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The Major Effects On Catalase Biology Essay

Our experiment was based around one important question – What factors affect the reaction rate of catalase enzyme the most? The catalase enzyme is known as an antioxidant enzyme found in living organisms that breaks down hydrogen peroxide to water and oxygen (Sahelian, n.d.). There are two factors known to affect the reaction rate of catalase – change in substrate concentration and change in temperature. Heat does increases reaction rate, but too much heat denatures the enzyme and the effects on the reaction rate are reversed. This was also tested in the catalase experiment. We were instructed to find the effects of varying substrate concentration (0.10%-0.80%) and the effects of a difference in temperature and see how these affected the reaction rate. The purpose of the Catalase experiment is to find out what factors affect the reaction rate the most. As a class, we hypothesized that if the substrate concentrations were increased by increments, then the reaction rate would also increase. It was also hypothesized that the addition of heat to the reaction, from ice to boiled, would increase the reaction rate as well, up to boiling, where the enzyme would be denatured and have a very low reaction rate. The results found after the experiment shows that the hypothesis stated above was correct.

Introduction: Enzymes are protein catalysts, and the basic function of enzymes is to speed up the process of conversion of reactants into products (Ophardt 2003). A catalyst is a substance that is able to speed up the chemical reaction rate without being used up. By using enzymes, cells are able to control the rate at which chemical reactions occur. The overall purpose of this lab is to observe different factors that influence enzyme action in cells and being able to measure reaction rates for the enzyme idenified as catalase. Catalase is one of the most efficient enzymes inside the human body (Bradley 2010). This enzyme is known to breakdown hydrogen peroxide, which is the reactant, into water and oxygen gas. Catalase is also known for protecting cells from harmful or intoxicating effects of hydrogen peroxide. Many enzymes are known to require cofactors and coenzymes to be able to catalyze reactions. Cofactors are known as metal ions which function at various oxidation states and coenzymes are known as non-protein organic molecules (Gilbert 2012). Enzymes are known for being highly specific. Because of the physical shape of the active site of enzymes, they usually attract only one reactant. The active site is the part of the enzyme where catalysis is known to take place. This leads into the transition state, which is the point in time during a chemical reaction where the reactant is transitioning between states (Hester and Degenhart 2012). The transition state is formed by bringing substrate molecules together inside the active site, which promotes reaction progress. There are two factors that influence the rate of reaction and these two factors are substrate concentration and change in temperature. When using a fixed concentration, the reaction rate will increase until the enzyme becomes saturated (Hester and Degenhart 2012). When the enzymes all become saturated, the reaction will continue as fast as that concentration of enzymes can catalyze the reaction, but if the substrate concentration is increased, however, no further increase in reaction rate will occur unless there is an addition of enzyme molecules in the system. An increase in temperature will increase the reaction rate because the substrates and enzymes start to move faster and run into each other, which makes them bind more quickly. But if the temperature is increased beyond a certain point, then the enzyme will become denatured. If an enzyme is denatured, the hydrogen bonds and other weak interactions that determine the enzymes structure are broken (Hester and Degenhart 2012). The structure of the active site is demolished, and the enzyme loses its catabolic activity. There are some proteins that can get back to their proper conformations after denaturation, but there are some that cannot. Enzymes can also become denatured by freezing or by extreme pH conditions. pH is a measure of how much hydrogen ion concentration is in a solution. Inhibitors can prevent enzyme catalysis from occurring in two ways. Competitive inhibitors can bind directly to an enzyme’s active site due to similarities of structure with the substrate, while non-competitive inhibitors do not engage with the active site and attach to an enzyme at another location (Clark 2007). With this lab, we hypothesized that if the substrate concentrations were increased by increments, then the reaction rate would also increase. It was also hypothesized that the addition of heat to the reaction, from ice to boiled, would increase the reaction rate as well, up to boiling, where the enzyme would be denatured and have a very low reaction rate.

Methods: We were separated into 8 groups of 3-4 people and in order to effectively observe the catalase in this experiment, we have to have the proper materials. The materials needed in this lab are a 500 milliliter beaker, a 10 milliliter graduated cylinder, a 50 milliliter Erlenmeyer flask with a rubber stopper, tubing, a U-shaped glass tube, disposable pipettes, 0.0, 0.1, 0.2, 0.4 and 0.8% concentrations of hydrogen peroxide, and room temperature, boiled, warm and ice cold catalase. Once obtaining all the materials, we filled the 500 milliliter beaker with 10 milliliters of water. After, one person must place their finger over the opening of the graduated cylinder and submerge it upside down into the water. This was one of the hardest parts of the experiment because for this to work correctly, the group has to watch the graduated cylinder and make sure no air bubbles escaped. This careful step will make sure the results are more accurate. After finishing that difficult step, a U-shaped glass tube was placed inside the bottom of the graduated cylinder under the water and connected to a plastic tube. The tube connected to a rubber stopper that was at the top of a 50 milliliter Erlenmeyer flask. For the control experiment, ten milliliters of 0.8% hydrogen peroxide was added to the Erlenmeyer flask, and also ten milliliters of a phosphate buffer solution that contained no catalase was added to the Erlenmeyer flask. The stopper was secured efficiently and each group member had a different job. One group member should gently swirl the flask while the others watched for observations and making sure the experiment was being done properly. When the group member starts swirling the flask the production of oxygen is very visible, and the group was instructed to watch the production and record how long it took for ten milliliters of oxygen gas to form inside of the graduated cylinder. However, if if no oxygen gas was produced, the group was to record this as zero milliliters of oxygen gas produced per minute. Once all the data was recorded, the first part of the experiment was complete. We then washed out all of our materials and prepared for the second part of the experiment. The second part of the experiment is exactly like the first in regards to procedures and materials, but for this experiment we only used the 0.8% concentration of hydrogen peroxide. We placed ten milliliters of 0.8% hydrogen peroxide solution into the Erlenmeyer flask. We were given four different temperatures of catalase to experiment with. The temperatures consisted of catalase at zero degrees Celsius, catalase at room temperature, catalase in warm bath water (40 degrees), and boiled catalase. Each temperature was added to the ten milliliters of 0.8% hydrogen peroxide and we as a group watched and observed how long it took for reaction to occur and how much water was displaced during the reaction. We recorded these results and cleaned up all lab materials used during the experiment, which concluded the second part of the experiment.

Results: After finishing our lab, we collected our data from our personal lab group and then we collected the data of every group in the class so that we had more trials to compare. We plugged all of our data into excel and made a table of all our raw data we collected for each substrate percentage. Then, we found the average time and standard deviation for each percentage of substrate we observed (Figure 1). Using the average times we found, we converted them into a line graph (Figure 2) to look for any type of trend. We also created a bar graph (Figure 3) which represented the mean temperatures of room temperature enzyme, warm enzyme, cold enzyme, and boiled enzyme. We then made three two-sample t-tests of equal variances to show the effect of time with temperature difference between room temperature and ice enzyme (Figure 5), boiled enzyme (Figure 6), and warm enzyme (Figure 4). The mean time (in sec) we found for room temperature enzyme was 38.345, ice enzyme was 24.72, warm enzyme was 61.87, and boiled enzyme was 1.09833. All the mean times increased from the colder temperature to the warmer temperature, with the exception of the boiled enzyme, which was the least time recorded. The p-value of room temperature enzyme vs. warm enzyme was .064474, room temperature enzyme vs. ice enzyme was .228201, and room temperature enzyme vs. boiled enzyme was .000375. The significance level is .05, so the only p-value that was statistically significant was the room temperature enzyme vs. boiled temperature enzyme.

Substrate %

0.00%

0.10%

0.20%

0.40%

0.80%

 

0

0.963

4.33

11.64

15

 

0

0.84

2.262

24.36

55.5

 

0

0.825

3.529

15.789

31.58

 

0

0.714

2.8

6.67

26.31

 

0

0.81

2.73

9.38

42.4

 

0

0.87

3.68

20.17

60.23

 

0

0.857

4.4

19.9

64.5

 

0

1.5

8.04

13.85

37.5

Average

0

0.922375

3.971375

15.21988

41.6275

STD

0

0.24337915

1.811934

6.002019

17.41873

Figure 1: This table shows the data we collected for each percentage of substrate and it also shows the average of the percentages and the standard deviation of each percentage.

Figure 2: This line graph represents the average time for each substrate percentage.

Mean Temperature (oC)

Figure 3: This is the bar graph that shows the mean temperatures of room temperature enzyme, warm enzyme, cold enzyme, and boiled enzyme.

t-Test: Two-Sample Assuming Equal Variances

 

Variable 1

Mean

38.345

Variance

298.7188

Observations

6

Pooled Variance

384.7539

Hypothesized Mean Difference

0

df

10

t Stat

-2.07744

P(T<=t) one-tail

0.032237

t Critical one-tail

1.812461

P(T<=t) two-tail

0.064474

t Critical two-tail

2.228139

Figure 4: This is the two-sample t-test of equal variances for the room temperature enzyme vs. warm enzyme.

t-Test: Two-Sample Assuming Equal Variances

 

Variable 1

Mean

38.345

Variance

298.7188

Observations

6

Pooled Variance

337.9722

Hypothesized Mean Difference

0

df

10

t Stat

1.28368

P(T<=t) one-tail

0.1141

t Critical one-tail

1.812461

P(T<=t) two-tail

0.228201

t Critical two-tail

2.228139

Figure 5: This is the two-sample t-test of equal variances for the room temperature enzyme vs. ice enzyme.

t-Test: Two-Sample Assuming Equal Variances

 

Variable 1

Mean

38.345

Variance

298.7188

Observations

6

Pooled Variance

151.1962

Hypothesized Mean Difference

0

df

10

t Stat

5.246596

P(T<=t) one-tail

0.000188

t Critical one-tail

1.812461

P(T<=t) two-tail

0.000375

t Critical two-tail

2.228139

Figure 6: This is the two-sample t-test of equal variances for the room temperature enzyme vs. boiled enzyme.

Discussion: Based on the above results shown in the two parts of the catalase enzyme lab, both experimental hypotheses were proven to be correct. The higher the concentration of hydrogen peroxide, the quicker the reaction will take place. Furthermore, the increase of the temperature of the enzyme from ice to boiling also increased the reaction rate up to boiling when the enzyme went through denaturation. For the T-tests in the second part of the experiment, the p-values led to interesting assumptions. The first two P-values are less than the alpha value of 0.05, which shows that’s the results were not statistically different. Therefore, based on the P-values, the only statistically different results were those of the ice catalase. This could be interpreted that any addition of heat (above a freezing temperature) increases the reaction rate of an enzyme in association to the understood temperature.

The results obtained could have been significantly impacted by means of human error. We had complications with Groups 5 and 8 when we were collecting data as a class. The data collected from these two groups had to be omitted because they had one set of data points that were exceptionally high and another set of data points that were exceptionally low. Because these two group’s data was excluded from all the data collections, it could have greatly impacted the overall averages and caused our graphs to be misunderstood. Another way human error could have factored in was with the different temperatures of catalase. The different temperatures were set out on a table during experimentation and after a certain amount of time, the ice catalase, boiled catalase, and warm catalase could have moved more in a direction to room temperature catalase, which would cause a miscalculation.

It is apparent that temperature affects the reaction between catalase and the substrate reaction. For future experiments, in order to see what else affects the reaction rate between the substrate and catalase, the pH could be tested and compared to the results of the temperature experiment. Likewise, the next experiment could use inhibitors, which normally prevent catalysis from occurring (Hester and Degenhart 2012). These were two factors we didn’t observe as a class, but they are known to affect the reaction rate like change in substrate concentration and change in temperature did.

Finally, the hypothesis of the group was tested and proven to be factual. The higher the concentration of hydrogen peroxide, the quicker the reaction will take place. Also, the increase of the temperature of the enzyme from ice to boiling also increased the reaction rate up to boiling when the enzyme went through denaturation.


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