Enzimatic Activity

ENZIMATIC ACTIVITY
BIOL 2013 Skill Laboratory II Dr. Ash A. Matchett

Christian J. Gonzlez Vargas

Interamerican University of Puerto Rico Bayamon Campus A00319644

Enzimatic Activity

1. Summary The purpose of the experiment was to test for enzymatic activity and how it is affected by different changes in pH, temperature and dilution. Five different experiments were created to test for enzymatic activity and quantify the results of those experiments. Results showed that the enzyme catalase reacts to the substrate H2O2 by breaking it down and releasing oxygen and water molecules with a rate that is dependent on the condition of the catalase. Exposing it to different temperatures showed that the catalase had an optimum condition of 37C to 45C were it reacted with the substrate the fastest and released more oxygen. Also, when exposed to different pH values the enzymatic activity was altered, with pH vales of 6.8 to 7.5 showing to be the most efficient condition for the catalase. Dilution of the enzyme or the substrate had a linear relation where the more diluted either of them was the less enzymatic activity was given. 2. Introduction The object of study in this laboratory experiment is enzymatic activity. Proteins are found within all living things, and they are the most versatile organic molecule present in them. Since they carry out so many different functions in organisms. For this reason they are under constant scrutiny from scientists and researchers alike. However, not all proteins are enzymes even when they are both made up of amino acid sequences. Lets establish the differences between enzymes and proteins before we go on further. Enzymes are globular proteins and as we know form affects function, especially in the tertiary structure. Whereas, some proteins are globular and the rest are fibrous. Unlike other proteins, enzymes can act as catalysts, to catalyze and regulate biological reactions. Moreover, enzymes are functional proteins, whereas proteins can be either functional or structural. Also, unlike other proteins, enzymes are highly substrate specific molecules. Finally, proteins can be digested or broken down by enzymes, whereas enzymes are unaffected by the reactions that they catalyze. Enzymes are proteins that act as catalyst for chemical reactions, in other words they diminish the activation cost for the reaction to begin. Enzymes act by binding the substrate upon which they act in their active site. However, they are very specific on which substrates they act on, as in other factors like pH and temperature. In this lab were testing the enzymatic activity of the enzyme catalase, which is found in all aerobic organisms. The purpose of this enzyme is to

Enzimatic Activity

hydrolyze by catabolic pathway the buildup of H2O2 and release H2O and O2 in the process. In this experiment the catalase in the liver reacts with the H2O2 Hydrogen Peroxide and releases oxygen as bubbles. Enzymes dont get consumed in the reaction that they catalyze so as long as they are kept within their acceptable levels of temperature and pH theyll keep functioning. Catalase is an extremely efficient enzyme that has a turnover rate of catalyzing 40 million H 2O2 molecules per second. With this being said I conclude my introduction of the enzyme catalase. 3. Materials and methods 3.1. Effect of the presence of the enzyme A liver extract was in the prepared and placed on a 150mL beaker. In preparation for the first part of the experiment, 6 test tubes were separated into 3 groups, group A, B & C in order to maintain result fidelity. The test tubes were labeled from 1 to 6 and placed in groups of two on a single rack. Each of the test tubes were added a 100uL of liver extract using the 1000 micropipette. Immediately after the test tubes had the liver extract, one by one the tubes were added 1mL of Hydrogen peroxide using the serological pipette of 1mL, as showed on table 1. Test Tube # Cell Volume Catalase 1 2 3 4 5 6 Table 1 After the substances were placed in the test tubes, the enzymatic activity was recorded for each test tube individually. First upon adding the substrate, then on 1 minute of having added the substrate and finally on 5 minutes after. Once the process was done for all the test tubes, the tubes were moved to another rack and placed under observation. 3.2. Effect of the temperature on the enzyme C B A 100uL 100uL 100uL 100uL 100uL 100uL Substrate(H2O2) 1mL 1mL 1mL 1mL 1mL 1mL

Enzimatic Activity

In this part of the experiment, we had a total of 10 test tubes separated in 5 different groups based on temperature, each group has the given temperature of the enzyme and the substrate accordingly. To do this we labeled the test tubes as either A or B, A for the liver extract and B for the substrate, and then placed a number depending on the group from 1 to 5. The test tubes from group A were added 100uL of liver extract with the 1000uL micropipette each and 1mL of water. Next the test tubes from group B were prepared by adding 1mL of peroxide with the serological pipette of 1mL to each one. The scheme of organization is as showed in table 2.
Group A B Temperature 0 C 1 1 25 C 2 2 37 C 3 3 50 C 4 4 100 C 5 5

Table 2 Once the test tubes were prepared, the test tubes from group 1 were placed on ice, group 2 was kept at room temperature, group 3 was placed on heated water, and group 4 & 5 were grouped with 3. However, the test tubes from group 3 were removed once 5 minutes were elapsed at their given temperature, same as group 4 & 5 respectively. Table 3 shows the groups, the volume of the individual test tubes and the temperatures at which they were exposed. Test Tube # A1 B1 A2 B2 A3 B3 A4 B4 A5 B5 Table 3 Substance Liver extract + Water Peroxide Liver extract + Water Peroxide Liver extract + Water Peroxide Liver extract + Water Peroxide Liver extract + Water Peroxide Volume 1.1mL 1mL 1.1mL 1mL 1.1mL 1mL 1.1mL 1mL 1.1mL 1mL Temperature
0 C 0 C 25 C 25 C 37 C 37 C 50 C 50 C 100 C 100 C

Enzimatic Activity

Once the test tubes had been at their specific temperature for 5 minutes test tubes containing the peroxide at its specific temperature was added to the liver extract of the same group systematically. First test tubes A1 & B1 were removed from the ice and B1 was added to A1, data was compiled according to the results. In every instance of the different temperatures the same was done respectively until every group had been done for the standard time intervals of 0, 1 & 5 minutes. 3.3. Effect of the concentration of the enzyme activity In preparation for this part of the experiment, 2mL of the liver extract was set aside and diluted with 4mL of distilled water. This gives up a dilution of the liver extract of , all the other variables remain the same. In order to prove how the concentration of the enzyme affects its activity 4 test tubes were prepared with 100uL of the liver extract each. One additional tube was prepared to be the control group with the same 100uL of liver extract. After the test tubes were prepared the dilutions were calculated and added to the test tubes as showed in table 4. Test Tube # 1 2 3 4 5 Liver extract 100uL 100uL 100uL 100uL 100uL Water added 10uL 20uL 30uL 40uL 0uL Dilution % 10% 20% 30% 40% 0

Once the individual dilutions were calculated, the first dilution of the liver extract was added to the calculations using the initial dilution of as presented in table 5. Test Tube # 1 2 3 4 5 Table 5 Individual Dilution 10% 20% 30% 40% 0 Initial Dilution 50% 50% 50% 50% 50% Final Dilution 60% 70% 80% 90% 50%

Enzimatic Activity

After all this information had been taken into account, 1mL of peroxide was added to the test tubes and observed for reactions one by one, until each tube had been added the peroxide and the data was annotated at 0 minutes, 1 minute and 5 minutes. 3.4. Effect of the pH on the enzyme activity To demonstrate the effect of the pH in the catalase activity in this section of the experiment the liver extract was not diluted. First 5 test tubes were labeled and added 100uL of liver extract each as showed on table 6. Test Tube # 1 2 3 4 5 Table 6 From there 5ml of phosphate buffer was added to each of the tubes, starting from test tube one with the most acidic buffer and so on until the most basic phosphate puffer. Five different phosphate buffers were used with the following pH; 2, 4, 6, 8 & 10. Table 7 shows the pH and the volume in the test tubes. Test Tube # 1 2 3 4 5 Table 7 Once the liver extract and the buffers were in place, 1mL of peroxide was added to each of the test tubes to test for reactions at the intervals of 0 minutes, 1 minute and 5 minutes. 100uL 100uL 100uL 100uL 100uL 5mL 5mL 5mL 5mL 5mL 2 4 6 8 10 5.1mL 5.1mL 5.1mL 5.1mL 5.1mL Liver extract Buffer Volume pH Final Volume Liver extract 100uL 100uL 100uL 100uL 100uL

Enzimatic Activity

3.5. Effect of the concentration of the substrate In preparation for this part of the experiment, 2mL of the liver extract was set aside and diluted with 4mL of distilled water. This gives up a dilution of the liver extract of , all the other variables remain the same. In order to prove how the concentration of the substrate affects the catalase activity. Test tubes were set aside to make for substrate dilution, in this case we labeled 4 test tubes and added a fifth for the control group which would remain unaltered. After the test tubes were prepared the dilutions were calculated and added to the test tubes as showed in table 8. Test Tube # 1 2 3 4 5 Table 8 Once the substrate was prepared and diluted the 5 test tubes were placed on a separate rack and added 100uL of liver extract each. This liver extract is already diluted by , so each liver extract test tube was added a different concentration of substrate. To which the enzymatic activity was quantified by measuring in millimeters how much bubbling it created at the standard times. 4. Results The results were compiled from every test for each substance in order to properly identify its composition and the organic molecules that it contains. The graphic represents the reactivity to the given reagent from 0 to 5, 0 being no reaction and 5 being a significant reaction with the reagent. More than a 3 in the chart indicates that the substance tested positive with the given reagent. Substrate 1mL 1mL 1mL 1mL 1mL Water added 100uL 200uL 300uL 400uL 0uL Dilution % 10% 20% 30% 40% 0

Enzimatic Activity

Result Tables
4.1. Effect of the presence of an enzyme

Time(Min) 0

Reaction(mm) 42mm

Time(Min) 0

Reaction(mm) 44mm

Time(Min) 0

Reaction(mm) 45mm

1 5

36mm 21mm

1 5

31mm 19mm

1 5

35mm 25mm

Average of two values showed in each group

Presence of Enzyme
50 45 40 35

Reaction in millimeters

30 25 20 15 10 5 0 0 1 5

Time Standard
0 1 5

Enzimatic Activity

4.2. Effect of temperature in catalase

0 C Time 0 1 5 25 C Reaction Time 04mm 35mm 24mm 0 1 5 37 C Reaction Time 45mm 35mm 25mm 0 1 5 50 C Reaction Time 50mm 38mm 25mm 0 1 5 100 C Reaction Time 04mm 0mm 0mm 0 1 5 Reaction 05mm 45mm 20mm

Measurement of the reaction of catalase after peroxide was added.

Effect of Temperature on Catalase

60

50

Reaction in millimeters

40

30

20

10

0 0 C 25 C 37 C 50 C 100 C

C
0 minutes 1 minutes 5 minutes

Enzimatic Activity

10

4.3. Effect of the concentration of the enzyme

Dilution 10% Dilution 20% Dilution 30% Dilution 40% Control

Time

Reaction

Time

Reaction

Time

Reaction

Time

Reaction

Time

Reaction

42mm

033mm

27mm

10mm

46mm

35mm

25mm

15mm

07mm

37mm

06mm

05mm

6mm

05mm

09mm

Liver extract diluted to initially and then individually diluted and added peroxide.

Effect of the Concentration of the Enzyme

50 45 40 46 42 37 35

Reaction in millimeters

35 30 25 20 15 10 5 0

33 27

25

15 10 7 9 6 5

Minutes Elapsed
0% 10% 20% 30% 40%

Enzimatic Activity

11

4.4. Effect of the pH in the enzyme catalase

pH 2 Time 0 1 5 08mm 0mm 0mm pH 4 Time 0 1 5 40mm 28mm 15mm pH 6 Time 0 1 5 53mm 32mm 20mm pH 8 Time 0 1 5 54mm 35mm 22mm pH 10 Time 0 1 5 20mm 08mm 0mm

Reaction of the catalase mixed with phosphate buffer and then added peroxide.

Effect of pH
Measurements in millimeters
60 50 40 30 20 10 0 2 4 6 8 10 y = 3.8x + 23.6

pH

4.5. Effect of the concentration of the substrate

Dilution 10% Time 0 1 5 50mm 10mm 10mm Dilution 20% Time 0 1 5 50mm 05mm 05mm Dilution 30% Time 0 1 5 60mm 05mm 05mm Dilution 40% Time 0 1 5 60mm 05mm 08mm Control Time 0 1 5 45mm 35mm 25mm

Enzimatic Activity

12

Substrate diluted to those factors and the catalase diluted to , substrate added to catalase.

5. Discussion In this lab several things were observed. Firstly, once the substrate (H2O2) was added to the enzyme oxygen bubbles were formed and led to an increased rate of reaction based on the concentration, pH and temperature of the catalase that was closer to the optimum condition values. According to this the closer the optimum value is the better the reaction would be. However, the anywhere before or after that value would produce less enzymatic activity. Catalase has an optimum temperature condition of 37C to 45C and an optimum pH of 6.8 to 7.5. In the first experiment of the presence of the enzyme catalase, we used the standards of 100uL of liver extract and 1mL of hydrogen peroxide. These values are reused throughout the whole experiment, with some variables changing but for the sake of result fidelity everything else remains under control. So hydrogen peroxide was added to the liver extract and enzymatic activity unveiled in the form of bubbling which means that oxygen was released from the reaction. Since the values used remained the same not much variation was produced from this part of the experiment. However it did set precedent for the rest of the experiment. When the enzyme in the test tube was placed in a hot bath, the enzyme denatured due to the temperature being too high, which was observed from the graph produced from the results of the effects of temperature in the catalase, showing a decline in reaction as the temperature shifted from its optimum value. In this lab the hypothesis my hypothesis was that if the substrate (H2O2) was added to the enzyme, the reaction slope will double if the temperature is within optimum value and if not the rate will decay. This happens because the enzyme is works within specific ranges and when outside of those temperature ranges damage to the protein can occur. As seen in the results when the enzyme is placed in ice, the rate of the reaction is noticeably slower. This happens because cooling the enzyme slows the enzymatic activity but doesnt cause damage to the protein unless it reaches freezing. However the same cant be said for the heating of the enzyme, since heating to extreme temperatures denatures the proteins. Which means that they are rendered useless since their tertiary structure has been damaged. As it is showed in the results for

Enzimatic Activity

13

the effect of temperature in the enzyme, reaction slowed when the temperature was cooler, doubled when in optimum and ceased for high temperatures. All the measurements from the release of oxygen were taken in millimeters to account for accuracy. Changing the concentration of enzyme affects the rate of decomposition of (H2O2) because the rate should be highest when the concentration of enzyme is highest. With higher concentration of enzyme, there is a higher chance of an effective collision between the enzyme and H2O2 molecule. The same is true for the inverse proportion which is the method that Im going to use to prove that the concentration of the enzyme affects the enzymatic activity. In this part of the laboratory I had 4 test tubes and one control, each of the test tubes had catalase in it that had been diluted to different factors in increasing order. The tubes were diluted from 10 percent to 40 percent and once they were added the substrate the reaction occurred as they always did but in lesser proportion relative to the concentration of the enzyme. As it is showed on the results for the concentration of the enzyme, it has a liner proportion that decreases as the enzyme is diluted. Thus proving that the concentration of the enzyme affects its activity. The enzyme activity was measured by using phosphate buffers to change the pH of the liver extract or more exactly the catalase. Enzymatic activity, just as in temperature, has its optimum conditions on which they operate. For the catalase, this optimum range is fromm6.8 to 7.5 pH lower or higher than that inhibits the enzyme activity. As it is showed in the data table for pH and on the graph, the pH and enzymatic activity have a curved proportion that rises when near to the optimum conditions for the catalase and drops when out of that range. This happens for various reasons, first the enzyme catalase denatures when the pH are at extremes. However, other changes in the protein structure can occur with different pH, for example, the polarity of the enzyme can change which would inhibit the binding on the active site to H2O2, giving us less reaction and release of oxygen. Clearly, the data shows that this just what optimum range the catalase works on. The dilution of the substrate doesnt creates much variation in the results because as long as the catalase remains the same the reactions will occur. Only slightly would some change be seen due to the fact that there would be less oxygen to release from the reaction. Which in my observations is what happened, the rate of reaction stayed the same but the bubbles seemed less and a lot more separated. As the data in the dilution of the substrate shows, there were less

Enzimatic Activity

14

residual bubbles at the five minute mark for the more diluted ones. Which means that there was less oxygen released. This concludes that, more dilution means less oxygen to release in the reaction. To conclude my analysis of this experiment, I have proven that the enzymatic activity happens when H2O2 is added to the liver extract which contains catalase. Enzyme activity has optimum ranges or conditions in which they operate. The range for optimum enzyme activity for temperate happens from 37C to 45C and the range for optimum pH condition is 6.8 to 7.5 pH. Dilution of the enzyme affects the rate of breaking down of H2O2 into water and oxygen whereas, dilution of the substrate affects the oxygen released in the reaction since there is less. Enzymatic activity is the most when the enzyme is within its optimum conditions and quickly slows of stops when out of it. 6. References

Brunelli L., Yermilow V., Beckman J.S. (2001). Modulation of catalase peroxidase and catalytic activity by nitric oxide. Free Radical Biology & Medicine 30(7), 709- 714. Bartoszek M., Kciuczyk M. (2005). Study of the temperature influence on catalase using spin labelling method. J. Mol. Struct., 744-747, 733-736. Takeda A., Hirano K., Shiroya Y., Samejima T. (1983). On the denaturation of porcine erythrocyte catalase with alkali, urea, and guanidine hydrochloride in relation to its subunit structure. Journal of Biochemistry (Tokyo) 93(4), 967-75. Samejima T., Mijahara T., Takeda A., Hachimori A., Hirano K. (1981). On the acid denaturation of porcine erythrocyte catalase in relation to its subunit structure. Journal of Biochemistry (Tokyo) 89(4), 1325-32.

Enzimatic Activity

15