Theresa Ierardi
Dr. Williams
Mar 106L
4 April 2018
The Effects of pH on Enzyme Rates of Reaction
Abstract
Amylase is found in a wide variety of organisms from bacteria to plants to animals (including humans) and its specific function is to break down the carbohydrate amylose (starch) through hydrolysis. Amylase is an important enzyme in the human body as it allows for the consumption of starch by breaking the polysaccharide down into maltose units. All enzymes, including amylase, function best at a certain optimal pH. PH changes affect the structure of an enzyme molecule and therefore affect its ability to bind with its substrate molecules. Changes in pH affect the ionic bonds and hydrogen bonds that hold the enzyme together, which naturally affects the rate of reaction of the enzyme with the substrate. On top if this, the hydrogen ions neutralise the negative charges of the R groups in the active site so that the substrate and the active site do not attract and therefore do not react. The optimum pH for most enzymes is pH 7.0. For this experiment, the rate at which amylase, a digestive enzyme, catalyzes a reaction is monitored through extraction of amylase from a marine invertebrate, the soft shell clam, Mya arenaria, and from human saliva. Therefore, in this experiment, the effect of different pHs on the reaction rate of amylase is studied.
Introduction
Enzymes are very important macromolecules made of protein. They can catalyze reactions in the body, meaning that they lower the activation energy of reactions and allow them to proceed without being consumed in the reaction itself. This is important because when the activation energy is lowered for a reaction, the reaction can happen more quickly. Without these crucial macromolecules, organisms would not be able to function very efficiently, and reactions would take an extremely long time to happen.
To catalyze reactions, the substrate, the reactant the enzyme performs on, must first bind to a specific area on the enzyme called the active site. It is currently believed that there are several different methods in which an enzyme lowers the activation energy of a reaction and therefore speeds up the process. To begin with, by binding with the substrates, the enzyme can bring the different reactions closer together, making it easier for a reaction between them to occur. In addition, sometimes the enzyme changes its shape around the substrate in an induced fit to stretch or bring out certain groups on the substrate. This makes it easier for the enzyme to catalyze a reaction. Sometimes, the active site even participates directly in the reaction between reactants by creating brief covalent bonds between the enzyme and its substrate. Each enzyme has a specific amino acid sequence. This sequence allows the protein to fold in a very specific three-dimensional configuration. This structure must be very precise, or the substrate will not be able to bind on the active site. This is an example of how the structure of a system ultimately influences its function. pH affects molecular structures of proteins through the manipulation of bonds within the amino acids. This rearrangement of bonds ultimately affects the charge of the molecules, therefore shifting the molecules and as a result altering the shape of the enzyme. Amylase is studied in this experiment. This enzyme helps digest carbohydrates; it hydrolyzes starch into its constituent glucose units. In humans, this enzyme is produced in the pancreas and the salivary glands. Without these enzymes, metabolism pathways would be very inefficient and difficult, and many reactions would take indefinitely long to occur.
Amylase is also a very important enzyme to any organism that ingests starch. Without it, organisms would not be able to utilize the energy in food consumed. It is important to study the effect of pH on enzyme activity, so it can be learned when amylase will function with maximum efficiency. Enzymes are known to be affected by changes in the internal cellular environment. In this experiment, the effect of three different buffer pHs (6.0, 7.0, and 8.0) and starch (Azure substrate) on the efficiency of an amylase reaction with human saliva and of Mya arenaria was tested to study the general pattern of enzyme efficiency as a result of the pH of the environment.
It was hypothesized that the optimal pH for the human saliva will be 7.0 because amylase is generally found in saliva of many animals, and the optimal for Mya arenaria would be 8.0 because the ocean has an average pH of 8.2. Therefore it would make sense for the enzyme to function best at the typical pH of those organisms. If amylase was placed at pH 7.0, then the rate of starch disappearance would be greater than that of the reaction at pH of 6.0. Because increasing pH, up to the optimal pH, generally causes a faster reaction, it was also expected that the reaction occurring in the solution with pH 8.0 would occur faster than the reaction in the 6.0 pH environment.
Methods and Materials
For this experiment, twelve soft shell clams, Mya arenaria, were to have one valve (shell) carefully removed with a sharp scalpel. This was by cutting the posterior and anterior adductor muscles as close to the shell as possible. Once the valves were removed, the stomach was exposed and the scalpel was used to make a small incision on the dorsal surface of the stomach. While wearing gloves, pressure upon the stomach made it so the crystalline style was exposed. When each crystalline style was obtained, they were then placed in a beaker of sterilized 3% NaCl solution (seawater). Afterwards, all twelve styles were removed and placed on a kimwipe to absorb excess water. Upon being removed from the seawater, all twelve crystalline styles were weighed and the value was recorded. Then the styles were placed into a mortar with a pinch of sterile sand to be grinded together. After two minutes, 5.0mls of sterile NaCl solution was added and altogether transferred into a conical centrifuge tube and chilled on ice for 10 minutes. Once the 10 minutes were finished, the tube was spun in a centrifuge for 2 minutes at 300xG (1500rpm) and carefully decanted the supernatant only into a new centrifuge tube, covered, and placed back on ice.
During the beginning part of the experiment, 7 new centrifuge tubes and 7 cuvettes were labeled with the numbers “1-7” with a sharpie. From a designated “saliva contributor”, 3mls of human saliva was collected as another amylase to be tested. Using the labeled centrifuge tubes, 2.5mls of buffer pH 6.0 was added to tubes #2 and #5, 2.5mls of buffer pH 7.0 was added to tubes #1, #3, and #6, and 2.5mls of buffer pH 8.0 was added to tubes #4 and #7. For all tubes, 2.5mls starch-Azure substrate was added. Using tube #1, 0.5mls of 3%NaCl solution was added to make the control tube. For the experimental tubes 2-4, 0.5mls of the clam amylase was added, for tubes 5-7, 0.5mls of the saliva amylase was added, and all tubes were inverted to be mixed continuously for 10 minutes. When the 10 minutes finished, all centrifuge tubes were spun in the centrifuge for 3 minutes at 1100xG (3500rpm) and beginning/end times were recorded. Once the 3 minutes were finished, the supernatant was pour from each tube into the appropriate numbered cuvette and qualitative observations were recorded. Using the Spectrophotometer, the wavelength was set to 620nm and with nothing in the sample chamber, absorbance was set to infinity. Using the control tube #1, it was placed in the chamber to set the absorbance to 0. For all the experimental centrifuge tubes, the absorbance was found and recorded.
Upon finishing the lab, data was collected from the other groups also conducting the experiment. All the data was averaged, and using a t-test, the average absorbance for clam and human amylase was found.
Results
pH Mya arenaria Human
6.0 0.00048g/min 0.03777g/min
7.0 0.0011g/min 0.0521g/min
8.0 0.00616g/min 0.00709g/min
Table 1. The Effect of pH on Amylase Reaction Rate. The following table depicts the results from the experiment and shows the amount of time each amylase reaction, put in buffers of varying pH, took to complete along with their corresponding tube numbers.
Graph 1. The following graph shows the effect of different pHs on the absorbance of reactions between the two amylase solutions of human and Mya arenaria. The data was obtained through an experiment in which several amylase reactions were placed in environments of different pH’s and the absorbance at which the reaction occurred was recorded.
From the data collected, the results of the human saliva amylase showed that the rate of absorbance within the reaction was best achieved in a pH of 7.0. For the clam amylase, it showed that the rate of absorbance within the reaction was best achieved in a pH of 8.0. When each amylase was in a pH of 6.0, the rate of absorbance within the reaction had decreased for clam amylase and increased for human. Upon observing the increased pH from 6.0 to 7.0 for both amylase, there was a consistent increase in enzymatic activity for clam and a consistent decrease for human saliva. With these results, it can be concluded that enzymatic activity for clam amylase is less efficient in pHs lower than 8.0 because of the acidity affecting the clam’s enzymes from catalyzing a reaction. For enzymatic activity for human saliva amylase, it can be concluded that it is less efficient in pHs higher than 7.0 because saliva is generally slightly acidic and not basic.
Discussion
It was predicted that the reaction between the two amylase solutions occurring in the 7.0 and 8.0 pH environment would occur most efficiently because that is the environments that both amylase normally functions in. The results of this experiment supported the hypothesis that had been made (stated above). The optimal pH of an enzyme varies depending on where it is normally found. Human amylase is found in saliva, which has a pH range from slightly acidic (6.0) to neutral (7.0) and clam amylase is found in the ocean, which has a pH of 8.2. Amylase enzymes are proteins made up of amino acids. Because pH affects the molecular structure of proteins through the making and breaking of bonds, if amylase is placed in a strongly acidic or strongly basic solution, its molecules would lose or gain electrons or protons which affects structure and charge of the amino acids, and hence can influence structure and function of an enzyme. At pHs higher and lower than that of the optimal point, the structure of amylase begins to straighten out due to the lack of bonds and the shape of the active site becomes distorted, making it difficult for the substrate to bind.
When the pH reaches a certain high or low point, the structure of the enzyme completely transforms which causes it to denature. Therefore, to a point (to be discussed in more detail below) increasing pH will cause substrates to bind to active sites more frequently. This relationship can be seen as the pH of the reaction increases from 6.0 to 7.0, the reaction rate of absorbance increases. In this experiment, the reaction between clam and human amylase that occurred in the 6.0 environment absorbed 0.00048g/min and 0.03777g/min, while the reaction that took place at 7.0 absorbed 0.0011g/min and 0.0521g/min, and then the reaction that took place at 8.0 absorbed 0.00616g/min and 0.00709g/min. This data is very consistent to the idea that points leading up to the optimal pH will show an increase in enzyme efficiency. The data also shows that at points above the optimal pH, the basicity will disrupt the structure of the enzyme, decreasing efficiency and resulting in an ultimately slower reaction rate, or a greater reaction absorbance.
In conclusion, our hypothesis was supported because it was found that pH 7.0 is the optimal for the enzyme human amylase and pH 8.0 is the optimal for clam amylase. pHs lower or higher than that would result in slower reaction rates.
References
Persoone, Guido, and E. Jaspers. Proceedings of the 10th European Symposium on Marine Biology, Ostend, Belgium, Sept. 17-23, 1975. Institute for Marine Scientific Research, 1976.
Herries, Dg. “Understanding Enzymes.” Biochemical Education, vol. 10, no. 1, 1982, p. 35., doi:10.1016/0307-4412(82)90029-2.
Fabry, Victoria J., et al. “Ocean Acidifications Effects on Marine Ecosystems and Biogeochemistry: Ocean Carbon and Biogeochemistry Scoping Workshop on Ocean Acidification Research; La Jolla, California, 9–11 October 2007.” Eos, Transactions American Geophysical Union, vol. 89, no. 15, 2008, p. 143., doi:10.1029/2008eo150004.
