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Cody Bearden Biology 101 Lab Report

The Effects of Change in Enzyme Concentration and pH Levels Have on Enzyme Activity

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Enzymes: The Biological Catalysts

Abstract In the biological world, catalysts are known as enzymes. Catalysts are very useful in many ways. Enzymes role in society is to lower the activation energy of reactions. Activation energy is the amount of energy a reaction has to have for it to occur. Enzymes are known as biological molecules. Enzymes are sensitive to changes in temperature and pH. Enzymes are a vital part of life because enzymes control all metabolism and metabolic processes that occur. Understanding how enzymes work and finding what affects them are essential to understanding how to keep good health and how to keep the body running soundly. Experiments like the one that was conducted in this lab, can gather the information to answer these questions. Ten test tubes were used in finding the results. In the first five test tubes, there were different levels of pH and without any enzymes. The other five test tubes have the same levels of pH but with an enzyme known as catechol. When looking on the results, the test tube containing a pH of six, displayed the high rate in activity. The results proved how any little change in pH can drastically alter the productivity of enzymes. Introduction Enzymes have many functions. Amongst those include speeding up a reactions rate. Enzymes are macromolecules that act as a catalyst (Campbell, 2009). Catalysts are chemical agents that are able to speed up the rate of a reaction, and not be consumed by the reaction (Campbell, 2009). Enzymes are also usually proteins. As proteins, enzymes allow many reactions happen within their homeostatic constraints (DeHay, 2009). In order for a reaction to happen, there needs to be a precise amount of activation energy that the cell has obtained. Cells may consume

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activation energy or make their own. The special part about enzymes is they will allow a reaction to occur with less energy. This allows for the reactions to happen and happen faster than normal. Enzymes have a specialized area composed of amino acids known as an active site (Campbell, 2009). The active site is specific to each substrate. A substrate is a reactant that the enzyme acts on (Campbell, 2009). Since every enzyme is shaped specifically for a certain substrate, the rates of reactions are able to happen faster and with less energy. Enzymes are able to recognize their specific substrate among many other related compounds (Campbell, 2009). Enzymes are often used to break and combine substrates. Enzymes are useful with making reactions easier and faster within a cell. However, enzymes are affected by outside factors and effect how well the enzymes are able to do their job (Campbell, 2009). Temperature, pH, as well as enzymes concentration, are a few factors amongst the many that effect enzyme capability (Yoshikuni). Temperature affects enzymatic reactions positively up to a certain point. This happens because with increasing temperature, substrates move more rapidly. Subsequently, substrates are able to run into the active site on the enzymes. Once temperature becomes too high, the heat agitates the enzyme and breaks the hydrogen and ionic bonds as well as any other reactions that stabilize the shape of the enzyme (Campbell, 2009). When this happens, enzymes are said to be denatured or no longer able to do its job. The reason for doing the experiment is to demonstrate how the differences in pH and the concentration of enzymes ultimately affect the reaction rate. The experiment may go either way. The concentration of enzymes as well as pH may or may not have any effect on how active the enzymes are. Experiments with enzymes are important because enzymes affect more than just reactions. Enzymes and their efficiency are a vital part in maintaining homeostasis.

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In order to get clear results on the effect of how pH and concentration of enzymes affects enzyme activity, two experiments were conducted. The first experiment was to test how pH affects enzyme activity. The hypothesis, if there is pH present, then there will be a change in the absorption rate. The null hypothesis, if there is pH present, then there will not be any change in the absorption rate. The variables are pH level, and enzyme activity. The pH level is the independent variable and enzyme activity is the dependent variable. The second experiment tests how the concentration of enzymes affects enzyme activity. The hypothesis is, if substrates are unlimited in availability, then the rate of the reaction of the enzyme will increase. The null hypothesis is, if substrates are unlimited in availability, then the rate of the reaction of the enzyme will decrease. The variables are enzyme concentration, reaction rate and Test Tube A. The independent variable is the enzyme concentration which is displayed in time. The dependent variable is the reaction rate. The controlled variable is Test Tube A which contains 11 mL of pH 7 Phosphate Buffer, 1 mL of water and no potato juice. Materials and Methods Exercise A Table 6.1: Enzyme pH Blank Setup Test Tube pH 4B pH 6B pH 7B pH 8B pH 10B pH Buffer 9 mL of pH 4 9 mL of pH 6 9 mL of pH 7 9 mL of pH 8 9 mL of pH 10 Potato Juice 1 mL 1 mL 1 mL 1 mL 1 mL 1 mL 1 mL 1 mL 1 mL 1 mL Water Total Volume = 11 mL = 11 mL = 11 mL = 11 mL = 11 mL

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For exercise A prepare 5 blanks according to Table 6.1. Label each blank from with the correct pH buffer with the letter B. Each blank should contain 9 mL of a different pH buffer, 1 mL of potato juice, and 1 mL of water. The solution in each should equal a total of 11 mL. Table 6.2: Enzyme pH Experimental Setup Test Tube pH 4 pH 6 pH 7 pH 8 pH 10 pH Buffer 9 mL of pH 4 9 mL of pH 6 9 mL of pH 7 9 mL of pH 8 9 mL of pH 10 Potato Juice 1 mL 1 mL 1 mL 1 mL 1 mL 1 mL 1 mL 1 mL 1 mL 1 mL Water Catechol 1 mL 1 mL 1 mL 1 mL 1 mL Total Volume = 12 mL = 12 mL = 12 mL = 12 mL = 12 mL

Prepare five blanks according to Table 6.2. Label the blanks according to the pH. Add 1 mL of catechol to each of the test tubes. Use Parafilm to cover the opening of each tube and invert the tube three to four times each. Let each tube sit for five minutes, but invert each tube at one minute intervals. Once tubes have sat for 5 minutes, fill a curvette with the test tube solution pH 4B. Open the spectrophotometer and set the absorbance to 420 nm. Use this solution to blank the spectrophotometer. After the spectrophotometer is at 0, fill a different curvette with the pH 4 test tube solution. Measure and record the absorbance of the solution. Repeat this process for each of the remaining test tubes. Use the B test tubes for each corresponding pH solution. Blank out the spectrophotometer before each solution is measured. Record results in table 6.3.

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Exercise B Table 6.4: Blank Test Tube Setup Test Tube pH 7 Phosphate Buffer Blank A Blank B Blank C Blank D 11 mL 10.5 mL 10 mL 9 mL 0 mL 0.5 mL 1 mL 2 mL 1 mL 1 mL 1 mL 1 mL = 12 mL = 12 mL = 12 mL = 12 mL Potato Juice Water Total Volume

Begin by labeling four test tubes. Label each test tube by placing a B before the appropriate letter. Take each test tube and add the corresponding contents in Table 6.4. Each test tube should equal a total of 12 mL of solution. Mix the solution up in each tube thoroughly. Prepare four experimental test tubes just like the ones in Table 6.4 but label these as A, B, C, and D. DO NOT ADD CATECHOL UNTIL INSTRUCTED TO DO SO!! Table 6.5: Experimental Test Tube Setup Test Tube pH 7 Phosphate Buffer Potato Juice Catechol (Add Last) A B C 11 mL 10.5 mL 10 mL 0 mL 0.5 mL 1 mL = 1 mL = 1 mL = 1 mL = 12 mL = 12 mL = 12 mL Total Volume

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9 mL

2 mL

= 1 mL

= 12 mL

Open the lid to the spectrophotometer and change the wavelength to 420 nm. Use Blank A to blank the spectrophotometer. Once the spectrophotometer is at 0, add catechol to test tube A and mix it up by putting paraffin over the opening and inverting rapidly. Once mixed, pour some solution into a curvette and quickly measure the results within 5 seconds of the solution in the curvette. Leave the curvette in the spectrophotometer and take reading every minute for exactly 6 minutes. Take your results and record them in Table 6.6. Use this process for each experimental tube and record the results.

Results Exercise A Table 6.3: pH Effect on Enzyme Activity Absorbance at 420 nm pH 4 0.14 pH 6 .20 pH 7 .34 pH 8 .241 pH 10 .216

Table 6.3 concluded the optimal absorbency occurred at pH 7. This proves that enzymes function the fastest and with the greatest efficiency when the pH is 7. If the pH is not at 7 or optimum pH, the enzymes form products at slower rate.

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Graph 6.3: Graphed results of Absorbance Level

0.40 0.35 0.30 0.25 0.20 0.15 0.10 0.05 0.00 pH 4 pH 6

Exercise A

Absorbance (420 nm)

pH 7

pH 8

pH 10

pH

. Graph 6.3 represents how absorbance increased from pH 4 to pH 7, and then decreased from pH 7 to pH 10. This table and graph clearly shows that pH level can affect enzymes activity.

Exercise B Table 6.6: Enzyme Concentration Effect on Reaction Rate Absorbance at 420 nm Minutes 0 1 2 3 A -.05 .085 .092 .086 B -.17 .080 .135 .157 C -.155 .070 .140 .180 D -.175 .085 .160 .188

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4 5 6

.073 .055 .035

.155 .140 .115

.2 .250 .190

.185 .160 .130

Graph 6.6: Graphed Results of Enzyme Concentrations

Enzyme Concentration Effect on Reaction Rate

0.6 0.5 Absorbance (420 nm) 0.4 0.3 0.2 0.1 0 0 1 2 3 Time (minutes) 4 5 6 y = 0.0098x + 0.0824 A B C D Linear (B)

Discussion Exercise A proves that enzyme activity is affected by pH levels. The hypothesis was, ,If there is pH present, then there will be a change in the absorption rate. The results are close enough to determine that the hypothesis is correct. The optimal pH is 7. This is not basic or acidic but neutral on the pH scale. If the pH is lower than 7 then the enzyme is most likely to denature due to the reactions not happening fast enough. If the pH is greater than 7 the enzyme is most likely going to be changed in some other way. The results have proven the research to be correct and adequate. The null hypothesis stated, If there is pH present, then there will not be any change in

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the absorption rate. Which means the null hypothesis can be accepted. Although all data fit into what is expected, many of things could have gone wrong. When the solution for exercise A was mixed, I believe we were not precise enough and that may be why the results are as close together as they are. For exercise B the null hypothesis stated, if substrates are unlimited in

availability, then the rate of the reaction of the enzyme will decrease. The results show that the null hypothesis can be accepted. In both experiments conducted, it was apparent that change in enzyme concentration and pH levels does have an affect on enzyme activity.

Literature Cited Campbell, N. A., Urry, L. A., Cain, M. L., Wasserman , S. A., Minorsky, P. V., Jackson, R. B., & Reece, J. B. (2009). Biology. (9th ed. ed.). San Francisco: Benjamin Cummings.

Dehay, G. 2009. Biology 101 Lab Manual, . Tri-County Technical College Press, Pendleton, SC, pp. 58-62.

Yoshikuni, Yasuo. Designed Divergent Evolution of Enzyme Function : Abstract : Nature. Nature Publishing Group : Science Journals, Jobs, and Information. Nature publishing Group, 20 Apr. 2006. Web. 08 Oct 2011. <http://www.nature.com/nature/journal/v440/n7087/abs/nature04607.html>.

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