (note: centrifuges must be balanced)

4. After the centrifuge has come to a complete stop, carefully remove the tubes from the centrifuge and remove the supernatant with a Pasteur pipette so as to not disturb the pellet. Place the supernatant into a clean microcentrifuge tube, estimate the volume recovered, and keep the sample on ice. You may discard the pellet. The supernatant is the crude concentrated extract that you will use for the characterizations listed below.

This is the basic assay you will use throughout this experiment; however, there will be changes depending on what is being measured, and appropriate controls will need be carried out.  Each assay will follow a format similar to this one.

1. Use 13 x 100 mm test tubes or other tube appropriate for the total volume required - DO NOT use spec 20 tubes for the reactions.
2. Add 0.1 ml assay buffer (assay buffer contains 1.0 M sodium acetate and 0.1 M MgCl₂, pH 5.7).
3. Add desired amount of enzyme containing sample (0 - 0.35 ml)
4. Add H₂O to bring volume to 0.45 ml and allow the tubes to react at room temperature.
5. At approximately 30 sec  to one minute time intervals between sample tubes, initiate the reaction by adding 50 µl of substrate (50 mM p-nitro-phenylphosphate), vortex, and incubate at room temperature for the desired time.
6. Stop the reaction at the desired time by the addition of 3.0 ml 0.5 M KOH.
7. Read the absorbance at the wavelength maximum for p-nitro-phenol.

1. Immediately before use, dilute a small portion of the extract 1/50 in ice cold H₂O. This is your working concentration for this assay.
2. Prepare 10 assay tubes, each containing 100 µl of the diluted extract. Remember to add water to bring the total volume of buffer, extract and water to 0.45 ml.
3. Set up reactions so that individual reactions run for about 0, 5, 10, 15, 20, 25, 30, 40, 50, 60 min. Initiate the reactions by the addition of substrate as described previously.
4. Stop each reaction at the desired time with 0.5 M KOH, and read all absorbances when all time points are completed. For the zero time point sample, add the 0.5 M KOH first, then add the enzyme.
5. Carry out a control which is incubated for 60 min, but which contains no enzyme.
(you will use the 0 control for early time points and the 60 minute control for late time points)

1. Dilute a portion of your crude extract 1/50 in cold water, immediately before use.
2. Prepare assay tubes which contain 0, 0.05, 0.1, 0.15, 0.2, 0.25, 0.3, and 0.35 ml in individual tubes.
3. Carry out the assay with each tube, terminate the reaction after 5 min.
(the 0 extract is used as the blank for each assay)

1. Prepare a series of assay tubes which have buffer, substrate and water added, three tubes for each temperature. Keep all tubes on ice until you are ready to use them.
2. You will control the temperature using water baths. Plan to use temperatures of approximately 2, 6, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, and 70C. Be sure to record the actual temperatures used.
3. You will begin all reactions by the addition of 50 µl of crude extract diluted 1/10 in cold water, and will terminate by 0.5 M KOH addition exactly 2 minutes later.
(your controls will be the third set of tubes, one at each temperature, quenched with KOH before addition of the enzyme)

You will be supplied a series of citrate-Tris buffers which span a pH range from about 3 to 8.

1. For each buffer you will prepare a reaction tube which contains 300 µl of the buffer and 100 µl of 0.1 M MgCl₂ in place of the standard assay buffer.
2. Using 50 µl of the 1/50 diluted crude extract carry out assays at each pH. Make sure that the final volume in each tube is 0.45 ml before adding the substrate. For any one of the buffers, do the sample in duplicate and terminate one of the two reactions (a control) immediately before adding the p-nitrophenyl phosphate by the addition of 0.5 M KOH.
3. Incubate each sample tube for 15 minutes before terminating the reaction.
(you will need a control for each pH- i.e., each different tube will have its own blank)

You will carry out a series of reactions at different concentrations of p-nitrophenyl phosphate to determine the optimal [substrate] to carry out the reaction, and also to determine the K_m for the reaction.

1. Prepare a series of tubes which contain buffer, water, and various amounts of the 50 mM p-nitrophenylphosphate solution, so that the final concentrations in the tubes (0.5 ml reaction volume) are: 0.25, 0.5, 0.6, 0.75, 1.0, 1.25, 2.50, 5.0, 10, 25 mM. Prepare three tubes with each concentration of p-nitrophenyl phosphate. Add H₂0 to each tube so that the final volume of each tube is 0.450 ml.
2. In two sets of tubes at each [substrate], begin each reaction by adding 50 µl of the crude extract diluted 1/50 with H₂O. Terminate the reactions exactly 5 minutes later with 0.5 M KOH.  (This assay will be done in duplicate in an effort to obtain better data)
3. In the third set of tubes at each concentration, add the 0.5 M KOH followed by the 50 µl of the 1/50 enzyme solution. This set of tubes serves as a control.
4. After all reactions are completed, measure the absorbance of each tube vs. the appropriate control.

Each group will be supplied with two inhibitors of the acid phosphatase. It is your task to determine:
1. The type of inhibition (competitive, uncompetitive, non-competitive)
2. The inhibition (K_i) for the inhibitor (refer to the supplemental handout on reversible enzyme inhibition).
You must prepare your own appropriate experimental design

Supplemental Data

DATA FOR TEMPERATURE OPTIMUM/ACTIVATION ENERGY, pH OPTIMUM, AND MICHAELIS CONSTANT DETERMINATION SECTIONS OF ACID PHOSPHATASE EXPERIMENT

I. Temperature Effects Data:

Crude Extract Dilution: 1/5

Length of Assay: 1 minute

Temp (C)	A399 Trial 1	A399 Trial 2	Average  A399	Control	Actual1 A399	µmoles/  minute
3	0.019	0.025		0.006
6	0.041	0.016		0.028
12	0.048	0.047		0.012
16	0.096	0.043		0.061
23	0.010	0.057		0.0
25	0.103	0.127		0.009
28	0.085	0.095		0.0
34	0.257	0.215		0.009
37	0.168	0.335		0.022
43	0.240	0.194		0.004
47	0.257	0.161		0.047
60	0.084	0.408		0.044
74	0.097	0.122		0.051

¹Actual A₃₉₉ = Average A₃₉₉ - Average of the Control A₃₉₉ values (x = 0.022, n = 13)

II. pH Optimum Data:

Crude Extract Dilution: 1/15

Length of Assay: 10 minutes

pH	A400 Trial 1	A400 Trial 2	Average  A400	µmoles  p-NP1	µmoles/  minute
3.08	0.121	0.216
3.61	0.160	0.236
4.20	0.216	0.265
4.62	0.251	0.488
5.03	0.289	0.606
5.61	0.325	0.540
6.01	0.402	0.528
6.66	0.426	0.500
7.01	0.242	0.470
7.50	0.138	0.261
7.98	0.075	0.185
8.48	0.060	0.176
8.95	0.054	0.177

¹p-NP = p-nitrophenol (reaction product)
 

III. Effect of Varying Substrate Concentration; Determination of Michaelis Constant

Crude Extract Dilution: 1/10

Length of Assay: 20 minutes

p-nitrophenol  [ ]; M	A405	µmoles p-NP  formed	reaction velocity1
0.00025	0.340
0.00050	0.442
0.0010	0.584
0.0025	0.618
0.0050	0.785

¹Units for reaction velocity are µmoles product formed/ml rxn mixture/min

Data Treatment and Analysis and Post Laboratory Questions

There are no "formal" post laboratory questions for this experiment. The questions below should be discussed in your report data analysis.

Measurement of Product Formation with Time

1. Prepare a table and a plot of absorbance vs. time.

For what time range was the assay linear with respect to time?

What explanations do you have for any deviations from linearity?

Why does the line not pass through the origin?

2. We will define a unit of enzymatic activity as the amount of an enzyme which catalyzes the transformation of one µmole of substrate to product in one minute under the experimental conditions used. Using the absorbance vs. time values, calculate the units of acid phosphatase present in wheat germ as follows:

a. Calculate the slope of the linear portion of the plot: this is the velocity of the reaction as change in absorbance per min.
b. Convert the slope to µmoles p-nitrophenol formed per minute by converting the absorbance value to the number of µmoles of p-nitrophenol using Beer's law and the molar absorption coefficient (extinction coefficient) for p-nitrophenol which we previously determined.
c. The number of µmoles p-nitrophenol formed is the amount formed per minute in the 0.5 ml reaction mixture. This value is the units of acid phosphatase activity present in the volume of crude extract that you use in the assay. Calculate units of enzyme activity per gram dry weight, by taking all of the dilution factors, etc., into consideration.

Determination of the Effect of Amount of Extract on Observed Enzyme Activity

1. Prepare a table and a graph which contains the volume of diluted extract, and the absorbance observed after 5 minutes.
What is the maximum amount of extract to be included in the assay to be within the linear part of the curve?

Determination of the Temperature Optimum and Activation Energy (E_a)

1. Plot temperature vs. absorbance for each tube.
What is the temperature optimum of the measurement of this enzyme activity?
2. The activation energy (E_a) can be obtained for the reaction by this type of analysis. According to the treatment of Arrhenius, E_a can be defined by the equation:
ln k = - ( E_a / R T ) + ln Q
where k is the rate constant for a reaction, R is the gas constant, T is temperature (K), and Q is a pre-exponential factor. A plot of ln k vs. 1/T yields a slope of -E_a/R. Using your experimental data, graphically determine E_a by plot.
Plot your data in the form of an Arrhenius plot as described in the distributed handouts¹. For the value of k use velocity values (absorbance, corrected for the blank). You may obtain a biphasic plot for the enzyme-catalyzed reaction. Which part of the plot is indicative of the E_a of the reaction? From this part of the plot calculate an E_a for the reaction. What is responsible for the other part of the plot? (consider the effect of temperature on enzyme structure). From this analysis, what is the temperature optimum for the measurement of this enzyme activity?

Determination of the pH Optimum

1. Prepare a table and a graph of Absorbance vs. pH.
What is the pH optimum?

Determination of the Michaelis Constant (K_m) for p-Nitrophenyl Phosphate Hydrolysis by Acid Phosphatase

1. Since the time has been kept constant for all reactions, these absorbance values can be considered the relative initial velocity (v_o) of the reaction in units of change in absorbance/5 min. Prepare a table of [Substrate], 1/[S], v_o and 1/v_o. Plot [S] vs. v and 1/[S] vs 1/v_o. Graphically determine K_m from the Lineweaver-Burk (double reciprocal) plot.
3.  Compare these values to those obtained from the supplemental data.

Determination of the Catalytic Inhibition of the Enzyme

The report should contain a description of the rationale for your experimental setup, raw data, graphed data, and details of calculations of the kinetic parameters and type of inhibition with explanation.