Apparatus
0.5 % starch buffered to pH 6.7
1% NaCl solution
Fearons reagent
20% NaOH solution
pH 6.7 buffer
Amylase solution
Distilled water
Tongs
Boiling tubes
Boiling tube rack
100 cm3 measuring cylinder
1, 5 and 10 cm3 pipettes and safety pumps
Gloves
Goggles
Cuvettes
Lab Coat
Stop clock
Colorimeter
Ice Bath (0 oc), 21oc , 30 oc, 40 oc, 50 oc, 60 oc, 70 oc water baths
Bunsen burner
Tripod
Gauze
Large Beaker (500ml)
Heatproof Mat
Method: -
- Put lab coat, goggles and gloves on.
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Prepare 7 tubes of starch (labelled S1 to S7) each containing the following: - 5cm3 of buffered starch, 2cm3 of pH 6.7 buffer, 1cm3 of 1% NaCl
-
Using a pipette, measure out 5cm3 of the amylase solution into 7 separate tubes (labelled E1 to E7)
Allocate the tubes as follows.
Tubes S1 and E1 in to the ice bath
Tubes S2 and E2 in to the room temperature water bath
Tubes S3 and E3 in to a water bath at 30oc.
Tubes S4 and E4 in to a water bath at 40oc.
Tubes S5 and E5 in to a water bath at 50oc.
Tubes S6 and E6 in to a water bath at 60oc.
Tubes S7 and E7 in to a water bath at 70oc.
- Allow all the tubes to equilibrate at the chosen temperature for at least 5 minutes.
- At 0 time, add the contents of E1 to tube S1 and shake to mix. Now place the tube with contents of both the tubes in to the ice bath.
- After exactly 30 seconds repeat the mixing procedure with tubes E2 and S2
- Continue the mixing process at 30-second intervals with the remaining set of tubes until all the reactants are mixed and are at the right temperatures.
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When its 5 minutes on the stopwatch add 1cm3 of the 20 % NaOH to tube number and mix the contents thoroughly. This will terminate any further reaction, and the test tube can now be removed from the ice bath.
- At exactly 30-second intervals, repeat the step above, ensuring that each mixture has had 5-minute reaction time at the chosen temperature.
- Prepare a boiling water bath by filling a large beaker to 1/3 capacity and heat to boiling with a Bunsen burner.
-
Add exactly 0.5 cm3 of Fearons reagent to each tube. Mix thoroughly and heat in the boiling water bath for 2 minutes or until a ‘redish’ colour has developed. Remove the tubes and cool to room temperature.
- Using water as a reference and a 430 filter use the colorimeter to make a quantitative colour comparison of each tube (Note: initial reading should be 0 before the sample is placed in the colorimeter). Read and note down the absorbance. (The darker the red colour that has developed the higher concentration of maltose in the solution and the greater the absorbance).
Results
Conclusion
The results obtained were plotted on a graph, which can be seen on the enclosed graph. From the graph we can conclude that our hypothesis was correct. In ice the absorbance was only 0.14 units, which means that the rate of reaction was very slow in ice as the enzyme molecules had very little kinetic energy thus there weren’t many collisions between the enzyme and the substrate, in this case between starch and amylase. As the temperature increased to room temperature 21oc, the absorbance increased to 0.28, indicating an increase in the rate of reaction. The same occurred at 30oc, an increase in temperature meant more kinetic energy, thus a higher rate of reaction, hence a high absorbance value (arbitary unit) shown on the calorimeter. However at 40oc the absorbance level was the same as that of 30oc. This is because the rate of reaction is now starting to decrease (at 400c) and an optimum temperature has already been reached. The optimum temperature is normally at 37.5oc in humans.
At 40oc, the enzymes start to denature and begin to lose their tertiary structure, thus the rate of reaction decreases.
At 50oc the amount of absorbance decreases to 0.32, indicating a further decrease in the amount of maltose produced, which shows a decrease in the rate of reaction.
At 60oc there was a sharp decline in the level of absorbance, indicating that the tertiary structure of the enzyme amylase has denatured immensely and thus the rate of reaction is very slow indeed as the substrate is no longer able to fit in to the active site of the enzyme.
At 70oc same occurs, the absorbance is the exact same (0.05) meaning that the enzyme has denature to its peak and a plateau has been reached, and thus the rate of reaction will stay constant despite of any further increases in temperature.
Although the results match the proposed hypothesis, there could have been a number of errors which might have affected the results, i.e. inaccurate measuring – a number of solutions were measured and mixed together, the measuring may not have been accurate. To make the measuring more accurate, burettes could have been used; as they are stable (held by a clamp stand and a tap also controls the flow of the solution) thus the measurement and reading the burette can be interpreted accurately. Using a pipette meant that the pipette wasn’t held stable, as it was help by a human hand thus the solution fluctuated at all times hence the amount could not be read accurately on the scale.
The experiment was only conducted once; therefore the results were not reliable. The experiment should have been conducted at least three times to determine whether the results obtained in each experiment matched and therefore were accurate or not. An average could also have been worked out and a conclusion could have been based on them results.
The water baths used were not all at the exact temperatures required; also each contained a different amount of water. If quality of water baths was good and they had been used, and that each had exactly the same amount of water and was at the exact temperature required, anomalous results could have been eliminated.
As the optimum temperature was between 30-40oc, an additional water bath should have been set up (i.e. 35oc) to determine whether the rate of reaction increase between the two temperatures or not. This could have given a stronger backing to the proposed hypothesis. There may also have been time errors, as there was a lot of time taking/accuracy involved. I.e. in step 9 the solution had to be added at exactly 5 minutes, this was not possible as I was only able to do one test tube at a time. To overcome this it would have been useful to have several people conducting the experiment together. A digital stopwatch could have been used, as oppose to clock stopwatch, which wasn’t very accurate.
Additionally, the pipettes caused an error in another way. More accurate results could have been obtained by cleaning the pipette between each reading, or using a new pipette each time solutions were measured out, but this could not practically happen. There was always some solution left over in the pipette from the previous measuring out, thus the measuring out was inaccurate.
Furthermore the initial reading on the calorimeter was placed on 0, however this might not always have been the case, thus the final reading could have been effected, to overcome this we could have used a colorimeter with had a digital display.
Another cause of an error may be due to the fact that the cuvettes may have been dirty/have finger markings on them as they were handled improperly. Some of the cuvettes were also old thus the light transmitted through may have been obstructed further due to the undesired particles they may have been situated on the cuvette. To overcome this we could have used a spectrophotometer.
In conclusion, the accuracy of the results was encouraging enough to make a rational conclusion. If the experiment had been carried out under more strict conditions and with more sophisticated instruments, the conclusion would not have been that different.
Bibliography
Revise As Biology, Heinemann Educational Secondary Division, Helen Eccles
http://www.ocean.udel.edu/extreme2002/experiment/images/experiment.jpg