Effects Observed on Enzyme Activity Caused by Variable Enzyme Concentration, pH, and Temperature Ki mberl mberl y Wil li ams, ams, Kerr Kerr y Rosz Roszell , F aris Al Bakhat, and An drew drew VanDam
Biology Lab 213 section 000, George Mason University, Virginia 17 November 2013
Introduction
In a technologically advanced society it is known, all too well, the idea of being able to make tasks easier by speeding them up. Computer technology has progressed toward goals of making everything available at one’s fingertips by speeding up many processes. Suppose it was necessary to contact a friend who was in another country, before the advancement of our technologically saturated world, a messenger was necessary to travel to that person and deliver it. This process spanned d ays or even months and there were no guarantees it would arrive. Technology has taken this task and created a reality that seemed impossible in a time not long distant. Enzymes, similar to the analogy above, speed up processes in a cell. It catalyzes, or speeds up, reactions that would otherwise take too long to occur for the cell to survive. Enzymes are specialized protein structures that reduce the rate of a reaction without becoming altered or used up in the reaction. Enzymes accomplish this by effectively decreasing the activation energy of a reaction. Activation energy is the energy required to fo rce a reaction or reactants from its stable state to an unstable state, from which most reactions occur. Think of a ball on a hill. The ball sitting at the top of the hill is in an unstable state, where it has taken energy to push it up the hill. The reaction will occur as the ball rolls down the hill or as the ball reaches the top of the hill. By lowering the amount of energy it takes to roll the ball up the hill, logically the entire reaction will hap pen faster. (Sadava, 151) Another reason to understand how enzymes function is they play an important role in our own digestion as humans. In a study at Cambridge, an experiment was performed on how factors affect amylase’s ability to convert starch into energy. Th e hypothesis for the experiment was to understand how to gain the most amount of energy from what is consumed by an individual. As amylase is better
understood, the information that was collected could be used to improve individual’s health. (Tester; Karkalas; Qi, 186) The effects of enzymes can be witnessed through experimentation because specific enzymes catalyze reactions in different ways. Experiments call for certain proteins d epending on the observations that are desired to be studied. The experiment that was used was visibly seen by the color change in a solution; representative of chemical structures being broken down by catalysis. Amylase is an enzyme commonly found in human saliva and germinating seeds. Amylase breaks down starch into simple sugars. The end result is glucose, this sugar molecule fuels cell activity in nearly every living organism. To perform the experiment Amylase was extracted from germinating seeds. Two other items are necessary, a starch solution and a detecting reagent. Lugol’s Iodine is the detecting reagent used to signify the presence of starch. A blue-black color indicates starch is present in the solution. When Amylase is added to the solution containing starch and Lugol’s Iodine, Amylase catalyzes the starch molecules. As the enzyme is added a dark blue-black color will be present which will fade to colorless because of the absence of starch and Lugol’s Iodine only reacts with starch. If the enzyme is within its optimal environment, the hypothesis is, the reaction will proceed all the way through, leaving no blue color in the solution. (Fox Madden, pg. 63-64) It is important to note that enzymes have certain environmental factors that need to be met for optimal performance. Extreme temperature, pH, and harsh chemicals can unravel or denature protein structures of an enzyme which renders it ineffective. Even with futile enzymes chemical reactions occur, but at much slower rates. Knowing this truth, another hypothesis was proposed. The blue-black color from the Lugol’s Iodine would still fade, but at a decreased rate, as the enzymes become ineffective due to adjusted environmental factors. A colorimeter will be used to gather data on this proposal by passing a beam through the contents of a test tube to obtain an absorption value for the specific color it possesses (Fox, Madden, pg. 70).
The hypothesis presented was compared to the data gathered from a number of experiments that were performed in order to evaluate how amylase reacts to varying environments. To prove the first hypothesis positive a test will be performed that will create an ideal environment for amylase. It is expected when starch, the substrate, is added to the solution; amylase will catalyze the substrate to completion. This result will be visually observable by the absence of the dark blue tint from the Lugol’s iodine and will appear yellow. If this occurs, it will be evidence that the data supports the hypothesis. In addition to observing color chan ge, the reaction time will be quantified in order to understand how quickly this reaction is occu rring, which will be used to compare with the second hypothesis. In order to test the second hypothesis the environment will be changed by varying the concentration of amylase by dilution, pH and temperature. As the environment is changed it is predicted that the rate of production product will decrease compared to the rate of the enzyme in an ideal environment. If it is observed that the time m easured increases or does not go to completion compared to the amylase in an ideal environment, this will be evidence to support the second proposed hypothesis. Materials and Methods (Variable Enzyme Concentration)
Enzyme concentration was adjusted to understand its effect on a constant concentration of substrate. To vary the concentration of amylase a serial dilution was performed by measuring 30 mLs of active amylase into a flask. Then 10 mLs was pipetted out and transferred into a flask containing 20 mLs of water. After mixing, the previous step was repeated for the third flask, resulting in three different concentrations: undiluted, 1:3, and 1:9. Each flask should contain equal volumes of solution. The spectrophotometer (set at 540nm) was zeroed after a blank cuvette containing 1.5 mLs of water and 3 drops of Lugol’s iodine was prepared. Immediately after starch was added the time was noted and the solution was mixed. 3 ml of solution was removed and added to a test tube containing 3 drops iodine, mixed, poured into cuvette and absorbance recorded promptly from spectrophotometer. The previous step was repeated on 30 second intervals until 3 minutes was reached and thereafter on
1 minute intervals (approximately 13 minutes). For the remaining two flasks the process was repeated as described above, including the spectrophotometer being zeroed out for the specific flask being used. After the results were graphed the effect of varying the concentration of amylase became clear. In Figure 1 the raw data is shown from the experiment. To calculate the percent of amylose present for a specific absorbance, which was recorded from the experiment, a standard curve was used. The data in Figure 2 represents the absorbance converted to the correlating percent amylose. From Figure 2 the graph in Figure 3 was assembled. Referencing the graph from Figure 3, a qualitative comparison gives evidence that none of the concentrations have similar slopes when comparing percent amylose to time. This dissimilarity shows that changing the concentration of an enzyme clearly influences the enzyme activity with the substrate. The graph suggests that as the concentration of the enzyme is increased the presence of substrate will decrease faster when compared to weaker concentrations of enzyme. This trend holds true as well for decreasing the concentration of the enzyme, meaning the presence of substrate will decrease more slowly. A noticeable error occurred in the weakest concentration, to get a comparable value the starting value was comparted to the ending value which gave an estimated slope. Aside from the fact the value was estimated it is clear that the slope for the weakest concentration is significantly less steep than the other two lines of higher enzyme concentration which supports the results discussed above. Materials and Methods (Variable pH)
During this phase the effects that pH and tempe rature had on enzyme reactions were observed. A spectrophotometer was needed set at 540nm, water, Lugol’s iodine, four flasks, cuvettes, test tubes, and a vortexer were needed. To test the effects of pH, four flasks were used with varying concentrations. The
spectrophotometer was blanked after 3 mLs of water was mix ed with three drops of iodine in a cuvette. 22mLs of pH 4, 5, 6, and 7 was added to the four flasks, respectively. In flask one 2 mLs of substrate (starch) solution and 1 mL of enzyme ex tract was added to the flask then mixed with a vortexer. Immediately after 3 mLs of solution was transferred to a test tube containing 3 drops of iodine. The test tube was vortexed and transferred to a cuvette. Once the cuvette was placed in the spectrophotometer the absorbance was recorded. This process was repeated once every minute for approximately seven minutes. For each the remaining flasks this process was repeated. After the data in Figure 6 was evaluated, it was determined that the more acidic the solution was the less enzyme activity was observed. It is evident from the data gathered from this ex periment as the acidity approached the level of that found in the human body, a pH of 7, enzymatic activity increased. Materials and Methods (Variable Temperature)
To test the effect of temperature on enzyme activity four flasks were used filled with 22 ml of water and 2 mLs of starch solution then mixed. 1 mL of enzyme extract was added to each of the four test tubes. Flask and test tube together we re placed into an ice water bath at 4 degrees Celsius, for ten minutes. Flask 2 and test tube 2 were tested at room temperature, 22 degrees Celsius. The content of the test tube was added to flask 2 and swirled till mixed. The data was gathered by immediately taking a 3 mL sample and added it to a test tube containing three drops of Lugol’s iodine. After vortexed the solution was put into a cuvette and placed in the spectrophotometer and the absorbance was recorded. This process was repeated ever y minute for seven minutes. After removing flask one from the ice bath, the 1 ml of enzyme extract was added, mixed, and placed back into the ice bath. Next a sample was immediately taken by extracting 3 mLs of solution and added to a test tube that contained three drops of Lugol’s iodine. The solution was vortexed and poured into a cuvette and placed in the spectrophotometer where the absorbance was determined. This procedure was completed every minute for the next seven minutes. Results were then recorded in the appropriate
table. Flask three and four were incubation at 37 degrees Celsius and 70 degrees Celsius, respectively, the same procedure was performed to take samples on set intervals and data recorded. From the observations recorded in Figure 9, it was seen that as the temperature was lowered below normal human body temperature enzyme activity slowed. It was also seen enzyme activity was inactive in the 70 degrees Celsius flask. This was most likely due to the broken down tertiary structure of the enzyme. Conclusion
The point of the variable enzyme concentration experiment was to see how concentration of enzyme would affect enzyme activity. The absorbance read from the spectrophotometer measured how much starch was still present at a specific time after enzyme activity began. It was seen in the table Figure 2 that the percentage for the undiluted and 1:3 enzyme dilutions decreased over time, which was expected to happen. The undiluted enzyme concentration decreased at the highest rate, whereas the 1:3 dilution decreased most gradually. This decreased rate of enzyme activity could be attributed to the enzyme saturation theory, which i mplies that reaction rate slows as more enzymes are used up and all active sites are occupied. In the 1:9 dilution in Figure 2, it was seen that the percent of starch appeared to move in a wave instead of a steady downward curve, which can be seen in Figure 3. This error was caused when the solution was extracted at the incorrect time from the flask and put into the cuvette. While waiting to record the intervals the solution reacted inside the cuvette instead of the flask which did not give an accurate account of the percent of starch for each specific time. The variable pH experiment was conducted to show the difference in enzyme activity when changing the pH of the solution in which the reaction is occurring. The absorbance, like in the previous experiment was read by the spectrophotometer to show the percent of starch still remaining over time. Unlike the previous experiment the time only went to seven minutes instead of eight. As
seen in Figure 5, the percent of starch decreased as time went on for each experiment. The pH 4 solution started off with a higher percent of starch, 0.0 19, and did not lower beyond 0.019. This shows that there was little to no activity that happened when amylase was exposed to a slightly acidic environment. When the pH was increased to 5 more enzymatic activity was observed. The most activity was seen when the pH was between 6 and 7. Although activity decreases slightly when the pH was increased to 7, both exhibit increased enzyme activity in relation to pH 4 and 5. This solidifies the theory that this particular amylase is specific to the human species because human saliva operates most efficiently at a pH range of 5.6 to 7.9. The effect of enzyme activity due to temperature shared a similar trend with pH. Recalling the procedure for the variable pH experiment, four different temperatures were observed and absorbance measured for the percentage of starch still present in the solution. The temperatures were 70, 37, 22, and 4 degrees Celsius. Figure 8 represents the percent starch for each solution. At 70 degrees Celsius the least amount of activity was observed, which can also be seen in the graph in Figure 9. At this high temperature activity ceases. This was most likely due to the denaturing of the enz yme. At 37 degrees Celsius, the average temperature of the hu man body, the most activity was observed. This observation was expected. During the experiment, as seen in Figure 7 but not easily seen in the graph or table Figures 9 and 8, respectively, is a blank section for the fourth minute for the absorbance at 37 degrees Celsius. This was due to a wasted cuvette. Even though this reading was lost it was not significant enough to distort the readings or interrupt the results for the other readings at 37 degrees Celsius. There was little enzymatic activity at 4 degrees Celsius due to a decrease in collisions of molecules because of a lower kinetic energy in the solution as the temperature was decreased. The flask in this experiment was taken out of the ice bath while extracting the solution for readings, which explains the consistent decrease in percentage of starch. As the solution warmed activity was able to occur. If the solution was kept at 4 degrees Celsius enzymatic activity would have possibly mirrored the activity at 70 degrees Celsius.
Appendix
(Figure 1) Absorbance vs. Time Table Time (min)
Undiluted Enzyme
1:3 Enzyme Dilution
1:9 Enzyme Dilution
0
2.661
2.903
2.83
0.5
2.383
2.808
2.528
1
2.138
2.663
2.865
1.5
1.84
2.489
2.743
2
1.559
2.194
2.317
2.5
1.193
1.99
2.38
3
0.91
1.833
2.734
4
0.68
1.774
2.727
5
0.479
1.693
2.647
6
0.358
1.608
2.582
7
0.294
1.534
2.554
8
0
1.529
2.017
(Figure 2) Amylose % vs. Time Table Time (min)
Undiluted Enzyme
1:3 Enzyme Dilution
1:9 Enzyme Dilution
0
0.042
0.046
0.045
0.5
0.038
0.045
0.040
1
0.034
0.042
0.046
1.5
0.029
0.040
0.044
2
0.025
0.035
0.037
2.5
0.019
0.032
0.038
3
0.015
0.029
0.044
4
0.011
0.028
0.043
5
0.008
0.027
0.042
6
0.006
0.026
0.041
7
0.005
0.024
0.041
8
0.000
0.024
0.032
(Figure 3)
Effects of Altering Enzyme Concentration 0.050 0.045 0.040 0.035 0.030 Undiluted Enzyme
% Amylose 0.025 0.020
1:3 Enzyme Dilution
0.015
1:9 Enzyme Dilution
0.010 0.005 0.000 0
0.5
1
1.5
2
2.5
3
Time (min)
4
5
6
7
8
(Figure 4) Absorbance vs. time
Time (min.)
pH 4
pH 5
pH 6
pH 7
0
1.219
1.213
1.128
0.964
1
1.222
1.091
0.788
0.587
2
1.218
0.969
0.465
0.218
3
1.21
0.839
0.235
0.091
4
1.205
0.774
0.122
0.069
5
1.197
0.669
0.098
0.059
6
1.184
0.669
0.057
0.041
7
1.187
0.662
0.029
0.052
(Figure 5) % Amylose vs. time
Time (min.)
pH 4
pH 5
pH 6
pH 7
0
0.019471
0.019376
0.018027
0.015425
1
0.020
0.017
0.013
0.009
2
0.019
0.016
0.008
0.004
3
0.019
0.013
0.004
0.002
4
0.019
0.012
0.002
0.001
5
0.019
0.011
0.002
0.001
6
0.019
0.011
0.001
0.001
7
0.019
0.011
0.001
0.001
(Figure 6)
Effect of Variable pH on Enzyme Activity 0.025
0.02 pH 4
0.015
% Amylose
pH 5 0.01
pH 6 pH 7
0.005 0 0
1
2
3
4
5
6
7
Time (min.)
(Figure 7) Absorbance vs. time
Time (min.)
4 degrees C
22 degrees C
37 degrees C
70 degrees C
0
1.599
1.311
1.008
1.342
1
1.477
1.259
0.153
1.297
2
1.43
1.136
0.054
1.295
3
1.373
1.101
0.024
1.35
4
1.333
1.022
5
1.237
0.966
0.064
1.307
6
1.16
0.915
0.022
1.303
7
1.075
0.867
0.031
1.261
1.286
(Figure 8) % Amylose vs. time
Time (min.)
4 degrees C
22 degrees C
37 degrees C
70 degrees C
0
0.026
0.021
0.016
0.021
1
0.024
0.020
0.003
0.021
2
0.023
0.018
0.001
0.021
3
0.022
0.018
0.001
0.022
4
0.021
0.016
0.000
0.021
5
0.020
0.015
0.001
0.021
6
0.019
0.015
0.000
0.021
7
0.017
0.014
0.001
0.020
(Figure 9)
Effect of Variable temperature on Enzyme Activity 0.030
0.025
0.020 4 degrees C
% Amylose 0.015
22 degrees C 37 degrees C
0.010
70 degrees C 0.005
0.000 0
1
2
3
4
Time (min.)
5
6
7
References
Madden, Charles R. "Ch 6 Enzyme Activity." Cell Structure and Function: A Labratory Manual . By Donna M. Fox. Revised Third Edition ed. Dubuque: Kendall Hunt, 2001. 63-63,70. Print. Sadava D.,Berenbaum, May R., H. C. Heller, and D.M Hillis. Life. The Science of Biology. Tenth Edition ed. Sunderland, MA: Sinauer Associated, 2011. Print. Tester, J. Karkalas and X. Qi (2004). Starch structure and digestibility Enzyme-Substrate relationship. World's Poultry Science Journal, 60, pp 186-195. doi:10.1079/WPS200312.
