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vicster vicster
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10 years ago
Set up an experiment at 50° C with a buffer pH of 4.0 and a [ S ] of 0.0 mM. Add enzyme. Find the slope of the line and record data for this first measurement. Continue to take measurements by carrying out separate experiments with increasing concentrations of [ S ]. Take a measurement at 2.5 mM, 5 mM, and 10 mM, then continue in 10 mM increments (e.g., 20 mM, 30 mM, 40 mM, 50 mM, 60 mM) until you have reached a [ S ] of 400 mM. Find the slope and record data after each measurement. Once you have finished these measurements, click the Plot Data tab.

Follow the directions below to use these data to create some of the plots described in assignment 1. Each plot is selected from the popup menu for Plot Type. Refer to these directions if necessary when creating plots for other experiments that you are working on.

Plotting VO vs. [ S ]: Click in the Title box and type in the title "Experiment 1 —

Substrate Concentration." VO vs. [ S ] should appear as the default plot in the Plot Type box. Shift-click on each row of measurements from 0 to 100, then click the Plot Selected Data button to produce a plot of VO versus [ S ].

Before you can determine Vmax and KM from this plot, you must first find the best-fit line for these data points. Click on the black KM arrow at the x-axis. Slide this arrow until you have found the best-fit line for these points. Adjust this line as needed by clicking and sliding the black Vmax arrow at the y-axis. A numerical value for Vmax and KM will appear in the Vmax and KM text boxes.

Setting Up a Lineweaver—Burk Plot: Return to the Data view and plot your data as a Lineweaver—Burk plot. Find the best-fit line for these data points by clicking and sliding the arrows at the x-axis and y-axis. The arrow at the intersect of the black plotted line and the y-axis indicates 1/Vmax. Notice that a value for 1/Vmax will appear in the 1/Vmax text box beneath the curve box. The arrow at the intersect of the plotted line and the x-axis determines —1/[ S ].

For a Lineweaver—Burk plot, Vmax and KM will not be calculated for you; you must calculate these values yourself. Based on the values you determined for 1/Vmax and —1/[ S ] calculate Vmax and KM from your plot. For your convenience a calculator can be accessed by clicking the Calculator tab at the top of the screen.

Setting Up an Eadie—Hofstee Plot: Return to the Data view and plot your data as an Eadie—Hofstee plot. Find the best-fit line for these data points by clicking and sliding the arrows at the x-axis and y-axis. The black arrow at the intersect of the black plotted line and the y-axis indicates Vmax. Notice that a value for Vmax will appear in the Vmax text box. The arrow at the intersect of the plotted line and the x-axis indicates Vmax/KM.

For an Eadie—Hofstee plot, KM will not be calculated for you. Calculate KM from your plot. For your convenience, a calculator can be accessed by clicking the Calculator tab at the top of the screen.

What was the relationship between [ S ] and invertase activity? Was this relationship what you expected? Why or why not? Explain your answers. What was the Vmax and KM for invertase for this experiment? What do these values tell you about invertase and its affinity for sucrose as its substrate?

How did the values for Vmax and KM derived from each of the three plots compare? Were these values similar or different? If they were different, explain possible reasons for these differences. Which plot did you find easier to use for determining these values?

What do you think would happen if you carried out an experiment with concentrations of sucrose greater than 400 mM? Formulate a hypothesis, then carry out an experiment to test your hypothesis. What did you observe? Describe your results.
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