Affect of temperature on enzyme peroxidase

Materials

• Safety goggles
• 70mL test tube
• 10mL graduated cylinder (±0.1 mL)
• Two hole double stopper (Size 4)
• 8cm glass tube with rubber tube on
• Red water; prepared by mixing water with red food colour
• Quarter inch diameter rubber tube
• ~120mL 3% hydrogen peroxide
• Potato
• Strainer

• Cork borer (size 6)
• Knife
• Thermometer( ± 0.5℃)
• Water bath (±1℃) filled with water
• Two retort stands
• Two test tube clamps
• Tube clamp
• Ice
• Timer (± 0.1s)

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Procedure

Figure 1: Diagram of Manometer

                                                                   Water bath

                                                                    Glass tube (with rubber

                                                                    Stopper   tube on it)

                                                                    Test tube

        Rubber tube

                                                                    Red water

                                                                    Test tube clamp

                                                                     

                                                                       

                                       

                                                                           

                                                                        Retort Stand

1. Put on safety goggles.
2. Set up manometer as shown in Figure 1.
3. Pour 10mL of 3% hydrogen peroxide into the graduated cylinder
4. Slice the potato using the cork borer.
5. Cut the tube of potato acquired from step 4 into equal ten slices using a knife.
6. Pour the measured 10mL of hydrogen peroxide into the test tube.
7. Measure the room temperature using the thermometer. Record the temperature.
8. Place the ten potato slices into the test tube.
9. Quickly place the stopper in the test tube and the tube clamp on the glass tube and start the     timer.
10. Stop the timer when the red water reaches the marked distance, and take off the tube clamp. Record the time.
11. Reset the timer, place the tube clamp on the glass tube and start the timer.
12. Stop the timer when the red water reaches the marked distance and take off the tube clamp. Record the time.
13. Repeat steps 11 and 12.
14. Take off the stopper and clean out the test tube.
15. Repeat steps 3 to 6.
16. Pour water into the water bath and pour the ice cubes in the water bath. Place the thermometer in the water.
17. When the temperature of the water reaches 5℃, place the ten potato slices into the water bath. Leave the potato slices in the water for 5 minutes to ensure that the slices are at the same temperature of the water. Record the temperature, 5℃, and remove the potato slices using  the strainer.
18. Repeat steps 8 to 15.
19. Heat the water in the water bath up to 40℃, and then place the potato slices in the water bath.
20. Once the potato slices have been placed in the water for 5 minutes, record the temperature 40℃ and repeat steps 8 to 15.
21. Heat the water in the water bath up to 60℃ and then place the potato slices in the water bath.
22. Once the potato slices have been placed in the water for 5 minutes, record the temperature 60℃, and remove the potato slices using a strainer.
23. Repeat steps 8 to 14.
24. Clean up working area.

Observations

Table 1: The time, in seconds, taken for the liquid in the manometer to rise to 5cm from the initial point marked on the tube in four different temperatures; 6 °C, 23°C, 30°C, 40 °C, with five trials done for each temperature.

Table 2: The qualitative observations of the potato slices and 3% hydrogen peroxide when reacted in four different temperatures; 6 °C, 23°C, 30°C, 40 °C.

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Analysis

The mean for the time taken for the liquid in the manometer to reach 5 cm above the initial point at two different temperatures can be calculated using the following equation:

                         

Let A represent the average time for the liquid in the manometer to reach 5 cm above the initial point at 6.0°C.

Let B represent the average time for the liquid in the manometer to reach 5cm above the initial point at 23°C.

A = 96.5s                                        B = 23.1s

        

The standard deviation for the time taken for the liquid in the manometer to reach 5 cm above the initial point at two different temperatures can be calculated using the following equation:

                                

 

Let A represent the data for the average time taken for the liquid in the manometer to reach 5 cm above the initial point at 6.0°C.

Let B represent the data average time taken for the liquid in the manometer to reach 5cm above the initial point at 23°C.

sA = 47.4                                                sB = 4.08

A Student’s t-test can also be used:

            

           t = 3.45

 

The degrees of freedom are calculated:

        d.f. = nA + nB – 2

           = 5 + 5 – 2

           = 8

Table 3: number of trials, mean, range, standard deviation, t-value, degrees of freedom and critical value for a comparisons of the average time taken for the liquid in the manometer to reach 5 cm above the initial point at four different temperatures, where each time value at a temperature was compared to other time values at other temperatures .

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Figure 1: The average time taken for the liquid in the manometer to reach 5cm above

the initial point at four different temperatures; 6°C, 23°C, 30°C, 40°C,

 where the time values at each temperature was compared to

other values at differing temperatures.

*All error bars represent ±1 standard deviation.

Conclusion

        The average time taken for the liquid in the manometer to reach 5cm above its initial point at four different temperatures; 6°C, 23°C, 30°C, and 40°C were compared through six different ways. The average time at 6°C, which was the temperature of the ice water, was compared with the average time at 23°C, 30°C, and 40°C. The average time at 40°C was compared with the average time 30°C, and 23°C, and the average time at 30°C was compared with the time value at 23°C, hence producing six different comparisons, which are displayed in the graph, Figure 1. As shown in Table 3, the t-value for the comparisons of average time between 6°C and 23°C, 6°C and 30°C, and 6°C and 40°C, which are 3.45, 3.30, and 3.33, respectively, exceed the critical value for p= 0.05, which is 2.308. This demonstrates that there is a significant difference between the time value at 6°C and the time value at the other temperatures. This is also depicted in Figure 1, as a distinct difference is displayed between the two bars in each comparison. The average time values at 30°C, and 40°C, which are 26.3s, and 25.5s, respectively, are higher than the value at 23°C, which is 23.1s as shown in Table 3, which suggest that as temperature increases, the time taken for the liquid in the manometer tube to reach a marked distance increases as well. The t-test however, displayed that there were no significant difference when comparing 30°C and 40°C, 23°C and 40°C, and 23°C and 30°C. This is evident in the t-values for the three comparisons, which are 0.250, 0.737, and 1.22, respectively, that do not exceed the critical value, 2.308, as shown in Table 3. This is also shown in Figure 1, where the bars in the last three comparisons do not show clear difference as the first three comparisons.

        Although the last three comparisons in Figure 1, suggested that the average time increases as temperature increases, no significant difference was shown according to the t-test as shown in the analysis and Table 3. The significant difference shown in the first three comparisons in Figure 1, which were the average time at 6°C and 23°C, 30°C, and 40°C, supports the hypothesis, which was that as the temperature to which peroxidase is exposed increases, the average rate of time taken for the line to rise a fixed distance in the manometer tube will decrease. As well, the average time at each temperature as shown in Table 3, support the hypothesis, as the average time at 30°C, which is 26.3 seconds, is lower than the value at 6°C,which is 96.5 seconds, and the value at 40°C, which is 25.5 seconds, is lower than the value at 30°C. This ultimately proves that the as temperature increases, the average time increases because as temperature increases, the kinetic energy increases, and more effective collisions of particles occur with the required activation energy and with greater force, thus increasing the rate of reaction, or the enzyme activity (Damji and Green, 2001). Also, the collisions between substrate and active site happen more frequently at higher temperatures due to faster molecular motion, and this also resulted in increase in enzyme activity (Allot, 2007). However, the average time value at 23°C, which is 23.1 is lower than the values at 30°C and 40°C, which suggest that errors occurred during the experiment.

Evaluation

        One source of error in the experiment was due to the tube clamp that was used to control the oxygen gas entering the plastic tubing. Each of the five trials for each temperature was timed from the moment when the tube clamp was clipped on to the rubber tube to the moment when it was removed from the rubber tube once the liquid in the manometer tube reached the 5cm distance above the initial point. The problem occurred between each trial, where the oxygen gas continued to be produced and escape through the glass tubing since the clamp was removed when a trial was done. In addition, the continuous production of oxygen gas from the reaction between the potato slices and hydrogen peroxide decreased between each trial and throughout the experiment as well. This caused the time taken for the liquid in the manometer tubing to reach 5cm above the initial point to continuously increase as time taken between each trial (the time taken to put on the tube clamp back on the rubber tube) led to decrease and escaping of the oxygen gas produced. This is evident in Table 1, where the time values measured at 6°C, for example, increased from trial 1 to 5. Due to the fact that the times taken between trials meant the escaping or decrease in the production of oxygen gas, the varying time taken between trials at four different temperatures led to greater increase in the time values in certain trials, and inconsistency in the time values in some trials. This caused the collection of inaccurate data since the time values showed inconsistency, and this led to inaccurate average time per temperature also. The continuous decrease in the production of oxygen gas, however, did not significantly affect the outcome of the experiment because by using the t-test, the significance in the first three comparisons in Figure 1 was proven, and conclusions were drawn from the data presented. This error could be improved by restarting the set-up; more specifically, using 10mL of hydrogen peroxide and 8 potato slices for each trial. Although it would take longer time, this will ensure that no oxygen gas will escape and decrease between trials, hence enhancing the collection of more accurate data.

        

        

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