3B_Investigating+the+effect+of+temperature+on+enzyme+action

__**INVESTIGATING THE EFFECT OF TEMPERATURE ON ENZYME ACTION**__

__**Aim:**__ To find out the effect that various temperatures have on enzyme action, hence determining the most ideal temperature for enzyme action to occur.

__**Hypothesis:**__ The tubes (A1 and A2) placed in ice, and the tubes (C1 and C2) placed in boiling water would turn the iodine solution blue-black, indicating the presence of starch in the mixture. Whereas the tubes (B1 and B2) placed in a 37 ºC water bath, would leave the iodine solution brown when dropped in the mixture, suggesting that the starch originally in the mixture had been digested by the amylase. This shows that the enzyme action (amylase digesting the starch) occurred the most effectively in tubes B1 and B2, hence implying that enzyme action is most effective at temperatures around body temperature (37 ºC). __**Method:**__ __**Apparatus and materials**__
 * 1) Label 8 test tubes A1, A2, B1, B2, C1, C2, D1 and D2 using the white labels provided.
 * 2) Add 5cm 3 of starch solution to tubes A1, B1, C1 and D1 using the dropper provided in the starch solution (taking care not to use droppers from other solutions so as to avoid contamination of the solutions) to drop the amount into a measuring cylinder before pouring it into the test tubes. This will ensure more accurate readings.
 * 3) Add 3cm 3 of distilled water to test tube D2, by first pouring the amount into a measuring cylinder (this will ensure more accurate readings) to measure the amount being used, this will act as a control for the experiment.
 * 4) Add 3cm 3 of amylase solution into tubes A2, B2 and C2, using the dropper provided in the amylase solution, (taking care not to use droppers from other solutions so as to avoid contamination of the solutions) to drop the amount into a measuring cylinder before pouring it into the test tubes. This will ensure more accurate readings.
 * 5) Light a bunsen burner and place a beaker of hot water over it, on a tripod stand and gauze. This is to allow the water to boil, creating a hot water bath, while preparing other water baths.
 * 6) Collect half a beaker of ice. (Remember to use goggles to light the bunsen burner)
 * 7) Fill two beakers with warm water, and with the aid of a thermometer, add tap water to the beakers until the temperature read on the thermometer is 37 ºC.
 * 8) At 15second intervals, place the test tubes into the various water baths, starting the stopwatch concurrently to ensure accurate time readings. Place A1 and A2 into the beaker of ice at 15s on the stopwatch, B1 and B2 into the 37 ºC water bath at 30s on the stopwatch, C1 and C2 into the boiling water bath at 45s on the stopwatch, and D1 and D2 into the other beaker of 37 ºC water at 60s on the stopwatch.
 * 9) Leave the tubes for 10 minutes to allow the solutions in the tubes to reach the temperature of the water bath.
 * 10) Pour the contents of A2, B2, C2 and D2 into the starch tubes A1, B1, C1 and D1 respectively. (Use a test tube holder to pour the contents of C2 into C1) Record the time immediately. Keep the tubes A1, B1, C1 and D1, containing the mixture in the respective water baths throughout the experiment and ensuring that the temperatures are maintained.
 * 11) Leave the mixtures in the water baths for another 10 minutes.
 * 12) After 10 minutes, test the mixture in each tube by dropping a drop of iodine solution on a white tile and using droppers to drop a drop of each mixture (A1, B1, C1 and D1) onto the iodine solution. (Be sure to use either new droppers for each solution, to avoid contamination, or to wash the droppers thoroughly before inserting them into a new solution)
 * 1) Bunsen burner, tripod stand and gauze
 * 2) 3 water baths
 * 3) Thermometers
 * 4) Measuring cylinders
 * 5) 8 test tubes and test tube rack
 * 6) 4 beakers
 * 7) 1 white tile
 * 8) 4 droppers
 * 9) Ice cubes (0ºC)
 * 10) Distilled water
 * 11) Hot water (100 ºC)
 * 12) Warm water
 * 13) Tap water
 * 14) Test tube holder
 * 15) Stop watch
 * 16) Goggles
 * 17) 2% starch solution
 * 18) 1% amylase solution
 * 19) Iodine solution

__**Results:**__ __Table of results of starch digestion using amylase at different temperatures__ __**Analysis:**__ The rate of enzyme-catalyzed reaction increases as the temperature is raised. However, many enzymes are adversely affected by high temperatures. The reaction rate increases with temperature to a maximum level, then abruptly declines with further increase of temperature. Because most animal enzymes rapidly become denatured at temperatures above 40°C, most enzyme determinations are carried out somewhat below that temperature. This can be seen from the graph below.
 * **Tube** || **Amt.: 5cm** **3** || **Tube** || **Amt.: 3cm** **3** || **Temp. (ºC)** || **Time placed in solution (s)** || **Time (tube X2) removed from solution (min)** || **Time (tube X1) removed from solution (min)** || **Colour of iodine when tested** ||
 * A1 || 2% starch || A2 || 1% amylase || 0 || 15 || 10:31 || 20:36 || Iodine turned blue-black ||
 * B1 || 2% starch || B2 || 1% amylase || 37 || 30 || 10:48 || 20:54 || Iodine remained brown ||
 * C1 || 2% starch || C2 || 1% amylase || 100 || 45 || 11:03 || 21:12 || Iodine turned blue-black ||
 * D1 || 2% starch || D2 || 1% amylase || 37 || 60 || 11:20 || 21:34 || Iodine turned blue-black ||

Source: [|__http://www.worthington-biochem.com/introbiochem/tempEffects.html__]

From the results, it can be seen that the solution in tube B1 left the iodine brown, indicating that there was no starch present in the solution any more. This proves that starch digestion, and hence enzyme activity occurred. This suggests that the temperature of the solution in B1 (37ºC) was suitable for the starch to be digested by the amylase. Furthermore, 37 ºC is around body temperature, so enzyme action should occur effectively at this temperature, as enzyme activity occurs in our body all the time. Therefore, the starch solution that was originally placed in the test tube before the experiment was digested by the amylase solution when the two were mixed together, leaving the iodine solution brown (original colour) during the iodine test. This also proves that temperatures around body temperature 37 ºC are the most suitable and ideal for enzyme activity.

In tubes A1, C1 and D1, the solutions turned the iodine solution blue-black during the starch test, indicating the presence of starch in the solutions. This is because the temperature was not suitable for starch digestion and enzyme reaction to occur. Tube A1 was placed in a beaker of ice, and therefore had a temperature of 0 ºC, which is too low for enzyme activity to occur. Therefore, the solution turned the iodine blue-black during the iodine test, as the starch solution that was placed in the test tube at the start of the experiment did not get digested by the amylase when the two tubes of solutions (A1 and A2) were mixed together. Therefore, this shows that 0 ºC is not a suitable temperature for enzyme activity to occur. As for test tubes C1 and C2, they were placed in boiling water (100 ºC). When the solution in C1 (after mixing solutions from C1 and C2) was added to the iodine solution, the iodine turned blue-black, proving that starch was still present in the solution, and hence did not get digested by the amylase when the two solutions were mixed together. This shows that 100 ºC is too high a temperature for enzyme activity to occur, as the ideal temperature range for enzyme activity to occur is (as earlier mentioned and tested) body temperature (around 37 ºC). In the 100 ºC solution, denaturing has already taken place in the solution, therefore decreasing the rate of enzyme activity, making the starch remain in the solution and not be digested by the amylase. Hence, 100 ºC is not a suitable temperature for enzyme activity to be occurred. Tube D1 and D2 were used as a control for the experiment, and no amylase was added to the solution. Therefore, the starch would definitely remain in the solution as no digestion can occur (no enzyme is present). This acts as a control as the solution would turn the iodine solution blue-black, indicating that starch is present. The colour from the reaction can then be used as an indication to show when starch is present in the solutions.

In conclusion, 0ºC and 100 ºC are not suitable for enzyme activity to occur, as the temperatures are too low and high respectively. 37 ºC is a suitable and the ideal temperature for enzyme activity to occur.

__**Extension Questions:**__
 * 1) Which tube shows that starch digestion has occurred? Give your reasons.

Tube B1 shows that starch digestion has occurred. This is because it left the iodine solution brown, suggesting that no starch was present in the solution. Starch digestion has occurred as the solution originally contained starch, and did not contain anymore at the end of the experiment.


 * 1) Which of the test tube contained mixtures that gave a blue-black colour?

Tubes A1, C1 and D1.


 * 1) Why did the mixtures in tubes stated in (2) remain blue-black?

Starch was present in the solutions, and therefore turned the iodine solution blue-black, indicating that the starch originally placed in the solution was not digested as the temperatures were not suitable for enzyme activity to occur. D1 did not contain any enzyme (amylase), and therefore definitely did not have starch digestion occurring.

It acts as a control for the experiment.
 * 1) What purpose does the tube D1 serve?


 * 1) What conclusion(s), based on the observations in this experiment, can be made about the effect of temperature on enzyme action?

The ideal temperature for enzyme action is around body temperature (37 ºC), and any temperature significantly higher or lower than that is not suitable for enzyme action to occur.