Biol 2281 Spring 2016, E10: Enzyme Assay Introduction, Procedure and Report
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Experiment 10: Acid Phosphatase Enzyme Assay Introduction*
*This background information is provided by John N Anderson. Permission is granted to reproduce this written material for educational purposes.
Biol 2281 Spring 2016, E10: Enzyme Assay Introduction, Procedure and Report
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Biol 2281 Spring 2016, E10: Enzyme Assay Introduction, Procedure and Report
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Biol 2281 Spring 2016, E10: Enzyme Assay Introduction, Procedure and Report
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Biol 2281 Spring 2016, E10: Enzyme Assay Introduction, Procedure and Report
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Materials Provided for Each Student
1. One box of blue pipet tips 2. Test tubes:
Small Test Tubes (12)
One big test tube (A*) 3. Cuvettes (12), holder and Kim wipes 4. Solutions Provided
a) a bottle of distilled H2O b) A bottle of 1.5% KOH Solution c) (Supply cart) 200 nmole/ml Nitrophenol Standard Solution d) (TA bench) Acid Phosphatase (on ice) e) (Supply cart) Substrate Solution (nitrophenol phosphate 1mM, pH 4.5) on ice
5. Micropipettes (P1000, P200) 6. Timer, masking tape and marker pen 7. Water bath set at 370C
Procedure I. Generate a Standard Curve
The Purpose: In order to determine the amount of nitrophenol produced in the enzyme reactions (part II), you will need to generate a standard curve of OD410 readings vs nmole/ml of nitrophenol. You will be able to convert the OD 410nm readings of product in the enzyme reaction into the concentration of nitrophenol based on the standard curve. (OD : optical density)
1. Label 6 small test tubes S1-S6. 2. Place 1ml of H2O using P1000 into tube S1 to S5.
3. Using P1000 to measure 2 ml of 200 nmole/ml of nitrophenol solution accurately and place
the solution into S6. 4. For S5-S2, do a serial dilution from S6 and each tube should contain 1ml final volume.
IMPORTANT: S1 will be used as blank. Do not transfer any solution out of S2 into S1. Discard 1ml from S2 into sink.
S6 S5 S4 S3 S2
200 100 50 25 12.5 (nmole/ml) 5. Add 1ml of KOH to all six tubes using micropipette P1000. DO NOT LET THE PIPET TIP
TOUCH THE SOLUTIONS IN TUBES S1-S6. You may use the same blue tip as long as it does not touch the liquid in test tubes and no liquid goes up into the barrel of the P1000.
6. Measure the OD for S1-S6 at 410nm. a) Make sure your spectrophotometer is correctly set at 410nm.
1. Turn on the spectrophotometer via the switch in the back.
2. Make sure the spectrophotometer is on Basic ATC mode. If unsure, pressing ESC will take you
back to the menu, and Basic ATC will be the option on the bottom right.
Biol 2281 Spring 2016, E10: Enzyme Assay Introduction, Procedure and Report
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3. After on Basic ATC mode, change mode to Absorbance via the Change mode button at the
bottom of the screen.
4. Set the wavelength to 410 nm by pressing Set nm and entering 410.
b) Autozero the spec using the solution in S1. (Clean cuvettes with Kim wipes, about 1 ml is needed for each cuvette). Make sure the arrow on the side of the cuvette is in-line with the circle, not facing into or out of the circle of slots. Press Measure Blank.
c) Measure the OD of S2 through S6 and record data in Table 1. Put S2 through S6 in slots 1-5 of the six-cell changer, accordingly.
Table 1: Standard Curve of Nitrophenol
Concentration of Nitrophenol (nmole/ml)
OD410
S1 0
S2 12.5
S3 25
S4 50
S5 100
S6 200
7. Plot the data of Table 1 onto the graph paper #1 and show it to your TA. (2 pts) 8. Do not dispose of your samples until your TA has checked your results. Rinse all cuvettes
thoroughly with water spray bottle and place them upside down on paper towel.
II. The Enzyme Assay 1. Label 6 tubes with masking tape A1-A6. Label 1 large glass tubes A*. 2. Set up the reaction in A*:
Step 1: Place 1 ml of KOH solution into each of the tubes A1 through A6
Step 2: Using 10-ml glass pipet, add 7 ml of phosphatase substrate solution to tube A*.
Take blue tips, P1000, test tubes (A1-A6 & A*), and timer to one water bath station.
Step 3: Set P200 to 50 ul. At TA bench, place yellow tip on P200 and use it to measure 50 ul of crude extract containing acid phosphatase. Go to your water bath station IMMEDIATELY.
Take no more than 15 seconds to complete steps 4 and 5:
Step 4: Use P200 to add 50 μl of acid phosphatase (about 1μg of pure enzyme) into tube A*. Use parafilm and shake the tube vigorously, without spilling, to mix. Using micropipette p1000, place 1 ml of solution from tube A* into tube A1. This solution will be used for determining the zero-time value of the reactions.
Step 5: Transfer A* tube into the 370C water bath. Start the timing immediately after the tube is placed into the water bath.
Step 6: At the times indicated in the table, remove 1ml of the reaction solution in tube A* and place it in the corresponding tubes while keeping your tube A* in the water bath throughout the 25 min reaction. Also IMPORTANT: Do not let your pipet tip touch the KOH solution in the tubes A2-A6, Why not?
The KOH in the tubes serves dual purposes. First it will stop the reaction because acid phosphatase will be catalytically inactive at alkaline pH. Secondly, the KOH will cause one of the products of the reaction (nitrophenol) to turn yellow.
Biol 2281 Spring 2016, E10: Enzyme Assay Introduction, Procedure and Report
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The Enzyme Reaction Setup-modified
A*
A1 A2 A3 A4 A5 A6
Step 2: Add 7ml of Substrate,
pH4.5
Step 4: Transfer
1 ml into A1
Step 3: Add
enzymeStep 5: Remove 1ml of
reaction mix to A2-A6
at different times
5.0 10.0 15.0 20.0 25.0 min0.0
Step 1: Add 1ml of
KOH into A1-A6
III. Measurement of the Products (nitrophenol) of the Enzyme Reactions
1. AutoZero the Spectrophotomer using A1 solution. Press Measure Blank. 2. Measure the OD410 for solutions in tubes A2 to A6. Record the readings in Table 2. . 3. Plot the data in Table 2 on the graph paper #2, and show your instructor. (2 pts)
Table 2. Acid Phosphatase Assay at 370C
Reaction Time (min)
OD410
A1 0 0
A2 5.0
A3 10.0
A4 15.0
A5 20.0
A6 25.0
IV. Cleaning Up
1. Remove all cuvettes from spectrophotometer. Rinse all cuvettes thoroughly with water spray bottle
and place them upside down on paper towel. 2. Turn off the spectrophotometer. 3. Take off the masking tape label from all of your test tubes. Thoroughly rinse the test tubes with tap
water at least 3 times for each tube. Replace them upside down in a metal basket. 4. Refill blue tip box and diH2O squirt bottle. 5. Empty trash container into regular trash.
Biol 2281 Spring 2016, E10: Enzyme Assay Introduction, Procedure and Report
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Post Lab Report (20 pts total) Title Page: Your name, date, the title of the experiment. Include the questions in your report.
1. A short summary paragraph of the experiment that describes the purpose, the chemical reaction, and how enzyme activity is measured. (4 pts)
2. Copy your completed Table 1 into your report. Use Excel to generate a standard curve of OD410 vs nmole/ml of nitrophenol. Make sure you include the equation and R2 value on your graph. If the R2 is less than 0.95, you need to drop some outlying data point and recalculate. Be sure to set your intercept to zero. Failure to do so will result in loss of points (4 pts)
3. What is the purpose of the standard curve generated in question 2? (1 pt)
4. Based on the equation generated by the standard curve (question 2), convert the OD410 values
in Table 2 into nmole of nitrophenol produced. Remember that the standard curve allows you to extrapolate concentrations (nmole/ml) of nitrophenol based on absorbance values. You need to calculate the amount (in nmole) by multiplying the concentration of nitrophenol by the volume measured (in the cuvette). The volume measured at each time point was 1 ml. (3 pts)
Reaction Time (min)
Original OD410 Reading from Table 2
nmole/ml based on Standard
Curve
Nitrophenol Produced in nmole (= nmole/ml x 1 ml)
0
5.0
10.0
15.0
20.0
25.0
5. Use Excel to plot a scatter diagram of nitrophenol produced (nmole) over time (min) for the
reaction using data in the above table in question 4. Perform linear regression analysis and determine the slope of the line. (You need to keep as many data points as possible in generating the linear relationship).
a. Turn in a printout of the graph with the equation and the R2 value. (2 pts) b. What is the reaction rate for the acid phosphatase? (2pts)
6. Staple your two graphs completed in the lab. You WILL NOT receive credit for them if they are
not attached! (4 pts)
For more graph paper see: w
ww
.vvi.com/graphpaper
© Copyright 2004; VVimaging, Inc. All Rights Reserved.
For more graph paper see: w
ww
.vvi.com/graphpaper
© Copyright 2004; VVimaging, Inc. All Rights Reserved.
Biol 2281, E10
Dr. Pickett, Dr. Lin -- Spring 2016 1
Experiment 10: Wheat Germ
Acid Phosphatase Enzyme Assay
What can you learn from this
experiment?
• Basic concepts of enzyme reactions
– Substrate, product, enzyme
– Initial velocity of an enzyme under specific conditions
• Making serial dilutions
• Prepare a standard curve
• Perform a simple yet complete set of enzyme assays
• Review graphing data and linear regression analysis
Enzymes Action
Figure 5.4
Measurement of the Rate of an
Enzyme-Catalyzed Reaction
What accounts for the decline in reaction rate with time?
What measures the true enzyme reaction rate?
The Initial Velocity: V0
1 ug of Pure Acid Phosphatase Activity at 370C
y = 14.582x
R2 = 0.9864
0
50
100
150
200
250
300
350
0 5 10 15 20 25
Time (minutes)
Nit
rop
hen
ol
Pro
du
ced
(nm
ole
s)
Factors that Influence the V0
• Temperature
• pH
• Amount of enzyme
• Substrate concentration
• Effects of inhibitors and/or activators
Biol 2281, E10
Dr. Pickett, Dr. Lin -- Spring 2016 2
• Temperature pH
Factors Influencing Enzyme Activity Amount of Enzyme affects V0
Effect of Substrate Concentration
on V0
• Km
• Vmax at a
specific
enzyme
concentration
Vmax
Km
Acid Phosphatase From Wheat Germ
• Crude Enzyme Extract – Extraction buffer contains
• MgCl2
• Tris-HCL, pH 8.0
• 0.05% NP-40
• Other sources – Prostatic acid phosphatase
– Alfalfa sprouts
– Beans
– Rice
The Reaction Catalyzed by Acid
Phosphatase
• Substrate: Nitrophenyl Phosphate (provided as 1mM
in Na-Acetate buffer of pH 4.5)
• Product: Nitrophenol + Phosphate
• 1.5% KOH: Serves what two purposes?
Experimental Procedures
• Prepare a Standard Curve for the Product Formed
• Perform the Enzyme Assay
• Cleaning Up
Biol 2281, E10
Dr. Pickett, Dr. Lin -- Spring 2016 3
The Standard Curve:
Determine the relationship between OD410
(absorbance) and the concentration of nitrophenol
• Plot the data on graph paper #1 ( 2 pts)
Standard Curve: OD vs. nmole/ml of nitrophenol
y = 0.01x
R2 = 0.9655
0
0.5
1
1.5
2
2.5
0 50 100 150 200 250
nitorphenol concentration (nmole/ml)
OD
or
Ab
so
rban
ce
How do you prepare a standard
curve? • _______________________________________
• Making a series of nitrophenol solutions:
– Stock solution concentration is _____________;
– Final volume of each solution is ______________;
– Final concentrations of each solution is:
– The dilution method used is _____________;
The Enzyme Reaction Setup-modified
A*
A1 A2 A3 A4 A5 A6
Step 2: Add 7ml of Substrate,
pH4.5
Step 4: Transfer
1 ml into A1
Step 3: Add
enzyme Step 5: Remove 1ml of
reaction mix to A2-A6
at different times
5.0 10.0 15.0 20.0 25.0 min 0.0
Step 1: Add 1ml of
KOH into A1-A6
Graphing OD410 vs Time
• Plot the of data on graph paper #2 ( 2 pts)
Acid Phosphatase Activity at 37C and pH 4.5
y = 0.0731x
R2 = 0.992
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
0 5 10 15 20 25
Time (min)
OD
at
41
0 n
m
At home: Graphing Data Using Excel
Post-Lab Report Question 5: Sample Graph for A* Reaction
1 ug of Pure Acid Phosphatase Activity at 370C
y = 14.582x
R2 = 0.9864
0
50
100
150
200
250
300
350
0 5 10 15 20 25
Time (minutes)
Nit
rop
hen
ol
Pro
du
ced
(nm
ole
s)
Quiz Review Questions • How is the initial velocity of an enzyme reaction determined?
• What contributes to the decline in reaction rate?
• What is the purpose of the standard curve used in this
experiment?
• Describe how the nitrophenol standard curve is generated in this
experiment. What parameters are used as X- axis and Y- axis?
• List three factors that influence the rate of enzyme catalyzed
reactions
• How is the reaction catalyzed by acid phosphatase stopped?
• What is the pH at which the acid phosphatase activity is
measured?
