Kinetics of alkaline phosphatase lab report

BCH 367 Elementary Biochemistry Lab Enzyme Kinetic Analysis of Alkaline Phosphatase

School of Molecular Sciences, Arizona State University 1

Experiment 4 Enzyme Kinetic Analysis of Alkaline Phosphatase

Introduction

Phosphatases are the common enzymes in numerous physiological actions, such as signal transduction. The enzyme catalyzes the hydrolytic cleavage of esters of phosphoric acid.

Figure 1. Hydrolysis of an ester bond to produce a phosphoric acid.

To measure the activity of the phosphatases, one can follow the liberation of the phosphate or of the other product released by hydrolysis reaction. The assay can be simplified using a substrate that is cleaved to give a highly colored phosphate free product. In this experiment, we will utilize 4-nitrophenylphosphate as the substrate. The hydrolysis releases phosphate to generate 4-nitrophenolate, which has a high molar absorptivity at 405 nm under alkaline conditions (ε405 = 1.88×104 OD M-1cm-1).

Figure 2. Hydrolysis reaction on the 4-nitrophenylphosphate.

This experiment will examine the effects of the concentrations of the enzyme and substrate, and the presence of an inhibitor upon the rate of the alkaline phosphatase catalyzed hydrolysis of 4-nitrophenylphosphate. The Part 1 will be done during the first lab period and the results obtained will be used for the Part 2 during the second lab

BCH 367 Elementary Biochemistry Lab Enzyme Kinetic Analysis of Alkaline Phosphatase

School of Molecular Sciences, Arizona State University 2

period. The student should study the chapter on enzyme kinetics from the BCH 361 text or other comparable information source, which will give good explanations of the plots that you will be required to make in order to determine the Vmax, Km, and Ki for alkaline phosphatase.

It is imperative that the students in this lab work in an efficient manner so that the lag time between the addition of reagents and start of the kinetic analysis is kept to a minimum. Since the most important information will be obtained in the second lab period, you should develop good techniques during the first period and use those same techniques during the second period.

Experimental Procedures

Part 1: Effect of enzyme concentration upon reaction rate.

Preparation of substrate solutions

The actual assay solutions must be prepared prior to diluting the enzyme since the enzyme must be used immediately if it is not on ice. Obtain your 96-well microplate. Fill the wells from B1 to B6 and C1 to C6 with 240 µl of substrate solution (5 mM 4- nitrophenyl phosphate in buffer). Use a multichannel pipette. These are the only wells that will be read by the instrument so do not put your substrate in different wells of your choice. Make certain that you do not introduce any air bubble into your solutions as it will cause erroneous readings from the plate reader.

Enzyme dilutions

Use your microplate to make the following dilutions. These dilutions should be made in the wells of A1-A5 of your microplate. Using the stock (1 mg/ml) alkaline phosphatase, make about 100-200 μl of each of the following concentrations: 250 μg/ml, 125 μg/ml, 50 μg/ml, 25 μg/ml, and 10 μg/ml. Since making a 10 μg/ml solution from a 1 mg/ml solution is a big jump in dilution factors, start by making the 250 μg/ml solution, then make your 125 μg/ml solution since this will only be a two-fold dilution from the 250 μg/ml solution. Proceed down to your lowest concentration in this manner. Use water as your diluting agent for each of your enzyme solutions. Also, add about 200 μl of water to the well A6 as this will be used as a control (0 μg/ml alkaline phosphatase).

Assays

DO NOT ADD YOUR ENZYME TO THE SUBSTRATE SOLUTIONS UNTIL IMMEDIATELY PRIOR TO RUNNING YOUR ASSAY! Make certain that the plate reader

BCH 367 Elementary Biochemistry Lab Enzyme Kinetic Analysis of Alkaline Phosphatase

School of Molecular Sciences, Arizona State University 3

is set up (λ = 405 nm) and ready for use prior to adding enzyme to substrate. Using a multi-channel pipette, add 10 μl of the enzyme solutions from the wells A1-A6 to the substrate solutions in the wells B1-B6. Gently stir the solution with the tip of the pipette after addition. Repeat this addition to the wells C1-C6. You must work rapidly since the reaction starts immediately. Quickly place your plate into the plate reader and start the assay. Transfer the data to an Excel worksheet and analyze your data prior to leaving lab to make certain that you have obtained data that makes sense. When using the plate reader, you do not zero the instrument and thus the data will be relative to the initial reading. You will collect data for 3 minutes and determine the actual increase in absorbance for each well by subtracting the time zero absorbance value from the absorbance reading at 3 minutes. You must convert from ΔAbs/min to Δ[product]/min using the extinction coefficient. Prepare a graph of initial velocity vs. enzyme concentration to show to your TA for next week.

You must decide the optimal enzyme concentration from the data obtained in the Part 1. You will use this enzyme concentration in the Part 2 of this lab next week. You want to pick an enzyme concentration that gives a fairly linear plot of Abs vs. time. Also, keep in mind that the substrate concentration that you are using is more concentrated than what will be used in the Part 2 so if the lower enzyme concentrations are not giving very good activity, you might want to select a higher enzyme concentration. If you have questions about which enzyme concentration to use, ask the instructor or your TA—have your data in graphical format (Δ[product] vs. Δ time) to make the conversation easier for all involved.

Part 2: Effect of substrate concentration and an inhibitor.

You will be doing an assay that is almost identical to what you did in the Part 1, with the change being that enzyme concentration will be held constant while substrate concentration will be varied. In this case, a constant amount of enzyme (determined from your data in part 1) will be used. The rate of reaction will be measured at six different concentrations of substrate in the presence and absence of inhibitor. You will do each reaction in duplicate.

1. Obtain your 96-well plate. You will use 36 wells on the plate, A1-A12, B1-B12, and C1-C12. The instrument is set to read only these wells and thus you must follow the instructions below.

2. Preparation of substrate solutions from the stock substrate (5 mM 4- nitrophenylphosphate in buffer) is performed by diluting the stock solution with buffer to the desired concentrations. Final concentrations of the diluted solutions should be 2.5 mM, 1.0 mM, 0.5 mM, 0.1 mM, and 0.05 mM. you will need a total

BCH 367 Elementary Biochemistry Lab Enzyme Kinetic Analysis of Alkaline Phosphatase

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volume of approximately 2 ml of each substrate dilution. Keep in mind that these are not the actual concentrations that will be present in the assays. Each assay will contain 230 µl of substrate in a final volume of 250 µl. Therefore, the actual concentration in the assay will be slightly less than the concentration of the solutions that you have prepared. You will need to calculate the exact concentration while doing your data analysis.

3. The instrument is programmed to scan the Row A to measure spontaneous hydrolysis of the sample, Row B for the effect of substrate concentration, and Row C for the effect of an inhibitor. The following tables provide information you can use to set up the assays for the experiment. Each group will work with only one inhibitor. Remember that since you are doing each analysis in duplicate each pair of wells will be identical. For example, the well A1 and A2 will be exactly the same. Start by adding the appropriate amount of water or inhibitor to the appropriate wells. You may use the multichannel pipettes to do this. Follow this with an appropriate amount of substrates to the proper wells. DO NOT ADD ENZYME AT THIS TIME! Take your plate to the microplate reader where your TA will let you know when to add enzyme.

Table 1. Analysis of substrate hydrolysis (Row A) Well A1-A2 A3-A4 A5-A6 A7-A8 A9-A10 A11-A12

Substrate concentration (mM) 0 0.05 0.1 0.5 1.0 2.5

Substrate volume (µl) 230 230 230 230 230 230

Water 20 20 20 20 20 20

Table 2. Kinetics of substrate concentration (Row B)

Well B1-B2 B3-B4 B5-B6 B7-B8 B9-B10 B11-B12

Substrate concentration (mM) 0 0.05 0.1 0.5 1.0 2.5

Substrate volume (µl) 230 230 230 230 230 230

Water (µl) 10 10 10 10 10 10

Enzyme (µl) 10 10 10 10 10 10

Table 3. Kinetics with inhibitor (Row C)

Well C1-C2 C3-C4 C5-C6 C7-C8 C9-C10 C11-C12

Substrate concentration (mM) 0 0.05 0.1 0.5 1.0 2.5

Substrate volume (µl) 230 230 230 230 230 230

Inhibitor (µl) 10 10 10 10 10 10

Enzyme (µl) 10 10 10 10 10 10

BCH 367 Elementary Biochemistry Lab Enzyme Kinetic Analysis of Alkaline Phosphatase

School of Molecular Sciences, Arizona State University 5

4. You will start by measuring activity of the Row A in the microplate reader. Once completed, the reader will eject the plate, at which time you will use the multi- channel pipette to add the enzyme to the Row B. Gently stir the solution with the tip of the pipette after addition. Since the multi-channel pipettes will only accommodate 8 pipette tips at a time, add the enzyme 6 wells at a time. Make certain to work quickly and accurately. Insert your plate into the plate reader and start the assay. Repeat for the Row C. 


5. When using the plate reader, you do not zero the instrument and thus the data will be relative to the initial reading. You will collect data for 90 seconds and determine the actual increase in absorbance for each well by subtracting the time zero absorbance value from the absorbance reading at 90 seconds. This is most easily done by exporting your data to the MS Excel. Make sure that your data is linear across the entire time course of the experiment. You must convert from ΔAbs/min to Δ[product]/min using the extinction coefficient. Prepare a graph of initial velocity vs. substrate concentration in the absence and presence of inhibitor for your final report, respectively. Generate values for the maximum velocity (Imax) and the Michaelis constant (Km) from the plot of velocity vs. substrate concentration. Also generate values for the Vmax, Km and the inhibitor constant (Ki) from the data using one of the linear forms of the Michaelis-Menten equation. Include this linear plot in your report. 


The two tutorial videos on the YouTube for enzyme kinetics graphs:

1. https://www.youtube.com/watch?v=6RMEokEDBaw 2. https://www.youtube.com/watch?v=0uYHnjjFcHw

Discussion Questions

1. Did you observe a direct proportionality between enzyme concentration and initial velocity?

2. Does the velocity of the reaction change with time? If so, why? 3. What type of inhibition is indicated by your data? Justify your answer. 4. Given the following reaction and equation for the initial velocity of the reaction:

k1 k3 E + S ⇄ ES ➛ E + P V = kcat [ES] = k3 [ES] k2 where kcat is the rate constant for the reaction which forms the product from the

BCH 367 Elementary Biochemistry Lab Enzyme Kinetic Analysis of Alkaline Phosphatase

School of Molecular Sciences, Arizona State University 6

ES complex. Explain in words why the velocity is directly proportional to the amount of enzyme added in the presence of saturating substrate levels.

5. Consider the Michaelis-Menten equation. Under what conditions will the Km be a measure of the substrate binding? v = Vmax [S] / (Km + [S]), where Km = (k2 + k3) / k1.

Materials

Stock enzyme: 1 mg/ml alkaline phosphatase in H2O with 1 mg/ml BSA added as a stabilizing agent

Stock substrate: 5 mM 4-nitrophenylphosphate in 0.05 M amidol, pH 9.0

Buffer: 0.05 M amidol, pH 9.0

Inhibitor: 75 mM phenylalanine and 15 mM Na2HPO4

Note that p-nitrophenol in basic solution has a constant ε405 = 1.88×104 M-1cm-1.

References

Bergmeyer, H., Editor, Methods in Enzymatic Analysis, 3rd edition, 4, 75-92 (1983).

Clark, J.M. Jr. and Switzer, R.L. Experimental Biochem 106 (1977).

Cox, R.P., Gilbert, P. Jr., and Griffin, M.J. Alkaline inorganic pyrophosphatase activity of mammalian-cell alkaline phosphatase. Biochem J 105:155-161 (1967).

Ghosh, N.K. and Fishman, W.H. On the mechanism of inhibition of intestinal alkaline phosphatase by L-phenylalanine. J Biol Chem 241:2516-2522 (1966).