Module 7: Enzyme Kinetics and Alkaline Phosphatase

PROCEDURE:

For this experiment you will be monitoring the effects of substrate concentration on the activity of Alkaline Phosphatase in the absence and presence of an inhibitor.

Materials provided:

-     0.600 mL of 1.30 µg/mL Alkaline Phosphatase

-     6 mL of Enzyme (dilution) Buffer (0.100 M Tris, 5 mM MgCl2, 0.1 mg/mL BSA, pH 8.0)

-     9 mL of 200 µM PNPP (in 0.100 Tris, pH 8.0)

-     4 mL each of various [PNPP] substrate without inhibitor: 100, 50.0, 10.0, and 4.00 µM PNPP in 0.100 M Tris buffer, pH 8.0

-     4 mL each of various [PNPP] with 0.100 mM NaH2 PO4 inhibitor: 600, 300, 150, 75.0, and 37.5 µM PNPP in 0.100 M Tris buffer, pH 8.0

-     10 PMMA cuvettes

-     2-3 clean microfuge tubes

Part A: Setting up and navigating the Cary60 Software with the No-Enzyme control:

1.   The Cary60 UV/Visible spectrophotometers should be setup as indicated below.

In the Windows screen, click on the Cary WinUV icon.

Click on the “Kinetics” application… NOT Enzyme Kinetics

Click on the Setup button and enter in the following settings under the various tabs, then click “OK” .

**Unless otherwise stated, this will be the same setup used for all Alkaline Phosphatase assays. Each run lasts 1 minute and the Cary60 will take absorbance readings every 1.2 seconds.

2.   To set the baseline absorbance to that of your buffer (without substrate or enzyme), add 1-2 mL of the assay buffer into a cuvette and click on the “Zero” button. You should see an absorbance value of approximately 0.000 on the top left of the screen. On the top right side of the screen you should see the wavelength set to 410 nm.

3.   When preparing to collect data, first dispense 100 µL of the enzyme buffer (NOT the Assay buffer) to the bottom of a clean cuvette. This will be the “No Enzyme” control.

4. On the Cary60, press Start at the top of the screen. A “Save As” window will pop-up. Make a new

folder on the Desktop with your name (or something that you will be able to identify with), open

your new folder and then save the run by entering a file name that can represent the reaction being monitored and click “Save” .

5. Another window will popup: “ Loading Guide” . Leave as is (Sample 1) and click “Okay” .

6. The last window to popup is called “Sync Start”. This window will start a count-down from 2 minutes. This indicates that you have 2 minutes before the computer will commence taking    absorbance readings.

- Before this time runs out: Pipette 1000 µL of the 200 µM PNPP substrate into the cuvette

containing the 100 μL enzyme buffer and quickly mix the contents by quickly and gently pipetting   the reaction volume “sucking up and pushing out” in the cuvette – do this pipetting motion at least three times. Immediately place the cuvette in the sample holder of the Cary60 and click “OK” to

start acquiring data.

** Once the reaction solution is mixed it is important that you start monitoring the reaction IMMEDIATELY! Do not wait for the 2 minute count-down to run out!**

7. The Cary60 software will proceed to record the absorbance reading (every 1.2 seconds) of the reaction mixture for the next minute.

8. Once you have finished monitoring the reaction for 1 minute, Zoom-in and Recalculate the rate for the entire minute.

-To Zoom-In on the rate profile, click on thebutton at the top of the reaction window. The reaction profile will look noisy. No worries…this is expected since it is magnified.

-Then to determine the rate of the reaction, you will need to “Recalculate”: Select the “Recalculate” button in the bottom-left of the window with your reaction profile. A window will popup – Set the     start and end time boxes to 0.00 and 1.00 minutes (for now) and select the Display Fit checkbox at    the top right of the pop-up window. Then click “OK” (or enter).

The software will present a line over your entire reaction profile that represents the average rate of the reaction over the period from 0 to 1 minute. Assess the line as it is drawn and decide whether it is a true representation of the rate. If so, then record the rate value for the reaction (Abs/min),

which will be displayed in the Text window, in a table - under the “Slope (Abs/min)” heading.

If you feel the line displayed does not match the data (due to some abrupt noise), then click on the  “Recalculate” button again and select the timeframe in which you think will avoid the abrupt noise but still give you a good representation of the reaction rate. Once you settle on a line that suits the rate you are seeing in your reaction profile, record the corresponding slope (abs/min) value in your table.

What should the rate be for this No enzyme control? Ask a TA if you’re unsure.

-Rinse out the cuvette and try to remove any residual liquid by inverting it and tapping it on to a piece of paper towel

Part B: Setup reactions for varying [PNPP] with and without inhibitor

1.    Obtain a set-up of small test tubes containing varying [PNPP]:

200 μM PNPP

100 μM PNPP

50 μM PNPP

10 μM PNPP

4 μM PNPP

2.    Obtain a set-up of small test tubes containing varying [PNPP] with 0.1 mM Sodium Phosphate inhibitor included:

600 μM PNPP + NaPi

300 μM PNPP + NaPi

150 μM PNPP + NaPi

75 μM PNPP + NaPi

37.5 μM PNPP + NaPi

3.    Make the “Working” dilution of the Alkaline Phosphatase enzyme:

In a clean microfuge-tube, make 1.5 mL of the “working” dilution - 0.130 µg/mL Alkaline Phosphatase

by diluting 0.150 mL of stock 1.30 µg/mL Alkaline Phosphatase with 1.350 mL of Enzyme Buffer (containing 100mM Tris (pH 8.0), 5 mM MgCl2  and 0.10 mg/mL BSA).

Preparing 1.5 mL of 0.130 µg/mL Alkaline Phosphatase:

0.150 mL of stock 1.30 µg/mL Alkaline Phosphatase

+ 1.350 mL of Enzyme Buffer

**It is IMPORTANT to MIX the working alkaline phosphatase dilution 2 - 3 times by complete inversion.

4. Setup the reaction cuvettes:

You will only need 10 cuvettes for this part. (Rinse the cuvettes, after use, with Milli-Q water   and let drain upside down (with the occasional tapping) on a paper towel. Do not discard. You will need them for the next section).

Set up the cuvettes by first neatly transferring 100 µL of the prepared “working” dilution – 0.13 µg/mL Alkaline Phosphatase to the bottom of each of the 10 cuvettes. Dispense

accurately and directly to the bottom of the cuvettes

5.    Follow the instructions as described in Part A starting from step 4.

Note: Instead of having 100 µL of the enzyme buffer, like previously instructed in Part A – step 6, you will now have 100 µL of 0.130 µg/mL Alkaline Phosphatase in the bottom of the cuvette.

Monitor the alkaline phosphatase reaction for each substrate concentration, one at a time in the     order as stated below. Remember that once the enzyme is mixed with the substrate it is important to quickly mix and IMMEDIATELY monitor the rate of reaction. Do not wait for the countdown to

reach zero.

-     200 μM PNPP

-     100 μM PNPP

-     50.0 μM PNPP

-     10.0 μM PNPP

-     4.00 μM PNPP

-     300 μM PNPP + Pi

-     150 μM PNPP + Pi

-     75.0 μM PNPP + Pi

-     37.5 μM PNPP + Pi

** For consistency purposes, it is important that you use the same enzyme preparation for both the control and inhibited set.

6.   To record initial rates for each reaction, Zoom-in and Recalculate as you did in Part A, step 8, except for the following:

Recalculate by selecting a smaller timeframe starting from 0 minutes - e.g. 0.00 to 0.20

minutes. This will allow you to catch the rate during the initial part of the reaction before a significant amount of the substrate becomes depleted.

Also make sure to ZOOM-IN on the reaction profile and assess whether the software is

determining the initial rate correctly. If not, re-enter your time range to suit what you think should be measured as the initial rate. Your initial rate should be linear. Please see the

example below.

The enzymatic rate will be presented in a table (below the reaction profile) under the heading “slope - Abs/min” Record the enzymatic rate on the Data Report Form.