The Effect of temperature on the enzyme β-Glucosidase
To investigate the effect of temperature on the enzymatic reaction you will need to set up an incubation mixture and blank for each of the temperatures. The selected temperatures are 4oC, 20oC, 37oC, 56oC, 65oC and 75oC (100oC boiling water results will be supplied). You will study THREE of the 6 temperatures in your group. You must work with another group to get a complete data set for the practical.
Before adding the enzyme allow the tubes to reach bath temperature and also bring the enzyme to the same temperature.
1. For each temperature set up three tubes, (two labelled ‘T (for test) + appropriate temperature' e.g. T37) are duplicates, containing 3 ml of 0.1 M salicin and 1 ml of buffer and the other (the blank, labelled ‘B + appropriate temperature' e.g. B37) containing 4 ml of buffer. Remember your initials on each tube.
2. Allow the tubes to reach bath temperature (10 mins) and add 1 ml of enzyme to all the tubes.
3. Mix and incubate for exactly 15 mins, then stop the reaction and develop the colour by adding 2 ml of 4-aminophenazone solution to each tube.
4. Add 1 ml of potassium ferricyanide to each tube, mix well and allow to stand for 10 mins for colour stabilisation.
5. Use the blank tube for the appropriate temperature to zero the spectrophotometer. Read the absorbance for each experimental tube at 515 nm.
6. Repeat steps 2-5 for each temperature.Treatment of data
1. Tabulate your data in an appropriate table below.
|
|
4°C
|
20°C
|
37°C
|
56°C
|
65°C
|
75°C
|
100°C
|
|
|
0.311
|
0.372
|
0.362
|
0.883
|
0.566
|
0.210
|
|
|
|
0.289
|
0.486
|
0.494
|
0.821
|
0.665
|
0.185
|
|
|
|
0.261
|
0.555
|
0.484
|
0.317
|
0.401
|
0.130
|
|
|
|
0.328
|
0.663
|
0.513
|
0.491
|
0.268
|
0.115
|
|
|
|
0.362
|
0.423
|
0.667
|
1.002
|
0.509
|
0.260
|
|
|
|
0.379
|
0.391
|
0.470
|
0.881
|
0.304
|
0.221
|
|
|
|
0.084
|
0.545
|
0.636
|
0.373
|
0.457
|
0.242
|
|
|
|
0.471
|
0.494
|
0.616
|
0.430
|
0.277
|
0.332
|
|
|
|
0.150
|
0.246
|
0.242
|
0.579
|
0.414
|
0.346
|
|
|
|
0.291
|
0.500
|
0.201
|
0.496
|
0.374
|
0.204
|
|
|
|
0.329
|
0.376
|
0.371
|
0.580
|
0.294
|
0.073
|
|
|
|
0.343
|
0.388
|
0.360
|
0.532
|
0.225
|
0.093
|
|
|
|
0.278
|
0.384
|
0.795
|
1.174
|
0.299
|
0.499
|
|
|
|
0.148
|
0.268
|
0.694
|
1.158
|
0.380
|
0.234
|
|
|
|
0.350
|
0.309
|
0.594
|
0.856
|
0.717
|
0.275
|
|
|
|
0.351
|
0.668
|
0.671
|
0.561
|
0.580
|
0.342
|
|
|
|
0.205
|
0.998
|
0.407
|
0.230
|
0.216
|
0.096
|
|
|
|
0.044
|
1.132
|
0.153
|
0.801
|
0.234
|
0.159
|
|
|
|
0.449
|
0.452
|
0.328
|
1.480
|
0.640
|
0.244
|
|
|
|
0.453
|
0.477
|
0.625
|
1.376
|
0.455
|
0.184
|
|
|
|
0.230
|
0.391
|
0.208
|
1.318
|
0.115
|
0.148
|
|
|
|
0.223
|
0.379
|
0.178
|
0.862
|
0.304
|
0.375
|
|
|
|
0.160
|
0.433
|
0.669
|
0.759
|
0.775
|
0.355
|
|
|
|
0.305
|
0.501
|
0.746
|
0.453
|
0.777
|
0.255
|
|
|
|
0.174
|
0.543
|
0.733
|
0.883
|
0.566
|
0.210
|
|
|
|
0.353
|
0.288
|
0.681
|
0.821
|
0.665
|
0.185
|
|
|
Mean
|
0.282
|
0.486
|
0.496
|
0.771
|
0.434
|
0.239
|
0.126
|
2. Plot the absorbance value PER MINUTE against temperature, comment on the shapes of the graph, indicate the optimum temperature.
3. A convenient expression for the relationship between the rate of reaction and temperature in the Q10 value. This is the ratio of the velocity of the reaction at the temperature of T + 10oC to its velocity at ToC, taken from the range where there is an increase in absorbance as the temperature increases (e.g. compare the absorbance/min values for 25oC and 15oC). The Q10 value for enzyme catalysed reactions usually fall within the range 1.5-2.5 while those for non-enzymatic reactions are usually in the range 2.0-4.0. It is useful to know the Q10 value for a given enzyme catalysed reaction if the activity is to be measured at a temperature other than the normal physiological temperature.
4. Using the maximum absorbance reading observed convert the reading into a rate of reaction (units of product (nM) / minute / mg of protein), (nM/min/mg). If the concentration of protein in your reaction is 2 mg/ml
Using Beers Law; Abs = cl
Where is the extinction coefficient; c = concentration of compound; l = the pathlength. The absorbance of quinoneimine chromogen assay product in a 1 cm path length cell at A515 is 12.2 mM-1 cm-1.
5. How does temperature affect enzyme activity?
As the temperature of a reaction increases from 0°C the enzyme will become active. The enzyme will be most active at its temperature. After this point the rate of reaction will decrease and eventually no activity will be observed, this is due to the enzyme becoming .
7. What is the optimum temperature for the ß-glucosidase used in the experiment (Using the graph)
8. What is the Q10 value for your reaction? (Use values at 30°C and 40°C)
9. What is the rate of reaction at the optimum temperature? (The starting concentration of the protein was 2mg/ml, the Extinction coefficient (ε) of the chromogenic product is A515 = 12.2mM-1cm-1