Instructions (because of difficulties we are experiencing with the simulation, expect this guide to be modified)
EnzymeLAB (available at https://learntech.imsu.ox.ac.uk/enzlab/)
- Simulate the behaviour of an enzyme over a range of substrate concentrations, at various pH values, and in the presence/absence of an inhibitor.
- Simulated enzymes obey Michaelis-Menten kinetics, as described above.
- Faster than a real time enzyme assay so it allows you to spend time designing your experiment and try our lots of different setting.
- Note: You must include the enzyme reference number in your report.
- It mimics real life! You need to eat between enzyme assay, and have tea (The simulation is from University of Oxford!)
First you need to choose a sensible enzyme volume. Did you get the specific activity (mmolmin-1mg-1) and the enzyme concentration (mgmL-1). You know the maximum rate measurable is 10 µmolmin-1. This should enable you to calculate the enzyme volume needed.Next! Substrate concentration – Hint! start with high substrate concentration at pH 7, so the enzyme will be near saturated and this gives an estimate of VmaxFinding a rough value for the optimum pH –plot activity versus pH.Finding Km and Vmax – take values of [S] varying by at least 10 fold, until rate drops below 0.5×Vmax. You will need about half the rates below 0.5×Vmax and half above.
- Find optimal enzyme amount (once set this will not need to be changed)
- Figure out a sensible substrate range (how are you planning this range?)
- Now you are ready to determine Km and Vmax
- The accuracy will depend on the spread of data points and number of data points.
- to get reasonable values for x (mean) and (standard deviation), need to repeat measurements at least 5 times
- Generally it is better to take values at a large number of [S] values rather than repeats at a smaller number of [S] values
- And you can check how pH affects Km and Vmax
- Determine the optimal pH
- Determine the determine Km and Vmax again at the optimal pH
- Using this link do the experiment and complete the following: you will have to use he unsecure web page to complete and use this software (advanced settings then proceed to link). https://learntech.imsu.ox.ac.uk/enzlab/
- Answer questions as you are directed, keep original formatting. The formatting to use is directly below in the table with grey, blue and red typing.
- Refer to the “BCEM2201 figure making guide” when constructing your figures, and figure caption! (below instructions on page 4)
- There are video links below to the class lectures that have some explanation also to help with the software experiment.
| 1. What is the optimal pH of the enzyme? (10 points) |
| Insert a Figure (including figure caption) here showing V0vs pH. |
| In a short paragraph, explain the process of acquiring the data set (amount of substrate used, number of trials), and its significance. |
| 2. Calculate the values of V0 at or close to Vmax and at or close to Vmax10 at the optimal pH, quoting your answers as mean +/- standard error. Comment on your answer. (10 points) Note after the lab on Sept 30, I got totally confused with this question when I tried to clarify it for you, I am sorry. The formula of course to calculate is V0 = Vmax or V0 = Vmax10 of course you do not need to use the MM equation… and the rest is to report individual values from the individual trials and the average, as seen below. |
| Insert a table here (including figure caption) showing the raw V0values from the 5 trials as well as mean of V0values +/- standard deviation. |
| In a short paragraph, explain the process of acquiring the data set (amount of substrate used, number of trials), and its significance. |
| 3. Calculate the values of Km and Vmax you obtained for this enzyme at its optimal pH or pH7, and indicate how you derived them. (20 points) |
| Insert a table here (including figure caption) showing Km and Vmax as determined by the simulation’s iterative fitting method. |
| Insert the Double reciprocal plot (Lineweaver-Burk plot, (include figure caption)) you generated from the raw dataset. |
| 4. Calculate the specific activity of this enzyme at its optimal pH or pH 7 in units of mmol product min-1 (mg of enzyme)-1, showing your working clearly. (10 points) |
| Type your answer here |
Title: Should capture the essence of the figure (experiment), Do not simply restate the axis labels with a “versus” written in between. For lab reports the figure title is part of the caption.
Caption: Should be concise but comprehensive. It should describe the data shown, draw attention to important features contained within the figure, and may sometimes also include interpretations of the data.
Labels: use to label different plots on the same graph. Only use if it is needed to aid the interpretation of the figure.
Axis titles: describe the dependent and independent variable of the experiment. Do not forget ti have the units included.
In a very plain language, a good figure Caption answers the following questions with one short sentence each.
What did you measure?
How did you measure it, what was the methodology?
What did you get, summarise the main results of the figure?
What does it mean, explain results if necessary?
Keep in mind every figure should “stand on its own” itmust be sufficiently clear, well-labelled, and described by its caption to be understood by your intended audience without reading the lab write up.
Questions frequently arise about how much methodology and what results to include in the caption. Figures are part of lab reports ( posters, manuscripts). Detailed methodology will be described in the Methods section. Specific results will be reported in the results text with a reference to the applicable Table or Figure. For BCEM 2201 worksheets imagine that your figures belong in a full lab report and report only the main result of the figure in the caption.
How to fit the linear part of the calibration curve on excel
When you make a calibration curve, sometimes towards the higher concentrations the curve will plateau. that part should be omitted when you find the line of best fit, but still, it should be presented on the figure.
Here is how you can manage to fit only the datapoint on the linear region with the line of best fit:
The set highlighted represents the orange points on the figure, and it was generated by “doubling up the A measurement of the linear points. Now the orange dot can be fitted with a linear threadline.
Make sure when you add the threadline you set intercept as 0 (this step of course assumes when you measured the A of your standard you used the blank appropriately and zeroed the spec.) If your line is not trending toward zero, it is an indication not blinking properly, or of a wrongly prepared blank.
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