Utilising the provided class data generate the following graphs: I) Michaelis Menten; II) Lineweaver-Burk; and III) Hanes-Woolf. Ensure that you clearly label each graph,and add the relevant trendlines with equations. Table 1: Class data demonstrating the Absorbance at 700nm obtained for the alkaline phosphatase enzyme reaction
Enzyme kinetics
In biochemistry, enzymes are proteins that act as biological catalysts. Catalysis is the addition of a catalyst to a chemical reaction to speed up the pace of the reaction. Catalysis can be categorized as either homogeneous or heterogeneous, depending on whether the catalysts are distributed in the same phase as that of the reactants. Enzymes are an essential part of the cell because, without them, many organic processes would slow down and thus will affect the processes that are important for cell survival and sustenance.
Regulation of Enzymes
A substance that acts as a catalyst to regulate the reaction rate in the living organism's metabolic pathways without itself getting altered is an enzyme. Most of the biological reactions and metabolic pathways in the living systems are carried out by enzymes. They are specific for their works and work in particular conditions. It maintains the best possible rate of reaction in the most stable state. The enzymes have distinct properties as they can proceed with the reaction in any direction, their particular binding sites, pH specificity, temperature specificity required in very few amounts.
Utilising the provided class data generate the following graphs: I) Michaelis Menten; II) Lineweaver-Burk; and III) Hanes-Woolf. Ensure that you clearly label each graph,and add the relevant trendlines with equations.
Table 1: Class data demonstrating the Absorbance at 700nm obtained for the alkaline phosphatase enzyme reaction
Table 1
| tube | Abs700mm |
| 1 | 0.000 |
| 2 | 0.060 |
| 2 | 0.090 |
| 4 | 0.140 |
| 5 | 0.190 |
| 6 | 0.250 |
| 7 | 0.290 |
The equipment we used are
• 20mM Tris Buffer pH 8.5
• 33mM MgCl2
• Alkaline Phosphatase (2mg/ml) in
20mM Tris Buffer pH 8.5
• 4mM Glucose-1-phosphate
• Acid Molybdate pH 5.0
• Reducing Agent
• Distilled Water
• Glass Test tubes
• Tube Rack
• Cuvette
• Pipettes and Tips
• Water bath set to 37oC
The method we used is
Method/Protocol:
1. Read the protocol in its entirety before starting. Take note of any additional information that appears
in subsequent steps that may influence how previous steps are performed.
2. Using glass tubes, generate the reactions mixtures as shown in Table 1. Important: Add the enzyme
last, and do not forget to label your tubes
a. Accurate pipetting is essential if you are to obtain good data. If unsure of your pipetting
technique, ask a demonstrator to show you.
3. With a gloved hand and using your thumb to seal the top of the tube, mix the contents of the tube by
inversion.
Table 1. Volumes (in ml) of reagents required to assemble the enzyme reactions
Tubes 1 to 7 below
| Reagent | 1 | 2 | 3 | 4 | 5 | 6 | 7 |
| 200mM Tris Buffer pH 8.5 | 0.1 | 0.1 | 0.1 | 0.1 | 0.1 | 0.1 | 0.1 |
| 33mM MgCl2 | 0.1 | 0.1 | 0.1 | 0.1 | 0.1 | 0.1 | 0.1 |
| Distilled water | 3.6 | 3.5 | 3.5 | 3.2 | 2.8 | 2.0 | 1.2 |
| Alkalaine Phosphatase (2.0mg/ml) | 0.2 | 0.2 | 0.2 | 0.2 | 0.2 | 0.2 | 0.2 |
| Total volume | 4 | 3.9 | 3.8 | 3.6 | 3.2 | 2.4 | 16 |
4. Incubate the tubes at 37oC for 10 minutes
5. Taking careful note of the instruction in step 7, add the substrate Glucose-1-phosphate (G-1-P) as
shown in Table 2
Table 2. Volumes (in ml) of 4mM glucose-1-phosphate to be added to the reactions assembled using
the information provided in Table 1
tubes below from 1 to 7
| Reagent | 1 | 2 | 3 | 4 | 5 | 6 | 7 |
| 4mM Glucose-1Phosphate | 0 | 0.1 | 0.3 | 0.4 | 0.8 | 1.6 | 2.4 |
| total volume | 4 | 4 | 4 | 4 | 4 | 4 | 4 |
mix the contents of the tube by inversion.
7. Incubate the tubes for exactly 30 minutes at 37oC. To achieve this, you will need to stagger the start
of each reaction – 30 second intervals should be sufficient
8. During the 30-minute incubation period, use another set of glass test tubes to make seven “stop”
tubes, each containing 3.8ml of distilled water and 0.5ml of acid molybdate. Label the tubes 1 -7 to
correspond with your seven reaction tubes.
9.Switch on thespectrophotometer, setwavelengthto 700nm
seven reaction tubes and add to the corresponding seven “stop” tubes.
a. Note: it is essential to stagger these additions at 30 second intervals in the same order
that you started the reactions. Therefore, each reaction tube will be stopped at exactly
30 minutes.
11. Add 0.2ml of reducing agent to each “stop” tube to enable the chromogenic reaction. With a gloved
hand and using your thumb to seal the top of the tube, mix the contents of the tube by inversion. The
final volume in these tubes will now be 5ml.
12. Allow reactions to stand at room temperature for 10 minutes.
13. Use a single cuvette to measure the absorbance of each chromogenic reaction at 700nm.
a. Transfer 2ml of Tube 1 to a cuvette and zero the spectrophotometer
b. Return the contents to tube 1, tap out excess from the cuvette onto paper towel
c. Transfer 2ml of Tube 2 to the cuvette and measure absorbance. Return the contents to
tube 2 and tap out excess onto paper towel
d. Repeat step c to measure the absorbance of the remaining reactions.
14. Tidy away the reagents and equipment you have used and clean down the bench area
Results and Data Analysis:
Complete Table 3 by calculating the substrate concentration ([S] mM) per tube and recording the
700nm absorbance for each reaction. Assuming the fixed 30-minute end-point assay is a good proxy for
the
| table | Abs 700nm |
| 1 | 0.000 |
| 2 | 0.060 |
| 3 | 0.090 |
| 4 | 0.140 |
| 5 | 0.190 |
| 6 | 0.250 |
| 7 | 0.290 |
determine the values of V and Km by generating Excel plots of:
1. Michaelis Menten plot: Absorbance (V) vs. substrate concentration [S]
a. Select logarithmic trendline and forecast by 8 periods
2. Lineweaver-Burk plot: 1/V vs. 1/[S]
a. Fit an appropriate trendline
3. Hanes-Woolf plot: [S]/V vs. [S]
a. Fit an appropriate trendline
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