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How Increased Temperature affects the Activity of Enzymes.

Introduction:
Enzymes are protein molecules present in cells which act as catalysts, controlling the rate of all chemical reactions that take within cells. Their effective functioning relies on their shape which is reciprocally shaped to the substrate molecule that it reacts with. In order to avoid any alterations in the shape and structure of the enzyme, the maintenance of an optimum condition is vital. This is because enzymes, due to their protein nature, are sensitive to various factors, in particular temperature (Reece, Urry et al, 2008, p.201). All enzymes have a certain temperature range in which they function best. Once the temperature goes beyond or below this optimum level, the hydrogen bonds that maintain the form of the enzyme break thus altering its composition (Chidrawi, Robinson, 2008. P.7). As a consequence, the substrate can no longer bind into the active site of the enzyme (that has now been altered) and so no further reactions are able to take place and thus the enzyme is said to have denatured. Unlike excessive heat, a change to the enzymes shape and structure due to extreme coldness is often reversible (if the temperature warms up again to its optimum level). The study was carried out using the enzyme Catalase (found in potato). These enzymes catalyse the substrate hydrogen peroxide, breaking it down into both water and oxygen. Hydrogen peroxide is a harmful and toxic by-product of metabolism and as a result the enzyme Catalase serves to break this substrate down, before damage is done to any of the cells and tissues, into less dangerous substances, that being oxygen and water. This breakdown is clearly indicated by a fizzing effect (bubbles) that arises once the reaction proceeds, thus a measure of bubble height serves as a clear indication of enzyme activity. The main aim of the study is to determine the effect of temperature on the breakdown of hydrogen peroxide (substrate) by the enzyme catalase. Considering that enzymes function best at a particular temperature optimum level and therefore anything below or above this optimum leads to a decline in enzyme activity (Reece, Urry et al, 2008, p.201), it has been hypothesised that; if temperature increases then the rate of reaction (enzyme activity) will also increase until it reaches a certain level (typically 37C however different enzymes function best at different temperatures) where enzyme activity will begin to slow down and eventually cease (denature).

Materials and Methods Equipment:


o o o o o o o o o Test tubes x15 Mortar and pestle Thermometer x4 Stop watch Marker for labelling test tubes Scalpels x2 Large test tube rack Metal spoon Knife o o o Ruler Lab coat Pipette

Reagents and solutions: o o o o Potato (Catalase) x2 3% Hydrogen Peroxide solution 18mL (1.5mL per test tube) Hot water baths (37C, 50C and 80C) Ice (0C)

The experimental procedure involved the following steps; 1. Gather all equipment required to complete the experiment and place it on your lab bench. 2. Divide the 15 test tubes into 5 groups by placing 3 test tubes into each group; Label the first set of three test tubes as Group 1 (1A, 1B and 1C). This will be the 0C temperature group. Now label the next three test tubes as Group 2 (2A, 2B and 2C), this will be the 37C group. Repeat this step for the remaining test tubes by placing three in each group and labeling them with their assigned temperature (i.e. group 3 = 50C and group 4 = 80C). This means that you should have 4 different temperature groups each with 3 test tubes as illustrated in Figure 1 below.

Figure 1. Experimental Set-up 3. Measure 2cm from the bottom of the test tube up (Fig. 1) using a ruler and this this point by drawing a horizontal line. Repeat this step for all the remaining test tubes from groups 1 to 4. Then place all test tubes into a large test tube rack so they can stand upright. 4. Peel and slice both potatoes finely using a knife (this way they will be easier to mash). Now place potato pieces in the mortar and pestle. Mash the potatoes until they become paste like and reach an equal consistency i.e. no bumpy or uneven pieces.

5. Using a metal spoon, carefully place some of the mashed potato into test tube 1A until it reaches the 2cm line that was previously marked. Repeat this step for all the remaining test tubes from Groups 1 to 4 (Fig. 1). 6. Fill up a beaker with ice and place all test tubes of Group 1 into this beaker. Place a thermometer into test tube 1A and wait until it reaches 0C (as Group 1 is the 0C temperature group).Once it reaches its allocated temperature, remove the thermometer and place it into test tube 1B. Take test tube 1A out from the beaker of ice and place it in the test tube rack. Using a pipette set to 1.5mL, vacuum and then dispense 1.5mL of hydrogen peroxide over test tube 1A and using a stop watch immediately time the reaction for 1 minute. You may be able to see bubbles this is a sign that the reaction is taking place between the enzyme catalase and substrate hydrogen peroxide (catalase is breaking down hydrogen peroxide into water and oxygen -> bubbles). Once the minute is over draw a horizontal line up the test tube where the bubbles have reached. Using a ruler, measure the height of the bubbles from the 2cm line up to the line that you have just marked and record your results in a table. Repeat this step for the remaining test tubes in Group 1. 7. Repeat step 6 for the remaining groups. This step will however, involve placing the test tubes in various water baths that have been set to the specific temperature allocated to the remaining groups (i.e. 37C, 50C and 80C water bath). When completing this step be sure not to burn yourself by gently placing/removing the test tubes in/from the water baths. 8. Organise all results into a table and calculate the average for the individual groups by dividing the total bubble height by the number of trials.

Results
Results for the experiment were obtained in terms of enzyme activity which was determined and clearly indicated by the height of bubbles that were produced (over 1 minute). These findings, along with averages, are presented in Table 1 below.

Table 1. Recorded bubble height and average bubble height (mm) for each of the temperature groups investigated. Group/temperature Test tube # 1A 1B 1C Height of bubbles (mm) 11 9 12 AVERAGE height of bubbles (mm)

1 = 0C

11

2 = 37C

2A 2B 2C 3A 3B 3C 4A 4B 4C

42 37 49 12 14 9 5 5 5

43

3 = 50C

12

4 = 80C

Additional notes and observations: The bubbles forming as a result of the reaction were exceeding the parameter of the test tube and falling outside the edges. This was a problem as it meant the height of the bubbles could not be accurately measured. As a result, the amount of hydrogen peroxide (substrate) was reduced from 5mL to 1.5mL, thus solving the problem. Bubbles formed almost immediately once the hydrogen peroxide was dispensed over the enzyme Catalase, especially for the 37C group. No bubbles were at all formed for the 80C group; the height taken down for each test tube in this group was the same as it was only the height of the hydrogen peroxide sitting in the test tube and not reacting (it could be said that the enzyme has denatured and hence no further reactions were taking place).
50 45

Average height of Bubbles (mm)

40 35 30 25 20 15 10 5 0 0C 37C 50C 80C

Temperature Groups

Figure 2. The average height of bubbles (mm) recorded over a 1 minute period, for each temperature group.

Discussion
Enzymes are highly efficient and are able to work rapidly, especially when placed in a favourable environment (Chidrawi, Robinson, 2008. P.7). Catalase in particular, is the fastest acting of all enzymes (Chidrawi, Robinson, 2008. P.7) with a very high turnover rate, that being the number of substrate molecules that one enzyme can act on in a minute. The experiment initially involved the usage of 5mL of Hydrogen peroxide per test tube. Although, once the hydrogen peroxide was dispensed over test tube 1A, the bubbles formed were exceeding the parameter of the test tube and thus bubble height was not able to be successfully measured. As a result, diversions were made to the method whereby the amount of hydrogen peroxide went from 5mL to 1.5mL, thus solving this problem (as substrate concentration was too high and this factor in itself can lead to increased enzyme activity). Besides this, no further problems or unexpected outcomes were encountered. The study was conducted in a group of 4, and the inputs of all members of the group were equivalent. Each member of the group contributed heavily to the study and the research behind it. When it came to designing the protocol, each person was assigned the task of researching enzyme activity and influencing factors. Temperature was then decided as the factor to be tested as we found it most easy to do, and interesting. The method behind the protocol was designed by all members of the group at a meeting that was organised. There were no problems encountered in the group, instead the experiment was performed well and according to plan as we were able to obtain the results we were hoping for, that being bubble height, as we felt this would give us clear insight into enzyme activity as the enzyme Catalase is responsible for the breakdown of hydrogen peroxide into water and oxygen. The results obtained in terms of bubble height, clearly depict the effects of temperature on enzyme activity, in particular, on the enzyme Catalase. Recent studies conducted also support this idea of how temperature displays a number of significant effects on enzyme activity by means of catalytic reactions and irreversible denaturisation, effecting both the structure and therefore function, of enzymes(Zheng, Y, Liu, J et al 2012). The results obtained and displayed in Table 1 show the bubble height (mm) for each test tube of each temperature group as well as, the overall average bubble height. Bubble height was recorded as a means providing insight into enzyme activity as they are an indication that the reaction is taking place as oxygen and water are being produced (as part of this catalytic reaction). Group 2, the 37C group had the highest enzyme activity as indicated by the highest bubble height with an average of 43mm (Table 1). This is relatively high, especially in comparison to Group 4, the 80C group which only had an average bubble height of only 5mm. This height however, was not the height of the bubbles released as part of the reaction. Instead it was the height of the hydrogen peroxide just sitting there in the test tube and not reacting with the enzyme catalase (hence why the height was the same for each test tube in this group i.e. 5mm). This could be due to the fact that the

temperature being 80C was too high for the enzyme to function properly as such heat is capable of severely deforming the shape of the enzyme, in particular the active site and thus the substrate molecule can no longer reciprocally fit into that active site. As a result the reaction is not able to take place and the enzyme is said to have denatured (indicate by hydrogen peroxide not reacting). Denaturation as a result of excessive heat is often irreversible. As the temperature increases, so too does enzyme activity. This trend continues until the temperature reaches a certain degree whereby the rate of reaction begins to slow down and eventually cease (Reece, Urry et al, 2008, p.201). This is because the rate of enzymatic reaction increases with increasing temperature up until a certain point and this is partly because substrates collide with the active sites more frequently with increasing temperature. This can be seen in Figure 2 whereby the height of bubbles increases with an increase in temperature. At 0C the average height was only 11mm but this soon jumps to 43mm once the temperature increases to 37C. 37C represents the peak of enzyme activity (Fig 2), after this point, enzyme activity drops rapidly. This is because the temperature has now surpassed the optimum level for this enzyme, that being 37C. This drop continues as the temperature continues to rise until the protein molecule eventually denatures (Reece, Urry et al, 2008, p.201). Figure 2 clearly reveals this drop as soon as the temperature exceeds 37C. The height of bubbles goes from 43mm at 37C to 12mm and then 5mm at 50C and 80C respectively.

Concluding Remarks
The quantitative and qualitative results obtained both support this idea that temperature is a significant factor that ultimately affects enzyme function as it alters the structure and shape of the enzyme and more importantly, its active site to which the substrate binds to. The shape of the active site on the enzyme is reciprocally shaped to the substrate molecule to which it binds to. The closer the affinity of enzyme and the substrate, the more rapidly the reaction is able to take place (Chidrawi, Robinson, 2008. P.7). Enzymes are protein molecules, and in being so they are sensitive to their environment (Reece, Urry et al, 2008, p.201). This means that alterations in their environment, such as temperature change, can lead to changes in enzyme activity. The results obtained support the hypothesis that if temperature increases then the rate of reaction will also increase until it reaches a certain level whereby enzyme activity will begin to slow down and eventually stop- at this point the enzyme is said to have denatured.

References

Books:
Chidrawi, G, Robson, M. 2008. Biology in Focus: Maintaining the Balance, McGraw Hill, Australia. Reece, J, Urry, L, Cain, M, Wasserman, S, Minorsky, P, Jackson, R. 2008. Campbell Biology: An Introduction to Metabolism, Pearson, San Francisco; America

Journal articles:
Zheng, Y, Liu, J, Zhima, Y, Xu Y, Chao ,T. 2012. Temperature Effects on Enzyme Activity of Chicken Liver Esterase Used in Calorimetric Biosensor. Artificial Cells, Blood Substitutes, and Biotechnology, Informa Healcare, China, pp. 125-131.

Websites:
Worthington. (2012). Enzymatic Reaction- Catalase, Retrieved May 5th 2012, from http://www.worthington-biochem.com/ctl/default.html Author Unknown. (2011). Effect of temperature on Enzyme Activity, Retrieved May 7th2012, from http://academic.brooklyn.cuny.edu/biology/bio4fv/page/enz_act.htm Blur it. (2009) What is the effect of Temperature on Enzyme activity? Retrieved may 12th 2012, from http://www.blurtit.com/q923677.html Pearson. (2010). Temperature and Enzyme Function, Retrieved May 12th 2012, from http://www.phschool.com/science/biology_place/labbench/lab2/temp.html