9 Kinetics of a Turnip Peroxidase PDF

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9 Kinetics of a Turnip Peroxidase...

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Kinetics of a Turnip Peroxidase

I.

II.

Pre-Laboratory. a) What scientific concept(s) will be learned in this lab activity?  The scientific concept of Michaelis-Menten equation and the Lineweaver-Burke (double reciprocal) plot will be learned in this lab. Also, the type of inhibitor will be identified through the double reciprocal plot. b) What laboratory techniques will be learned in this activity?  The laboratory technique that will be learned in this lab is measuring the wavelength at a certain absorbance using a spectrometer. c) What is the objective (or goal) for the lab?  The goal for this lab is to observe the effects of substrate concentration, temperature, pH, and the presence of inhibitors on the rate of reactions. This will provide a clear understanding of how the turnip peroxidase reaction works. This kinetics information could be used in real life such as drug development and design. Materials and Methods. Dr. Pattison’s BCHS 3201 protocol, pages 116-136. Part A was already made for the students and the data was shared for the class for parts C-G.

 Part B: Table 1 – Tabulation of the Data from Part B with the Setup of Each Test Tube Reagents Distilled Total Guaiacol Turnip 1% H2O2 (µL) Water (mL) Volume Tube (µL) Extract (µL) (mL) 1 50 500 4.45 5 2 50 100 2.35 2.5 3 500 2.0 2.5  Part C: Table 2 – Tabulation of the Data from Part C with Varying Concentration of the Turnip Extract Given that Each Group from A-I Has a Different Concentration Guaiacol Turnip Distilled 1% H2O2 Total Group Tube (µL) Extract (µL) Water Volume (µL) (mL) (mL) 1 50 500 4.45 5 A 2 50 100 2.35 2.5 3 500 2.0 2.5 1 50 300 4.2 5 B 2 50 100 2.35 2.5 3 300 2.2 2.5 1 50 250 4.7 5 C 2 50 100 2.35 2.5

D

E

F

G

H

I

3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1

50 50 50 50 50 50 50 50 50 50 50

250 200 200 150 150 100 100 75 75 50 50 25

100 100 100 100 100 -

2.25 4.75 2.35 2.3 4.8 2.35 2.35 4.85 2.35 2.4 4.875 2.35 2.425 4.9 2.35 2.45 4.925

2.5 5 2.5 2.5 5 2.5 2.5 5 2.5 2.5 5 2.5 2.5 5 2.5 2.5 5

2 3

50 -

25

100 -

2.35 2.475

2.5 2.5

 Part D: Table 3 – Tabulation of the Data from Part D with Varying Concentration of the Substrate H2O2 Given that Each Group from A-G Has a Different Concentration Total Distilled Guaiacol Turnip 1% H2O2 Volume (µL) Water Group Tube (µL) Extract (mL) (mL) (µL) 1 50 100 4.85 5 A 2 50 25 2.425 2.5 3 100 2.4 2.5 1 50 100 4.85 5 B 2 50 50 2.4 2.5 3 100 2.4 2.5 1 50 100 4.85 5 C 2 50 150 2.3 2.5 3 100 2.4 2.5 1 50 100 4.85 5 D 2 50 250 2.2 2.5 3 100 2.4 2.5 1 50 100 4.85 5 E 2 50 300 2.15 2.5 3 100 2.4 2.5 1 50 100 4.85 5 F 2 50 350 2.1 2.5

G

3 1 2 3

50 50 -

100 100 100

400 -

2.4 4.85 2.05 2.4

2.5 5 2.5 2.5

 Part E: Table 4 – Tabulation of the Data from Part E with Varying Temperature Given that Each Group from A-E Has a Different Temperature Guaiacol Turnip Distilled 1% H2O2 Total Group Tube (µL) Extract (µL) Water Volume (µL) (mL) (mL) 1 50 100 4.85 5 A 2 50 100 2.35 2.5 4 °C 3 100 2.4 2.5 1 50 100 4.85 5 B 2 50 100 2.35 2.5 26 °C 3 100 2.4 2.5 1 50 100 4.85 5 C 2 50 100 2.35 2.5 37 °C 3 100 2.4 2.5 1 50 100 4.85 5 D 2 50 100 2.35 2.5 66 °C 3 100 2.4 2.5 1 50 100 4.85 5 E 2 50 100 2.35 2.5 100 °C 3 100 2.4 2.5  Part F: Table 5 – Tabulation of the Data from Part F with Varying pH Values Given that Each Group from A-E Has a Different pH Guaiacol Turnip Distilled 1% H2O2 Total Group Tube (µL) Extract (µL) Water Volume (µL) (mL) (mL) 1 50 100 4.85 5 A 2 50 100 2.35 2.5 pH=3 3 100 2.4 2.5 1 50 100 4.85 5 B 2 50 100 2.35 2.5 pH=7 3 100 2.4 2.5 1 50 100 4.85 5 C 2 50 100 2.35 2.5 pH=9 3 100 2.4 2.5 1 50 100 4.85 5 D 2 50 100 2.35 2.5

pH=11 E DI H2O

3 1 2 3

50 50 -

100 100 100

100 0

2.4 4.85 2.35 2.4

2.5 5 2.5 2.5

 Part G: For this part, certain amount of the hydroxylamine inhibitor was used and the substrate hydrogen peroxide amounts were varies each time. Table 6 – Tabulation of the Data from Part G with Varying Concentration of the Substrate H2O2 in the Presence of an Inhibitor Given that Each Group from A-G Has a Different Substrate Concentration Guaiacol Turnip 1% 10% Distilled Total Group Tube (µL) Extract H2O2 Hydroxyl Water Volume (µL) (µL) - amine (mL) (mL) 1 50 75 25 4.85 5 A 2 50 75 25 2.35 2.5 3 25 2.475 2.5 1 50 75 25 4.85 5 B 2 50 75 25 2.35 2.5 3 50 2.45 2.5 1 50 75 25 4.85 5 C 2 50 75 25 2.35 2.5 3 75 2.425 2.5 1 50 75 25 4.85 5 D 2 50 75 25 2.35 2.5 3 100 2.4 2.5 1 50 75 25 4.85 5 E 2 50 75 25 2.35 2.5 3 125 2.375 2.5 1 50 75 25 4.85 5 F 2 50 75 25 2.35 2.5 3 150 2.35 2.5 1 50 75 25 4.85 5 G 2 50 75 25 2.35 2.5 3 200 2.3 2.5 III.

Results. Part B: Table 7 – Tabulation of the Absorbance in a Two-Minute Time Interval under Wavelength of 470 nm of Part B Time (seconds) 0

Absorbance at 470 nm -0.3

20 40 60 80 100 120 140 160 180

-0.25 -0.03 0.17 0.36 0.40 0.44 0.49 0.55 0.62

Slope ( ΔA /min ): (Δ A) Slope = x 60 s/min (time) (0.62−(−0.3 )) = x 60 = 0.306 absorbance unit/min (180)

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Graph 1 – Absorption versus the time in seconds of the data from Table 7 for Part B plotted in this graph. Part C: Table 8 – Tabulation of the Absorbance in a Two-Minute Time Interval under Wavelength of 470 nm of Part C with Different Turnip Extract (Enzyme) Concentration to Observe the Effect of Enzyme Concentration on the Rate of Abs470 nm Increase

Volumes of Turnip Extract (µL)

Time (sec) 0 20 40 60 80 100 120 140 160 180

500

300

250

200

150

100

75

50

25

0.058 0.23 0.386 0.53 0.648 0.72 0.784 0.838 0.884 0.932

0.12 0.24 0.36 0.43 0.51 0.59 0.64 0.7 0.73 0.76

0.06 0.26 0.32 0.39 0.43 0.47 0.52 0.56 0.58 0.62

0.07 0.13 0.19 0.26 0.32 0.37 0.41 0.45 0.5 0.53

0 0.02 0.1 0.15 0.2 0.24 0.3 0.34 0.4 0.43

0 0.03 0.06 0.09 0.13 0.17 0.2 0.24 0.27 0.3

0 0.01 0.03 0.05 0.07 0.11 0.15 0.17 0.21 0.24

0 0.01 0.02 0.04 0.07 0.09 0.11 0.13 0.15 0.17

0.01 0.02 0.02 0.03 0.04 0.05 0.06 0.07 0.08 0.09

Slope ( ΔA /min ): (Δ A) x 60 s/min Slope = (time) Table 9 – Calculation of Slopes of the Data from Table 8 and Graph 2. The Concentration 100 µL Has the Only Linear Format with the Slope Given in this Table Turnip Extract Concentration (µL) 500

Calculations (0.932−0.058) (180)

Slope (unti/min) x 60

0.294

s/min

300 250 200 150 100 75 50 25

(0.76−0.12) x 60 s/min (180) (0.62−0.06) x 60 s/min (180) (0.53−0.07) x 60 s/min (180) (0.43−0) x 60 s/min (180) (0.3 −0) x 60 s/min (180) (0.24−0) x 60 s/min (180) (0.17−0) x 60 s/min (180) (0.09−0.01) x 60 s/min (180)

0.216 0.186 0.156 0.144 0.102 0.078 0.054 0.024

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Graph 2 – Absorption versus the time in seconds of the data from Table 8 for Part C plotted in this graph. Part D: Table 10 – Tabulation of the Absorbance in a Two-Minute Time Interval under Wavelength of 470 nm of Part D with Different H2O2 Concentrations to Observe the Effects of Substrate Concentration on the Rate of Abs470 nm Increase Volumes of H2O2 (µL) Time (sec) 0 20 40 60 80 100 120

25

50

150

250

300

350

400

0 0.02 0.04 0.08 0.11 0.14 0.16

0.01 0.02 0.05 0.09 0.13 0.17 0.2

0.01 0.09 0.13 0.17 0.22 0.26 0.31

0 0.01 0.08 0.15 0.2 0.24 0.28

0 0.03 0.11 0.18 0.21 0.26 0.31

0 0 0.07 0.13 0.18 0.22 0.26

0 0.08 0.13 0.2 0.25 0.29 0.32

[Substrate0] of H 2O 2 µL

mM

25 50 150 250 300

1.47 2.94 8.82 14.71 17.65

ν0 (µmol/min) of tetraguiaco l formation 9.55 11.59 14.89 15.01 15.45

140 160 180

0.19 0.22 0.24

0.23 0.26 0.3

0.34 0.38 0.41

0.31 0.35 0.36

0.34 0.36 0.39

0.29 0.32 0.34

0.35 0.37 0.39

350 400

20.59 23.53

14.24 14.68

Slope ( ΔA /min ): (Δ A) Slope = x 60 s/min (time) Table 11 – Calculation of Slopes of the Data from Table 10 and Graph 3. The Concentration 125 µL Has the Only Linear Format with the Slope Given in this Table Volumes of H2O2 (µL) 25 50 150 250 300 350 400

Calculations (0.24−0) x 60 s/min (180) (0.3−0.01) x 60 s/min (180) (0.41−0.01) x 60 s/min (180) (0.36−0) x 60 s/min (180) (0.39−0) x 60 s/min (180) (0.34−0) x 60 s/min (180) (0.39−0) x 60 s/min (180)

Slope (unti/min) 0.080 0.100 0.133 0.120 0.130 0.113 0.130

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Graph 3 – Absorption versus the time in seconds of the data from Table 10 for Part D plotted in this graph.

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Graph 4 – Michaelis-Menten plot of the initial substrate concentration versus the initial velocity of turnip peroxidase exhibited saturation kinetics that is shown in Table 10. Table 12 – Tabulation of the Reciprocal of the Initial Substrate Concentration and the Initial Velocity of Turnip Peroxidase Taken from Table 10

Graph 5 – reciprocal Table 12. regression estimate KM

[Substrate0]-1 (mM-1) 0.68 0.34 0.11 0.07 0.06 0.05 0.04

V0-1 (min/ µmol) 0.10 0.09 0.07 0.07 0.06 0.07 0.07

Double plot of data from The linear will allow one to and Vmax.

Part E: Table 13 – Tabulation of the Absorbance in a Two-Minute Time Interval under Wavelength of 470 nm of Part E with Different Temperatures to Observe the Effects of the Temperature on the Rate of Abs470 nm Increase

Temperature of Reaction (C°)

Time (sec) 0 20

4

26

37

66

0 0.07

0 0.08

0 0.05

0.02 0.03

40 60 80 100 120 140 160 180

0.15 0.19 0.25 0.29 0.33 0.36 0.38 0.39

0.16 0.22 0.27 0.31 0.35 0.38 0.4 0.43

0.13 0.19 0.25 0.3 0.35 0.38 0.4 0.42

0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03

ν0 (µmol/min) of tetraguiacol formation 0 Initial rate was estimated as the slope 0.01 of the linear line that passed through the first three data points in each set 0.01 of measurements. 0.03 0.03 4 C°  25.80 0.04 26 C°  27.52 0.05 37 C°  22.36 0.05 66 C°  1.72 0.05 100 C°  1.72 0.06

100

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Graph 6 – Absorption versus the time in seconds of the data from Table 13 for Part E plotted in this graph.

Part F: Table 14 – Tabulation of the Absorbance in a Two-Minute Time Interval under Wavelength of 470 nm of Part F with Different pH Values to Observe the Effects of the pH on the Rate of Abs470 nm Increase pH of Reaction Time (sec) 0 20

3

7

9

11

0.01 0.01

0 0.03

0.01 0.01

0.03 0.03

40 60 80 100 120 140 160 180

0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01

0.06 0.09 0.13 0.16 0.18 0.2 0.23 0.26

0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01

0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03

DI H20

ν0 (µmol/min) of tetraguiacol formation

0 Initial rate was estimated as the slope 0.02 of the linear line that passed through the first three data points in each set of 0.04 measurements. 0.06 pH 3  0.09 0.00 pH 7  0.11 10.32 pH 9  0.14 0.00 pH 11 0.16  0.00 DI H20  0.18 6.88 0.2

Δ in Abs470 nm vs. Time 0.3

0.25

Abs470 nm

0.2 pH 3 pH 7 pH 9 pH 11 DI H20

0.15

0.1

0.05

0 0

20

40

60

80

100

120

140

160

180

200

Time (s)

Graph 7 – Absorption versus the time in seconds of the data from Table 14 for Part F plotted in this graph. Part G: Table 15 – Tabulation of the Absorbance in a Two-Minute Time Interval under Wavelength of 470 nm of Part G with H2O2 Concentrations with the Presence of an Inhibitor to Observe the Effects of the Inhibitor on the Rate of Abs470 nm Increase with Different Substrate Concentrations Volumes of H2O2 (µL) Tim e (sec) 0 20 40 60 80

25

50

75

0 0.01 0.02 0.04 0.07

0 0.02 0.04 0.05 0.08

0 0.04 0.07 0.09 0.12

100

0 0.07 0.12 0.18 0.23

125

0 0.09 0.15 0.2 0.25

150

0 0.12 0.16 0.19 0.28

200

0 0.13 0.24 0.27 0.35

[Substrate0] of H 2O 2 µL 25 50 75

mM 1.47 2.94 4.41

ν0 (µmol/min) of tetraguiaco l formation 5.32 8.86 10.93

100 120 140 160 180

0.08 0.1 0.14 0.25 0.26 0.09 0.12 0.19 0.27 0.28 0.11 0.16 0.22 0.3 0.32 0.12 0.2 0.25 0.32 0.36 0.13 0.24 0.3 0.34 0.4 Slope ( ΔA /min ): (Δ A) x 60 s/min Slope = (time)

0.31 0.34 0.36 0.37 0.41

0.38 0.39 0.4 0.42 0.43

100 125 150 200

5.88 7.35 8.82 11.76

12.51 13.74 14.43 14.78

Table 16 – Calculation of Slopes of the Data from Table 15 and Graph 8. The Concentration 125 µL Has the Only Linear Format with the Slope Given in this Table Volumes of H2O2 (µL) 25 50 75 100 125 150 200

Calculations (0.13−0) x 60 s/min (180) (0.24−0) x 60 s/min (180) (0.3−0) x 60 s/min (180) (0.34−0) x 60 s/min (180) (0.4−0) x 60 s/min (180) (0.41−0) x 60 s/min (180) (0.43−0) x 60 s/min (180)

Slope (unti/min) 0.043 0.080 0.100 0.113 0.133 0.137 0.143

Δ in Abs470 nm vs. Time 0.5 0.45

Absorbance (47 nm)

0.4 0.35

25 µL 50 µL 75 µL 100 µL 125 µL 150 µL 200 µL

0.3 0.25 0.2 0.15 0.1 0.05 0

0

20

40

60

80

100

120

140

160

180

200

Time (seconds)

Graph 8 – Absorption versus the time in seconds of the data from Table 15 for Part G plotted in this graph.

Michaelis Menten Plot of Turnip Peroxidase Kinetics 16.00 14.00 12.00

ν0(µmol/min)

10.00 8.00 6.00 4.00 2.00 0.00 0.00

2.00

4.00

6.00

8.00

[H2O2]0 (mM)

10.00

12.00

14.00

Graph 9 – Michaelis-Menten plot of the initial substrate concentration versus the initial velocity of turnip peroxidase exhibited saturation Kinetics in the Presence of 80 µL of the Inhibitor Hydroxylamine that is shown in Table 15. Table 17 – Tabulation of the Reciprocal of the Initial Substrate Concentration and the Initial Velocity of Turnip Peroxidase Taken from Table 15 [Substrate0]-1 (mM-1) Without inhibitor 0.68 0.34 0.11 0.07 0.06 0.05 0.04

V0-1 (min/ µmol) With inhibitor 0.68 0.34 0.23 0.17 0.14 0.11 0.09

[Substrate0]-1 (mM-1) Without inhibitor 0.10 0.09 0.07 0.07 0.06 0.07 0.07

V0-1 (min/ µmol) With inhibitor 0.19 0.11 0.09 0.08 0.07 0.07 0.07

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Graph 10 – Double reciprocal plot of data from Table 17. The linear regression will allow one to estimate KM and Vmax for assays with or without the inhibitor hydroxylamine.

According to the graph above, the inhibitor exhibits noncompetitive characters although it is supposed to mainly have competitive characteristics. IV.

Discussion/Post Lab Questions. Part B: Following the Activity of Turnip Peroxidase. 1. Explain the control in this experiment (the what and why).  Test Tube 1 is the control for this experiment. It is used to calibrate the spectrometer. Looking back at Table 1, test tube 1 lacks H2O2 which is the substrate that is needed for the reaction to take place. Without it, the substrate will not bind to the binding cleft on the enzyme turnip peroxide that is also present in the test tube. This control tube also contains distilled water and Guaicacol that is considered a donor that donates 2 hydrogens. This tube is used for calibration since it required no reaction to occur in order to get accurate results. 2. Can you be absolutely sure that you are testing peroxidase? What would you need to do to be sure turnip peroxidase is responsible for the color change of guaiacol and not some other turnip enzyme?  No it cannot be absolutely sure that turnip peroxidase is being tested. A brown product called tetraguaiacol is formed when the donor guaiacol is oxidized (reducing agent) and the turnip peroxidase is being reduced. This can be measured in a spectrometer at a wavelength of 470 nm. 3. What volume of turnip extract (or range of volumes) yielded a linear curve for your turnip peroxidase activity?  500 μl was only tested during lab and that did not yield a linear curve. 4. How might you determine how much peroxidase is ACTUALLY in the volume of extract used to create your linear curve? Think about the experiments you might need to do. Refer to your biochemistry textbook if you need ideas.  Peroxidase concentration to create linear curve can be measured by adding a specific amount of substrate hydrogen peroxide and measuring equilibrium very quickly when reaction takes place. An experiment can be done is to vary the factor of substrate concentration in different test tubes until a specific concentration is spotted to form a linear curve (trial and error). Part C: Varying the Amount of Enzyme in the Reaction. 1. Does the reaction go to completion for any of the enzyme concentrations (absorbance reaches a maximum threshold)?  All reactions go to completion except for the 25 μl of enzyme reaction does not go to completion by slowing down the rate near the end. 2. Calculate ∆A/min over a time period where the assay is linear for each level of H2O2 tested.  The calculations and slopes are shown in Table 9 in the Results section. The linear curve values are bolded. The turnip extract was 100 μl and the slope calculated per minutes was 0.102. 3. Are all the turnip peroxidase active sites saturated in any of the reactions? Which one and how do you know?



Only the 25 μl of enzyme reaction is saturated since it started slow at a low rate and leveled off at the end. Other enzyme concentrations were not saturated because they did not level off and they were at an increasing rate of absorbance.

Part D: Varying the Amount of Substrate in the Reaction. 1. What conclusions can you draw from your data about the impact of substrate concentration on reaction rate?  According to data and graph 3 in the Results section, increasing the amount of substrate in the reaction will increase the rate of the reaction until it is fully saturated and that is when it can no longer increase the rate. This is the overall conclusion of the graph although there were some errors with some concentrations due to human error with handling calculations of distilled water and pipetting techniques. 2. At what point does increasing the substrate concentration no longer have an effect on the reaction rate? Why is this so?  Increasing substrate concentration no longer has an effect on reaction rate when it reaches the maximum threshold. When this happens, the turnip peroxidase active site is fully saturated meaning that there no more active sites for the hydrogen peroxide substrate to bind and therefore the reaction rate levels off and stay constant. 3. Using the Michaelis-Menten curve you created, what is the Vmax? Wh...