The Effect Of Ph On Catalase Activity
We are studying the effect of pH on catalase activity. Catalase by scavenging hydrogen peroxide to water and oxygen is an important enzyme of cell defense mechanisms against oxidative stress in all living organisms (Dat et al., 2003). Cells use catalase because it is the most efficient enzyme as an antioxidative enzyme which lowers hydrogen peroxide or superoxide to accumulate to toxic levels in plant growth (Bowler et al. 1992). The formula that involves catalase as the enzyme is . pH is a measure of the Hydrogen ion concentration of a solution (What is pH). For this experiment, we are using pH to see its effect on enzyme activity in catalase solution.
Researchers conducted a study on the effect of catalase activity for bovine liver at high …show more content…
pH by adding 0.5 M of Sodium Hydroxide to the catalase. The natural pH of their catalase was at 7.0, and there was enzyme activity (Prajapati et al., 1998). However, the enzyme activity was found to be rapidly reducing from pH 9.5 onwards with a sharp decrease between pH 9.5-11.0 and a complete loss of activity from pH 11 onwards (Prajapati et al., 1998). These results indicate that significant structural and functional changes are associated with alkaline unfolding of catalase at (pH 11.5) catalase (Prajapati et al., 1998). This explains why catalase is not effective at a high pH. Another group of researchers looked at the effect of activity for the catalase at a pH of 3.0-11.4. They saw that activity was constant at a pH of 4-8.5 for the catalase solution. When the pH of the catalase solution was below 4 there was a decrease in activity because there was a formation of an inactive catalase-hydrogen peroxide compound (Chance, 1952). This explains why catalase does not work effectively at a low pH. Another group of scholars also manipulated the pH of the catalase, and they also had similar results to the experimenters above. At a pH of 7, there enzyme activity was at about 55%, but when there catalase was at a pH of 10 by adding Sodium carbonate, there enzyme activity was only 20% (Kono and Fridovich, 1982). A different group of researchers studied a different type of enzyme, urease, but they found that the same results occurred at a low pH. Exposure to buffer below pH 4 resulted in loss of intracellular urease activity (Bauerfeind 1997). From learning about the results of past experiments performed our hypothesis is if the optimum pH of the catalase solution is altered by adding a large amount of acid or a base which makes the catalase solution a pH of 1-4 or 10-14 then there would be less or no enzyme activity because the bonds of the catalase would denature or change.
Materials and Methods
Equipment and Supplies needed:
Catalase activity measuring apparatus
Potatoes
Blender
Scale
Knife
1000 ml plastic beaker 1% Hydrogen Peroxide Solution 6 50 ml beakers 1 M Sodium Hydroxide 1 M Hydrochloric acid Plastic cup pH test strips
To start off, we blended 350 grams of potatoes for 15 seconds on a low speed to make the catalase solution.
We then poured 30 ml of the catalase solution into three 50 ml beakers. We also added 10 ml of water to each of the 50 ml beakers containing the catalase solution. For the catalase activity measuring apparatus kit, we filled the container halfway with water, and we filled the graduated cylinder with water all the way to the 0 ml mark. We also made sure that the hose was under the graduated cylinder in the container of water. Next, the group poured in 40 ml of 1% Hydrogen Peroxide and catalase solution into the gas stopper. Once the reaction started every 5 seconds, we would record how much oxygen was produced for 30 seconds. We repeated that for two trials. We then used a plastic cup to lower the pH of the catalase solution from 6 to 3.5, by adding 1 M of Hydrochloric acid to the solution. To make sure it was a pH that was needed, we used a pH test strip. We added 40 ml of 1% Hydrogen Peroxide and the altered catalase solution into the gas stopper. Once the reaction started every 5 seconds, we would record how much oxygen was produced for 30 seconds. We repeated that process for two trials For the third experimental group we altered the pH of the catalase solution from a pH of 6 to a pH of 8 by adding 1 M of Sodium Hydroxide to the solution. To make sure it was a pH that was needed, we used a pH test strip. We added 40 ml of 1% Hydrogen Peroxide and altered catalase …show more content…
solution into the gas stopper. Once the reaction started every 5 seconds, we would record how much oxygen was produced for 30 seconds. We repeated this part for two trials. For the fourth experimental group we altered the pH of the catalase solution from a pH of 6 to a pH of 12 by adding even more 1 M of Sodium Hydroxide to the solution. To make sure it was a pH that was needed, we used a pH test strip. We added 40 ml of 1% Hydrogen Peroxide and altered catalase solution into the gas stopper. Once the reaction started every 5 seconds, we would record how much oxygen was produced for 30 seconds. Again, we repeated this process for two trials.
Results
The control catalase solution with a pH of 6 that was not altered with an acid or base had a rapid production of oxygen every 5 seconds for 30 seconds, and the final volumes for the three trials respectively of oxygen generated was 59, 56 and 50 ml (Figure 1). However, when a decent amount of 1 M Hydrochloric acid was added to the catalase resulting in a drop of pH from 6 to 3.5, there was no oxygen produced for the 30 seconds for the three trials (Figure 2). For the third trial, a slight amount of 1 M Sodium Hydroxide was added to make the catalase solution a pH of 8 and it rapidly produced oxygen every 5 seconds with the three trials producing 55, 50 and 60 ml of oxygen (Figure 3). A catalase solution with a pH of 12 did not produce any oxygen for the 30 seconds given, so the volume was 0 ml for all three trials performed (Figure 4).
Discussion
Our hypothesis was if optimum pH of the catalase solution is altered by adding a large amount of acid or a base which makes the catalase solution a pH of 1-4 or 10-14 then there would be less or no enzyme activity because the bonds of the catalase would change and denature the solution.
The results that we got from performing the experiment supports our hypothesis, because when the pH of the catalase was not changed or changed slightly oxygen was produced. Our data suggests that catalase solution that is not changed a lot by an acid and base is the most effective in enzyme activity. However, when the pH of the solution was altered by 3-5 levels of pH there was no enzyme activity. The control catalase without any acid or base added to it at a pH of 6 produced 8-12 ml of oxygen every 5 seconds for thirty seconds (Figure 5). However for trials 2 and 4, they were similar because the pH of the catalase solution was altered enough to change the structure and break the bonds which resulted in no oxygen production. For example, trial 2 had a decent amount of 1 M Hydrochloric acid added to the catalase resulting in a drop of pH from 6 to 3.5. There was no oxygen produced for the 30 seconds given (Figure 5). Similarly, a catalase solution with an addition of Sodium Hydroxide resulting in an increase from a pH of 6 to 12 did not produce any oxygen for the 30 seconds given, so the average final volume was 0 ml (Figure 5). The catalase solution that was changed by the Sodium Hydroxide to
a pH of 8 showed that it was not enough alteration to denature the enzyme activity, because on average every 5 seconds for 30 seconds 7-14 ml of oxygen was produced (Figure 5). It actually produced oxygen at a faster rate than the control with no alteration by an average of about 4 ml of oxygen, but ultimately the control catalase at a pH of 6 increased production for the last 5 seconds and produced the same amount of oxygen as the catalase that was altered to a pH of 8 at 55 ml for 30 seconds (Figure 5). This data is useful, because it tells us what conditions the catalase could react with hydrogen peroxide to make it not toxic for cells but make it in to safe oxygen. For future experiments, we could look at the effect of enzyme activity at a pH of 5 and 9 for catalase solution, because it could possibly produce oxygen but not as much when the pH is not altered by an acid or base.
Researchers conducted a study on the effect of catalase activity for bovine liver at high …show more content…
pH by adding 0.5 M of Sodium Hydroxide to the catalase. The natural pH of their catalase was at 7.0, and there was enzyme activity (Prajapati et al., 1998). However, the enzyme activity was found to be rapidly reducing from pH 9.5 onwards with a sharp decrease between pH 9.5-11.0 and a complete loss of activity from pH 11 onwards (Prajapati et al., 1998). These results indicate that significant structural and functional changes are associated with alkaline unfolding of catalase at (pH 11.5) catalase (Prajapati et al., 1998). This explains why catalase is not effective at a high pH. Another group of researchers looked at the effect of activity for the catalase at a pH of 3.0-11.4. They saw that activity was constant at a pH of 4-8.5 for the catalase solution. When the pH of the catalase solution was below 4 there was a decrease in activity because there was a formation of an inactive catalase-hydrogen peroxide compound (Chance, 1952). This explains why catalase does not work effectively at a low pH. Another group of scholars also manipulated the pH of the catalase, and they also had similar results to the experimenters above. At a pH of 7, there enzyme activity was at about 55%, but when there catalase was at a pH of 10 by adding Sodium carbonate, there enzyme activity was only 20% (Kono and Fridovich, 1982). A different group of researchers studied a different type of enzyme, urease, but they found that the same results occurred at a low pH. Exposure to buffer below pH 4 resulted in loss of intracellular urease activity (Bauerfeind 1997). From learning about the results of past experiments performed our hypothesis is if the optimum pH of the catalase solution is altered by adding a large amount of acid or a base which makes the catalase solution a pH of 1-4 or 10-14 then there would be less or no enzyme activity because the bonds of the catalase would denature or change.
Materials and Methods
Equipment and Supplies needed:
Catalase activity measuring apparatus
Potatoes
Blender
Scale
Knife
1000 ml plastic beaker 1% Hydrogen Peroxide Solution 6 50 ml beakers 1 M Sodium Hydroxide 1 M Hydrochloric acid Plastic cup pH test strips
To start off, we blended 350 grams of potatoes for 15 seconds on a low speed to make the catalase solution.
We then poured 30 ml of the catalase solution into three 50 ml beakers. We also added 10 ml of water to each of the 50 ml beakers containing the catalase solution. For the catalase activity measuring apparatus kit, we filled the container halfway with water, and we filled the graduated cylinder with water all the way to the 0 ml mark. We also made sure that the hose was under the graduated cylinder in the container of water. Next, the group poured in 40 ml of 1% Hydrogen Peroxide and catalase solution into the gas stopper. Once the reaction started every 5 seconds, we would record how much oxygen was produced for 30 seconds. We repeated that for two trials. We then used a plastic cup to lower the pH of the catalase solution from 6 to 3.5, by adding 1 M of Hydrochloric acid to the solution. To make sure it was a pH that was needed, we used a pH test strip. We added 40 ml of 1% Hydrogen Peroxide and the altered catalase solution into the gas stopper. Once the reaction started every 5 seconds, we would record how much oxygen was produced for 30 seconds. We repeated that process for two trials For the third experimental group we altered the pH of the catalase solution from a pH of 6 to a pH of 8 by adding 1 M of Sodium Hydroxide to the solution. To make sure it was a pH that was needed, we used a pH test strip. We added 40 ml of 1% Hydrogen Peroxide and altered catalase …show more content…
solution into the gas stopper. Once the reaction started every 5 seconds, we would record how much oxygen was produced for 30 seconds. We repeated this part for two trials. For the fourth experimental group we altered the pH of the catalase solution from a pH of 6 to a pH of 12 by adding even more 1 M of Sodium Hydroxide to the solution. To make sure it was a pH that was needed, we used a pH test strip. We added 40 ml of 1% Hydrogen Peroxide and altered catalase solution into the gas stopper. Once the reaction started every 5 seconds, we would record how much oxygen was produced for 30 seconds. Again, we repeated this process for two trials.
Results
The control catalase solution with a pH of 6 that was not altered with an acid or base had a rapid production of oxygen every 5 seconds for 30 seconds, and the final volumes for the three trials respectively of oxygen generated was 59, 56 and 50 ml (Figure 1). However, when a decent amount of 1 M Hydrochloric acid was added to the catalase resulting in a drop of pH from 6 to 3.5, there was no oxygen produced for the 30 seconds for the three trials (Figure 2). For the third trial, a slight amount of 1 M Sodium Hydroxide was added to make the catalase solution a pH of 8 and it rapidly produced oxygen every 5 seconds with the three trials producing 55, 50 and 60 ml of oxygen (Figure 3). A catalase solution with a pH of 12 did not produce any oxygen for the 30 seconds given, so the volume was 0 ml for all three trials performed (Figure 4).
Discussion
Our hypothesis was if optimum pH of the catalase solution is altered by adding a large amount of acid or a base which makes the catalase solution a pH of 1-4 or 10-14 then there would be less or no enzyme activity because the bonds of the catalase would change and denature the solution.
The results that we got from performing the experiment supports our hypothesis, because when the pH of the catalase was not changed or changed slightly oxygen was produced. Our data suggests that catalase solution that is not changed a lot by an acid and base is the most effective in enzyme activity. However, when the pH of the solution was altered by 3-5 levels of pH there was no enzyme activity. The control catalase without any acid or base added to it at a pH of 6 produced 8-12 ml of oxygen every 5 seconds for thirty seconds (Figure 5). However for trials 2 and 4, they were similar because the pH of the catalase solution was altered enough to change the structure and break the bonds which resulted in no oxygen production. For example, trial 2 had a decent amount of 1 M Hydrochloric acid added to the catalase resulting in a drop of pH from 6 to 3.5. There was no oxygen produced for the 30 seconds given (Figure 5). Similarly, a catalase solution with an addition of Sodium Hydroxide resulting in an increase from a pH of 6 to 12 did not produce any oxygen for the 30 seconds given, so the average final volume was 0 ml (Figure 5). The catalase solution that was changed by the Sodium Hydroxide to
a pH of 8 showed that it was not enough alteration to denature the enzyme activity, because on average every 5 seconds for 30 seconds 7-14 ml of oxygen was produced (Figure 5). It actually produced oxygen at a faster rate than the control with no alteration by an average of about 4 ml of oxygen, but ultimately the control catalase at a pH of 6 increased production for the last 5 seconds and produced the same amount of oxygen as the catalase that was altered to a pH of 8 at 55 ml for 30 seconds (Figure 5). This data is useful, because it tells us what conditions the catalase could react with hydrogen peroxide to make it not toxic for cells but make it in to safe oxygen. For future experiments, we could look at the effect of enzyme activity at a pH of 5 and 9 for catalase solution, because it could possibly produce oxygen but not as much when the pH is not altered by an acid or base.