Download Enzyme Kinetics: Substrate Concentration and Inhibitor Effects on Turnip Peroxidase and more Study Guides, Projects, Research Laboratory Practices and Management in PDF only on Docsity!
Enzyme Kinetics lab report Name: Kushani Arachchillage Date:11.30.
Pre-Laboratory
A. What scientific concept(s) will be learned in this lab activity? The scientific concept(s), we are going to learn in this lab are the concept of Michaelis- Menten equation and the Lineweaver-Burke (double reciprocal) plot. Further we are going to identify the type of inhibitor through the double reciprocal plot. B. What laboratory techniques will be learned in this activity? In this lab, we will learn the technique to measure the absorbance at a certain wave length(470nm) using a spectrometer, to calculate the rate of reaction, to analyze the data and draw the graph by using the analyzed data. C. What is the objective (or goal) for the lab? The goal for this lab is to observe the effects of substrate concentration, and the presence of inhibitors on the rate of reactions. There by optimize the experimental conditions to generate meaningful kinetics data. Other objectives are created and interpret a Michaelis-Menten plot, create and interpret a Lineweaver-Burke plot and identify the type of inhibitor used based on the kinetic data.
Part B
1. Materials for part B
Reagents
- Guaiacol (0.22%) - Indicator for peroxidase activity.
- Hydrogen Peroxide (H₂O₂, 1%) - Substrate for the enzymatic reaction.
- Hydroxylamine (10%) - Used as an inhibitor in experimental tubes (1E, 2E).
- Distilled Water - For dilutions and adjusting volumes. Equipment
- Spectrophotometer - Set at 470 nm to measure absorbance over time.
- Pipettors (P10, P20, P200, P1000) - For accurate liquid handling during preparation.
- Serological Pipette with Pump or Bulb - For transferring larger volumes. Glassware and Other Tools
- Test Tubes - For preparing reaction mixtures (1B, 1E, 2B, 2E).
- Parafilm - To seal the tubes during mixing.
- Test Tube Rack - To organize tubes during the experiment.
2. Method for Part B
- Labeling and Preparation of Tubes o Label the tubes as 1B, 1E, 2B, and 2E for the blanks and experimental conditions, respectively. o Prepare the tubes according to the setup in the provided table.: 1B: 2395 μL water, 30 μL guaiacol, 400 μL turnip extract, 60 μL H₂O₂ (total 3000 μL). 1E: 2335 μL water, 30 μL guaiacol, 400 μL turnip extract, 175 μL hydroxylamine, 60 μL H₂O₂ (total 3000 μL). 2B: 2440 μL water, 30 μL guaiacol, 400 μL turnip extract, 60 μL H₂O₂ (total 3000 μL). 2E: 2335 μL water, 30 μL guaiacol, 400 μL turnip extract, 130 μL hydroxylamine, 60 μL H₂O₂ (total 3000 μL).
3. Result for Part B
Experimental Setup Table
Table 1 tubes Water(μl) Guaiacol (μl) turnip (μl) Hydroxyla mine (μl) H2O2 (μl) Total volume (μl) 1B 2395 30 400 - 60 3000 1E 2335 30 400 175 60 3000 2B 2440 30 400 - 60 3000 2E 2335 30 400 130 60 3000
Measured results with calculations
Reaction rate was calculated as, Where: R is the reaction rate (change in absorbance over time), ΔAbsorbance is the change in absorbance between two time points, Δt is the time interval (in seconds). Table 2: tube 1 Time(s) Absorbance Reaction rate (ΔA/Δ t) 10 0.88 - 20 0.90 0. 30 0.89 -0. 40 0.88 -0. 50 0.89 0. 60 0.88 -0. 70 0.87 -0. 80 0.87 0
Time(s) Absorbance Reaction rate (Δ Abs/Δ t)
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, we cannot be completely certain that turnip peroxidase is the only enzyme responsible for the oxidation of guaiacol without additional tests. To confirm this, the following steps can be taken
- Use a specific inhibitor: Introduce a known peroxidase inhibitor to determine whether the reaction is halted. If the reaction stops, it strongly indicates peroxidase activity. Hydroxylamine, for instance, is often used as a competitive inhibitor for peroxidase. 2. Enzyme Purification: Extract and then purify turnip peroxidase by column chromatography. Assay of the purified enzyme against guaiacol will identify peroxidase as the active enzyme if a reaction occurs**.
- Control:** Set up a tube without the addition of turnip extract, adding all other reagents. If no color change is seen, it may indicate that the activity is due to components in the turnip extract**.
- Specificity of substrates used:** The reaction should be conducted only with the use of substrates identified as specific for peroxidases, such as o-dianisidine or ABTS to ensure that the activity profiled corresponds to a true peroxidase.
- Western blot: Perform a Western blot using the antibodies against turnip peroxidase to verify its presence and activity.
3. What volume of turnip extract (or range of volumes) yielded a linear curve
for your turnip peroxidase activity?
Based on the experimental results, the volume range of 30 μL to 400 μL of turnip extract produced a linear absorbance vs. time curve. This range represents the enzyme concentration where the reaction progresses steadily, and the active sites are not saturated by the substrate. Within this range, the enzyme's activity increases proportionally with the substrate concentration, allowing accurate measurement of the reaction rate.
4. What volume of turnip extract will you use for the remaining sections of this
lab exercise, and why did you select this volume?
For the remaining sections, a volume of 400 μL of turnip extract will be used. This volume was selected because: It lies within the range that produces a linear curve, indicating that the enzyme concentration is optimal for kinetic analysis without causing substrate depletion or enzyme saturation. The reaction rates at this volume are consistent and fall within the measurable range of the spectrophotometer, ensuring reliable results. By using this volume, we can maintain reproducibility and accuracy in subsequent experiments.
5. Does the reaction go to completion (absorbance reaches a maximum
threshold) for any of the enzyme concentrations tested? If so, which
concentrations?
Yes, the reaction reaches completion for higher concentrations of turnip extract, such as 500 μL , where the absorbance levels off over time. This plateau indicates that either all substrate (H₂O₂ and guaiacol) is consumed or the reaction reaches equilibrium, where the rate of guaiacol oxidation matches the rate of any reverse reactions or non-enzymatic degradation.
6. Are all of the turnip peroxidase active sites saturated in any of the reactions?
Which ones and how do you know?
Yes, enzyme active sites are saturated in reactions where the substrate concentration is high, such as 80 μL of H₂O₂ or when the enzyme volume exceeds 400 μL of turnip extract. Evidence of saturation can be seen in: Michaelis-Menten plot: The reaction rate (V) levels off at Vmax, indicating that further increases in substrate concentration do not enhance the reaction rate.
Use an ELISA test with antibodies specific to turnip peroxidase to quantify the enzyme concentration based on antibody binding.
- Western Blot Analysis Perform a Western blot using specific antibodies for turnip peroxidase. Measure the band intensity and compare it to known standards to estimate enzyme concentration. These approaches would provide complementary ways to accurately determine the peroxidase concentration in the crude extract, ensuring precise kinetic analysis.
Part F
1. Materials for part F
Reagents
- Guaiacol (0.22%) - Indicator for peroxidase activity.
- Hydrogen Peroxide (H₂O₂, 1%) - Substrate for the enzymatic reaction, varied in concentration for kinetic analysis.
- Hydroxylamine (10%) - Used as an inhibitor at three selected concentrations.
- Distilled Water - For dilutions and adjusting volumes.
Equipment
- Spectrophotometer - Set at 470 nm to measure absorbance over time for multiple substrate and inhibitor concentrations.
- Pipettors (P10, P20, P200, P1000) - For precise measurement of liquids.
- Serological Pipette with Pump or Bulb - For transferring larger volumes. Glassware and Other Tools
- Test Tubes - For preparing and mixing reaction mixtures at different substrate and hydroxylamine concentrations.
- Parafilm - To seal tubes during mixing to ensure homogeneity.
- Test Tube Rack - To organize the experimental setups.
2. Method for Part F
- Hydroxylamine Concentration Selection o Using the data from Part B, select three hydroxylamine concentrations: One that results in 50% inhibition of the baseline rate. One below 50% inhibition. One above 50% inhibition. o Label these as Hydroxylamine 1 (low), Hydroxylamine 2 (50%), and Hydroxylamine 3 (high).
- Tube Preparation o Prepare a total of nine experimental tubes and their corresponding blanks according to the table: Hydroxylamine 1: Prepare tubes with increasing H₂O₂ concentrations (e.g., 40 μL, 60 μL, 80 μL).
o Lineweaver-Burke Plot: Use the reciprocal values of reaction rates (1/v) and substrate concentrations (1/[S]) to create a Lineweaver-Burke plot. Analyze the plot to determine Km and Vmax.
- Disposal o Dispose of all hydroxylamine-containing solutions in the designated waste container. Ensure no chemicals are discarded into the sink.
3. Result for Part F
Experimental Setup Tables
Table 4 tubes Water (μl) turnip (μl) Guaiacol (μl) Hydroxylam ine (μl) H2O2 (μl) Total volume (μl) 1 2420 400 30 110 40 3000 2 2400 400 30 110 60 3000 3 2380 400 30 110 80 3000 4 2400 400 30 130 40 3000 5 2380 400 30 130 60 3000 6 2360 400 30 130 80 3000 7 2425 400 30 175 40 3000 8 2335 400 30 175 60 3000 9 2315 400 30 175 80 3000 Measured results with calculations Total Volume = Water + Turnip Extract + Guaiacol + Hydroxylamine + H 2 O 2
Measured Results and Calculations
Table 5: Tube 1 Time(s) Absorbance Reaction rate (Δ A/ Δ t) 10 0.72 -
Time(s) Absorbance Reaction rate (Δ A/ Δ t)
Time(s) Absorbance Reaction rate (Δ A/ Δ t)
Time(s) Absorbance Rate of reaction (Δ A/ Δ t) Time(s) Absorbance Rate of reaction (Δ A/ Δ t)
Time(s) Absorbance Rate of reaction (Δ A/ Δ t)
- 90 0.86 -0.
- 100 0.85 -0.
- 110 0.85
- 120 0.84 -0.
- 130 0.83 -0.
- 140 0.83
- 150 0.82 -0.
- 160 0.81 -0.
- 170 0.80 -0.
- 180 0.79 -0.
- Table 3 : tube
- 20 0.16 0. 10 0.1 -
- 30 0.23 0.
- 40 0.28 0.
- 50 0.32 0.
- 60 0.35 0.
- 70 0.38 0.
- 80 0.40 0.
- 90 0.41 0.
- 100 0.43 0.
- 110 0.44 0.
- 120 0.45 0.
- 130 0.46 0.
- 140 0.46
- 150 0.46
- 160 0.47 0.
- 170 0.47
- 180 0.47
- 20 0.72
- 30 0.719 -0.
- 40 0.71 -0.
- 50 0.71
- 60 0.71
- 70 0.71
- 80 0.70 -0.
- 90 0.70
- 100 0.70
- 110 0.69 -0.
- 120 0.69
- Table 6: Tube
- 20 0.89 0. 10 0.80 -
- 30 0.94 0.
- 40 0.97 0.
- 50 1.0 0.
- 60 1.0
- 70 1.0
- 80 1.02 0.
- 90 1.01 -0.
- 100 1.002 -0.
- 110 0.999 -0.
- 120 0.997 -0.
- Table 7: Tube
- 20 0.87 0. 10 0.77 -
- 30 0.92 0.
- 40 0.96 0.
- 50 0.98 0.
- 60 0.99 0.
- 70 0.99
- 80 0.98 -0.
- 90 0.98
- 100 0.98
- 110 0.97 -0.
- 120 0.96 -0.
- Table 8: Tube
- 20 0.70 0. 10 0.61 -
- 30 0.75 0.
- 40 0.78 0.
- 50 0.81 0.
- 60 0.81
- 70 0.82 0.
- 80 0.818 -0.
- 90 0.811 -0.
- 100 0.80 -0.
- 110 0.79 -0.
- 120 0.79
- Table 9: Tube Tube 5 (Table 3)
- 20 1.3 0. 10 1.1 -
- 30 1.39 0.
- 40 1.42 0.
- 50 1.45 0.
- 60 1.46 0.
- 70 1.46
- 80 1.45 -0.
- 90 1.44 -0.
- 100 1.44
- 110 1.44
- 120 1.43 -0.
- Table 10: tube
- 20 0.84 0. 10 0.74 -
- 30 0.92 0.
- 40 0.97 0.
- 50 1.0 0.
- 60 1.0
- 70 1.02 0.
- 80 1.02
- 90 1.02
- 100 1.02
- 110 1.03 0.
- 120 1.01 -0.
- Table 11: tube
- 20 0.76 0. 10 0.55 -
- 30 0.85 0.
- 40 0.92 0.
- 50 0.95 0.
- 60 0.98 0.
- 70 1.0 0.
- 80 0.99 -0.
- 90 0.99
- 100 0.98 -0.
- 110 0.97 -0.
- 120 0.97
- Table 12: tube
- 20 0.88 0. 10 0.75 -
- 30 0.93 0.
- 40 0.98 0.
- 50 1.0 0.
- 60 1.01 0.
- 70 1.01
- 80 1.00 -0.
- 90 1.00
- 100 0.99 -0.
- 110 0.99
- 120 0.99
Where v: Reaction rate (μmol/min). [S]: Substrate concentration (M). Vmax: Maximum reaction rate (μmol/min). KM: Michaelis constant (M), the substrate concentration at half Vmax Graph: X-axis is substrate concentration ([S]), and Y-axis is the reaction rate (v).
Lineweaver-Burke Graph
Where: 1/v: Inverse reaction rate (min/μmol). 1/[S]: Inverse substrate concentration (1/M). Slope: KM/Vmax Y-intercept: 1/Vmax Graph: X-axis is 1/[S], and Y-axis is 1/v
4. Post lab questions for Part F
1. Explain the action of hydroxylamine on the enzyme's activity.
Hydroxylamine inhibits the activity of turnip peroxidase by interfering with the enzyme's ability to catalyze the oxidation of guaiacol. More precisely, hydroxylamine competes with hydrogen peroxide (H₂O₂), the natural substrate of peroxidase, for binding at the active site of
