Acid Phosphatase Enzyme Assay Introduction

 

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.

cofactors (if needed).

 

 

 

 

 

 

 

 

 

Materials Provided for Each Student

 

  1. One box of blue pipet tips
  2. Test tubes:
  • Small Test Tubes (12)
  • One big test tube (A*)
  1. Cuvettes (12), holder and Kim wipes
  2. Solutions Provided
  1. a bottle of distilled H2O
  2. A bottle of 1.5% KOH Solution
  3. (Supply cart) 200 nmole/ml Nitrophenol Standard Solution
  4. (TA bench) Acid Phosphatase (on ice)
  5. (Supply cart) Substrate Solution (nitrophenyl phosphate 1mM, pH 4.5) on ice
  1. Micropipettes (P1000, P200)
  2. Timer, masking tape and marker pen
  3. Water bath set at 370C

 

Procedure

 

  1. Generate a Standard Curve

 

The Purpose: In 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)

 

  1. 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.
  2. Measure the OD of ~1 mL of solution for S1-S6 at 410nm.
    1. Make sure your spectrophotometer is correctly set at 410nm.
  3. Turn on the spectrophotometer via the switch in the back.
  4. Make sure the spectrophotometer is on Basic ATC If unsure, pressing ESC will take you back to the menu, and Basic ATC will be the option on the bottom right.

 

 

  1. After on Basic ATC mode, change mode to Absorbance via the Change mode button at the bottom of the screen.
  2. Set the wavelength to 410 nm by pressing Set nm and entering 410.
    1. 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.

 

  1. 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. (Data provided on page 9-13)

 

                     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  

 

  1. Rinse all cuvettes thoroughly with water spray bottle and place them upside down on paper towel.

 

  1. The Enzyme Assay

 

  1. Label 6 tubes with masking tape A1-A6.

 

  1. Set up the reaction in A*:

 

  • Step 1: Place 1 ml of KOH solution into each of the tubes A1 through A6.

 

Take blue tips, P1000, and tubes A1-A6 to a water bath station.

 

  • Step 2: Using 10-ml glass pipet, add 7 ml of phosphatase substrate solution to tube A* and place tube A* in a blue rack at your 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 timer immediately after the tube is placed into the water bath.

 

  • Step 6: At the times indicated in the table (next page), 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? (answer is on next page)

 

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.

 

 

 

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. (Data provided on page 9-12)

 

Table 2. Acid Phosphatase Assay at 370C

Reaction Time (min) OD410
A1 0  
A2 5.0  
A3 10.0  
A4 15.0  
A5 20.0  
A6 25.0  

 

  1. 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.

 

Post Lab Report (15 pts total)

 

Title Page: Your name, NetID, date, the title of the experiment.

 

Include the questions in your report.  

 

  1. A short summary paragraph of the experiment outlined in this protocol that describes the purpose, the chemical reaction, and how enzyme activity was measured. (3 pts)

 

  1. Using the data in Table 1, 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. (3 pts)

 

  1. What is the purpose of the standard curve generated in question 2? (1 pt)

 

  1. 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

 

  1. 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).
    1. Turn in the graph with the equation and the R2 value displayed. (2 pts)
    2. Based on this graph, what is the reaction rate for the acid phosphatase? (2 pts)

 

  1. Include the two data tables (Table 1 and Table 2 provided below).  (1 pts)

 

 

Section_______________Name __________________

 

 

 

 

Table 1: Standard Curve of Nitrophenol

 

Concentration of Nitrophenol (nmole/ml)  

OD410

S1 0 0
S2 12.5 0.10
S3 25 0.27
S4 50 0.43
S5 100 0.87
S6 200 1.65

 

 

 

 

 

 

 

Table 2. Acid Phosphatase Assay at 370C

 

Reaction Time (min) OD410
A1 0 0
A2 5.0 0.17
A3 10.0 0.29
A4 15.0 0.49
A5 20.0 0.60
A6 25.0 0.85

 

 

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