Effect of temperature and inhibition on the rate of pepsin digestion.
Substrate Concentration Effect of temperature and inhibition on the rate of pepsin digestion K.Mukherjee,D. Sarpangal, E. Hsu., A. Choi,C. HanMonta Vista High SchoolFebruary 11, 1994 AbstractThis experiment focused oil the role of temperature and inhibition in determining the digestion rate of the enzyme pepsin. In the experiment, pepsin in an HCl solution was used to digest the proteins found in egg whites. Different preparations, such as egg white and egg white with starch, were tested at three different temperatures. Our results yielded the conclusion that, of the temperatures tested, protein digestion using pepsin occurs most effectively at 37 C. Furthermore, we found that starch inhibits the activity of pepsin. IntroductionOne of the most important biological processes for animals is that of digestion. In humans, digestion begins in the mouth, where food is chewed and salivary amylase begins to digest carbohydrates. After food is swallowed, it is transported down the esophagus into the stomach, where digestion begins in earnest. The stomach's muscular walls help to break down food, as does the hydrochloric acid secreted by glands inside the gastric pits. Later oil, the intestines help to digest and absorb food. However, digestion Would not be possible without the presence of stomach enzymes. Various enzymes help to digest food in general, and in particular they are needed for the digestion of proteins. Proteins consist of chains of amino acids which form into three-dimensional shapes because of electrostatic attractions and repulsions between polar and nonpolar molecules. The enzyme pepsin, secreted by the chief cells in the stomach [2], is responsible for splitting proteins into peptides, or chains of fewer than 50 amino acids. However, many factors can affect the activity and rate of reaction of enzymes such as pepsin. pH, temperature, tile concentration of substrate, and the presence of inhibitors can alter the effectiveness of an enzyme [1]. It was already determined by Yang, et al, that pepsin requires the presence of HCl in order to adequately function [5]. In this experiment, we explored the effects of temperature and presence of starch and NaOH on the rate of digestion of egg whites by pepsin. Materials and MethodThe first necessity for conducting this experiment was a source of protein. Because egg whites are almost completely formed of protein, they were used. Three eggs were
cracked, and the whites were poured into three beakers, while the yolks were, kept in the shells and later discarded. 30 mL of 1M NaOH was poured into one beaker as an inhibitor, and starch was mixed with the contents of another beaker. The third beaker, or control, contained only egg whites. After being thoroughly mixed, the beakers were placed on a hot plate to solidify the egg white enough to put into capillary tubes which would then be placed in an HCl-pepsin solution. The nine capillary tubes were cut about 4 cm in length frorn a long tube. Three ...
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cracked, and the whites were poured into three beakers, while the yolks were, kept in the shells and later discarded. 30 mL of 1M NaOH was poured into one beaker as an inhibitor, and starch was mixed with the contents of another beaker. The third beaker, or control, contained only egg whites. After being thoroughly mixed, the beakers were placed on a hot plate to solidify the egg white enough to put into capillary tubes which would then be placed in an HCl-pepsin solution. The nine capillary tubes were cut about 4 cm in length frorn a long tube. Three were filled with plain egg whites, three were filled with egg whites and NaOH, and three were filled with starch and egg Whites. We used a combination of capillary action, pushing, and sucking through all eyedropper to fill the capillary tubes with egg whites. If the pepsin digested the protein, egg white would be eaten away from the ends of the capillary tubes, and thus the rate of digestion of pepsin under different conditions could be determined by how much egg white had disappeared. After this, the capillary tubes were placed in a hot water bath to cook them, and the length of egg white in each tube was measured. One of each type of capillary tube was placed in each of three beakers. One beaker was kept at 0 C in an ice water bath, another at room temperature, and a third at 37 C in an incubator. Each beaker contained 30 mL of 1.0 M HCl and 20 mL of all aqueous pepsin solution. (The concentration of pepsin was approximately 1g/50 mL, and was constant for all the beakers.) Each day, the tubes were removed from their HCl and pepsin baths and the amount of egg white remaining was measured. This part of the experiment went on for three days. ResultsThese graphs illustrate the results of our experiment. Some sets of results are not included because of error. First, no digestion occurred at 0'C, in view of the fact that our measurement was accurate to within ± 0. 1 cm, but the amount of egg white eaten away was less than 0.1 cm. Therefore, there is no graph showing the results of the experiment when conducted at 0'C. In addition, the data resulting from the use of NaOH as an inhibitor could not be included because the mixture of egg white and NaOH apparently dissolved in the pepsin-HCl. solution; thus the rate of digestion was impossible to measure in all cases except 0'C. However, this data was still not used because of the lack of adequate standards for comparison. From the graphs, it can be observed that egg whites were more easily digested at 37'C than at room temperature. It can be further observed that pepsin was approximately 50% less effective digesting the starch-egg white mixture than the plain egg white. DiscussionIt can be seen frorn our experiment that temperature and inhibition affect tile digestion rate of enzymes. Theoretically speaking, a graph of enzyme activity versus temperature looks something like a bell curve; at very low temperatures, there is little enzyme activity. This lack of activity occurs because of the lack of kinetic energy; at lower temperatures molecules have less energy which can be contributed to the making of activated complexes. Thus, at lower temperatures tile overall rate of chemical reactions is very low. However, as the temperature increases, so does the rate of reaction and the rate of enzyme activity. Most enzymes have a certain temperature at which they are most effective [1]. For example, the enzymes in our gut bacteria, such as Eschericha coli, are most probably adapted to work most efficiently at a temperature of 37'C; thus incubators are usually set to this temperature when E coli is present as part of an experiment. As the temperature increases beyond tile ideal temperature for an enzyme, the hear results in additional kinetic energy which changes the shape of an enzyme and the electrostatic bonds between various molecules. As a result, the enzyme becomes less effective. As the temperature continues to increase, irreversible damage is done to the structure of the enzyme; it is denatured in the same way that egg proteins are when they are fried sunny side up. In our experiment, it appears that the enzyme pepsin is more effective at 37'C than at room temperature or 0'C. However, since we did not conduct tests at temperatures higher than 37'C, it is impossible to determine whether that temperature is the ideal temperature for pepsin. At 0'C, it is possible that no digestion occurred at all, because none of the changes in the length of egg white present in the capillary tubes exceeded our measurement error (± 0.1 cm). At room temperature, some measurable digestion did occur; 0.3 cm of the starch-egg white mixture was digested, while 0.6 cm of the plain egg white was digested. However, both of these results were outstripped by far by the performance of pepsin at 37'C. At this temperature, 0.7 cm of the starch-egg white mixture and 1.4 cm of the plain egg white were digested. Further investigation into this area could use a number of different temperatures. If a relatively accurate graph of enzyme activity versus temperature were desired, it would be necessary to obtain many data points, perhaps starting from 0'C and ending at 50'C in 5' increments. This more detailed extension of our experiment would yield more definite conclusions about the most effective temperature for pepsin. Temperature is not the only factor affecting the effectiveness of enzymes. In the real world and in our experiment, various other substances are present which act as inhibitors and decrease the rate of reaction of enzymes. In our experiment, it is fairly certain that starch served as all inhibitor. On average, pepsin digested twice as much egg white when starch was not present. At room temperature, 0.6 cm of plain egg white was digested, but only 0.3 cm of egg white and starch was digested. At 37'C, 0.7 cm of the starch-egg white mixture was digested, but pepsin "ate" 1.4 cm of the plain egg white. However, from the tests we conducted it is not possible. to classify starch as either a competitive or noncompetitive inhibitor. A competitive inhibitor competes with the substrate for access to the active site of all enzyme. As a result, the enzyme constant, or kill, would increase, indicating the enzyme was less effective. However, if enough substrate is present, the enzyme wastes less time examining the inhibitor, and thus its maximum speed would be unaltered. Oil the other hand, a noncompetitive inhibitor decreases enzyme activity by binding to some other area oil an enzyme besides the active site. These inhibitors may thus alter the shape of the enzyme, decreasing its effectiveness. As a result, the enzyme constant would be unaffected, but the maximum speed would be decreased. The only way to determine whether starch is a competitive or noncompetitive inhibitor would be to conduct tests with different concentrations of starch and egg white and observe the rate of digestion. From this data, it would be possible to construct a graph of rate versus substrate concentration. The graphs with and without starch could be compared. If there was a change In slope, but not in maximum speed (Vmax), between the two graphs, we could conclude that starch serves as a competitive inhibitor. If there was a change in teh horizontal asymptote of the graph, we could conclude that starch serves as a noncompetitive inhibitor. It is even possible that starch could have both competitive and noncompetitive characteristics; perhaps starch, as a carbohydrate, could produce van der Waals forces which would attract it to the active site and other sites oil the enzyme, resulting in both competitive and noncompetitive inhibition. However, we do know from Our experiment that there exists some kind of electrostatic attraction or hydrogen bonding between starch and pepsin or between starch and egg white, because the pepsin is clearly being affected by the presence of the starch. The starch could either "stick" to or encase the protein, making it difficult for the pepsin to cleave the protein's peptide bonds, or it could "stick" to the enzyme itself, directly interfering with its ability to catalyze reactions. To determine which, if any, of these theories actually explains our observations would require research into the structure of pepsin, egg white proteins, and starch, as well as additional observations based Oil Our Current experimental design. In addition to expanding the research oil inhibition, this experiment could be improved in several other ways. First, the results for the egg white-Na0H mixture could not be used in the experiment because, In two out of three water baths, the mixture diffused our of the capillary tubes and Was thus lost. In general, the NaOH created severe difficulties; it tended to form a gelatin when mixed with egg white, and as a result tile mixture was exceedingly difficult to place inside capillary tubes. Most probably, a weaker base, such as Ca(OH)2, would have to be used if tile experiment were redone. We could also use wider tubes in a future experiment. While using wider test tubes would require more egg white, starch, and base, it could make the cooking process much easier; Saran wrap could be used to cover the ends during cooking, preventing leakage. Even though our experiment call clearly be improved, it did yield some Conclusions. We determined that pepsin was more effective at 37'C than at room temperature or 0'C. We also determined that starch acts as an inhibitor of pepsin, thus creating all interesting field for future study. References,[1] Arms, Karen, Biology: A Journey Into Life, Saunders College Publishing, 1988, pp. 482. [2] Bastian, Glenn F., An Illustrated Review of the Digestive System, Harper-Collins College Publishers, 1994, pp. 56-57.[3] Ja, William, personal communication, 1996.[4] Tocci, Salvatore, Biology Projects for Young Scientists, Franklin Watts, 1987, pp. 86-89.[5] Yang, Beverly, et al., "The Effects of Enzymes on the Digestive Process," Monta Vista Journal of Molecular Biology, vol. 1, number 1, pp. 59-69.