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Lineweaver-Burk Plot from Absorbance Data

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I carried out a practical last week and need to write it up but canot remember how to do the calculations from the Michaelis-Menten graph plotted which I guess helps me to plot a Lineweaver-Burk.
I have attached the practical requirements.
I have to produce a table (attached) but cannot fill in certain columns until I can work out calculations. Could someone please show me how to calculate using one example so I can complete table and write my practical?

I had 12 test tubes the 1st six were control second six had inhibitor added. The substrate concentration in each six test tubes were (for first six, then following six) 0.1mM, 0.2mM, 0.4mM, 0.8mM 1.6mM and 2.4mM. Absorbance was measured at 700nm and recorded in order as follows: 1st six - 0.038, 0.064, 0.094, 0.206, 0.199 and 0.238 2nd set was 0.039, 0.067, 0.064, 0.110, 0.119 and 0.230.

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Solution Summary

The calculations and graphs required for Lineweaver-Burk plots for enzyme data with and without inhibitor are shown.

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Experiment 1: Effect of Temperature on Amylase Activity
The data for this activity are in Table 1.
Table 1. V of the enzyme amylase at different temperatures, [S] and pH
Environ-mental Conditions: pH = 7
[S] = 90 mM pH = 7
[S] = 60 mM pH = 7
[S]=120 mM pH = 5
[S] = 90 mM pH = 9
[S] = 90 mM
Temper-ature (ºC) V (mM/min) V (mM/min) V (mM/min) V (mM/min) V (mM/min)
15 0.00096 0.00089 0.00095 0.00124 0.00060
30 0.00158 0.00137 0.00161 0.00205 0.00097
32 0.00222 0.00200 0.00230 0.00300 0.00138
35 0.00278 0.00245 0.00288 0.00357 0.00163
37 0.00300 0.00275 0.00311 0.00395 0.00180
39 0.00293 0.00261 0.00300 0.00375 0.00177
41 0.00267 0.00256 0.00283 0.00346 0.00162
43 0.00241 0.00231 0.00245 0.00306 0.00146
45 0.00212 0.00176 0.00209 0.00272 0.00123
48 0.00171 0.00147 0.00167 0.00224 0.00100
50 0.00131 0.00120 0.00138 0.00165 0.00078
55 0.00094 0.00090 0.00104 0.00121 0.00055

9. Develop a hypothesis to predict the effect of an increase in temperature on amylase activity. Test your hypothesis using the data provided in Table 1. State your hypothesis.

10. Using the data in Table 1.
a. What is the optimal temperature for amylase activity? __________

b. Would amylase isolated from any two organisms (for example, fish amylase vs. amylase from the small intestine of humans) show the same temperature optimum? Explain your answer.

11. Explain why a temperature lower or higher than the optimum would cause decreases in amylase activity. (What is happening to the enzyme to produce these decreases in activity?)
12. If you were to carry out these temperature experiments at a higher or lower [S], what effect would [S] have on the temperature optimum for amylase? Formulate a hypothesis. Using the data from Table 1, test your hypothesis. Explain your results. (Note: Do not submit your plotted graph.)
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Experiment 2: Effect of Substrate Concentration on Amylase Activity
The data for this experiment are in Table 2.
Table 2. V (mM/min) at different [S] (mM) of the enzyme amylase at 37ºC and pH = 7.0
[S] (mM) V (mM/min) [S] (mM) V (mM/min)
0.0 0 140 0.00432
2.5 0.00044 160 0.00433
5 0.00102 180 0.00432
10 0.00161 200 0.00464
20 0.00223 220 0.00447
30 0.00277 240 0.00455
40 0.00313 260 0.00454
50 0.00349 280 0.00469
60 0.00363 300 0.00472
70 0.00388 320 0.00467
80 0.00383 340 0.00471
90 0.00395 360 0.00487
100 0.00401 380 0.00477
120 0.00429 400 0.00473

14. Using the data in Table 2, examine the relationship between [S] and amylase activity. To answer the questions, plot the data: V vs [S] and 1/V vs 1/[S] (Lineweaver-Burk). (Note: Do not submit your plotted graphs.)
a. Explain the relationship and your results.
What are the Vmax and KM for amylase for this experiment?
15. Compare the values for Vmax and KM derived from each of the two plots (V vs [S] and Lineweaver-Burke).
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b. If the values were different, explain possible reasons for these differences.

c. Which plot did you find easier to use for determining these values?

16. What do you think would happen if you carried out an experiment with concentrations of starch greater than 400 mM?

17. Briefly compare the effects of a competitive versus a non-competitive inhibitor on an amylase-catalyzed reaction i.e. how would these inhibitors respectively affect KM and Vmax?

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