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The objective of this experiment was to determine the effect of temperature on the rate that enzymes work. The purpose was to determine whether increasing the temp made the enzymes more active, and if so, at what temperature does the activity start to decline. The experiment consisted of thirty test tubes, with 5 test tubes at each temperature. The temperatures used were 10, 20, 30, 40, 50, and 60 degrees Celsius.
For each temperature there were four test tubes with a sucrose substrate, a buffer, and an enzyme, and one test tube with just sucrose substrate, a buffer, and distilled water. After the liquids were mixed and left for exactly twenty minutes, DNS was added to each test tube and then each tube was boiled for 10 minutes, and finally the test tubes were removed from any heat and distilled water was added. Finally the blank test was placed in the photo spectrometer, and the results were compared the other four test tubes to determine the absorption rate for each temp. Compared with the best fit line for the given data, the average absorption was plotted and then calculated to determine the micro-moles of sucrose at each temp, and from there the rate of micro-moles of sucrose per minute.
Why Does Enzyme Activity Decrease At High Temperatures
The results were that at 10, 20, 30, 40, 50, and 60 degrees Celsius the average absorbance was .2895, .6880, .9100, 1.515, 1.670, and 1.345 respectively. This shows that from 10 to 50 degrees Celsius the enzyme activity increased, however at some point above 50 degrees Celsius the enzyme activity decreased. This implies that enzymes are more active around 40 and 50 degrees Celsius and less active either below or above those temperatures. The data provides grounds for a conclusion that enzymes are more active around 40 and 50 degrees Celsius, and less active on either end, with the activity declining sharply toward either extreme.
The purpose of the experiment was to determine the effect of temperature on enzyme activity, specifically Invertase. Invertase is an enzyme that catalyses the cleavage of Sucrose into Fructose and Glucose. Enzymes are catalytic proteins that are used to speed up reactions. Enzymes speed up reactions by lowering the activation energy needed to complete a reaction in four ways: by bringing the substrates close together, orientating the substrates correctly, promoting acid-base reactions, and excluding water from the reactive environment. In order for a chemical reaction to occur, the necessary components of the reaction must first interact with each other. In most cases, this interaction is orientation specific: one collision between 2 molecules will allow the reaction to proceed while another collision of different molecules will not.
The active site of an enzyme not only provides a specific environment for substrates to interact, but correctly orients the substrates involved, allowing the reaction to proceed. Acid-base reactions are a major component of many chemical reactions. Enzymes promote acid-base reactions by bringing proton-accepting and proton-donating R groups of amino acids in close proximity to substrates. Another way enzymes lower the activation energy is by shutting out H20. Enzymes bind substrates so tightly in their active site that some or all of the water molecules in solution are shut out. The absence of water molecules greatly lowers the activation energy for reactions that require a non-polar environment or reactions that occur between hydrophobic substrates.
While enzymes do lower the activation energy of reactions, the rate at which they do this depends on many factors. Temperature is one of the factors that determines at what rate enzymes will catalyze reactions. All enzymes have a temperature range at which they catalyze the most reactions. Also at either end of the temperature spectrum, enzymes will cease to work. Enzymes are held together by a combination of Hydrogen Bonds, Hydrophobic interactions, and Vander wall interactions.
These weak, non-covalent interactions can only hold enzymes together under very specific environmental conditions (temperature, PH, salt concentration). As any or all of these conditions become too harsh, the non-covalent bonds which hold the enzyme together are no longer able to do so. At the coldest temperatures, enzymes will not work because the particles in a specific solution would not move, and therefore the enzymes will not come in contact with any substrates with which to react. At the hottest temperatures the weak non-covalent bonds are not strong enough to hold the high energy components of the enzyme together.
This experiment, while important is in no way groundbreaking. The data collected will not surprise anybody, but it will help to reinforce the conclusion that temperature effects enzyme activity in the way that at extremes of temperature enzymes will not work, and somewhere in between the lack of activity will be the ideal temp for each specific enzyme. Also this experiment will help the class learn firsthand how temperature, and all the other factors that effect enzyme activity, actually do.
Each section of the experiment had a specific purpose, to aid in the formulation of a conclusion. The goal was to test the effect of temperature on enzyme activity. To test this, 5 test tubes were heated at temperatures at 10 degree intervals between 10 and 60 degrees Celsius, four with all the solutions present, and one constant with everything except the enzyme. The purpose of the control was to determine the color change (absorption rate) of the sucrose solution compared to a test tube without any enzyme. In case there was a change in color even without the enzyme, the control would determine how much change was due to enzyme activity, and how much was unrelated. After the heating at each specific temp for 20 minutes, DNS was added.
The purpose of DNS was to stop the reaction and provide data for how much enzyme activity took place. The DNS reacted with the glucose, and the solution with DNS would change color depending on how much sucrose was separated into glucose and fructose. The more enzyme activity the darker the color, and the darker the color the more light would be absorbed by the test tube while in the spectrophotometer. Without the DNS one would not be able to tell with such accuracy just how active the enzyme Invertase was. The test tubes were placed in boiling water when the DNS was added to speed up the particles and to make sure everything that could react, did.
Methods and Materials
Initially, fairly large beakers containing tap water were heated to temperatures between 10 and 60 degrees Celsius at 10 degree intervals. When the water in these beakers reached the desired temperature, using whatever method necessary, the water was manipulated to stay at the temperature for as long as necessary, at least 30 minutes. After the desired temp was reached, 5 test tubes for each temperature were prepared, and each test set of test tubes was numbered 1-4, and B. All 5 test tubes were initially filled with .5ML of the sucrose substrate, and .5ML of the buffer. After that four of the test tubes had .5ML Invertase added, while the other had .5ML of distilled water added. Once all the necessary solutions had been added, the set of 5 test tubes, (one control and four with enzyme) for each temperature level were added to the temperature specific bath.
The test tubes were placed in the bath in such a way that the test tubes would rest inside the beaker, with the heated or cooled water effecting the temperature inside the beaker. However there would be no contact between the heated water and the solutions inside the test tube. For the next 20 minutes each set of 5 test tubes was kept inside each temperature specific beaker, with the necessary adjustments being made to assure steadiness of temperature. When 20 minutes was up, each set of 5 test tubes was removed, and separated to avoid confusion of data. After the beakers were taken out, 1ML of DNS was added to each test tube in each temperature, then the tubes were covered with aluminum foil, and finally all the test tubes were placed in a beaker with boiling water for 10 minutes. After 10 minutes all the test tubes were removed from the boiling water bath.
Next .5ML of distilled water was added to each beaker, then aluminum foil was placed over the top, and finally each test tube was cooled under cold water. After all the test tubes were cooled, each set of 5 was separated and prepared for the spectrophotometer. For each temperature level the following description is the same. The OD was set to 540 nm, and then the temperature blank was used to then set the transmission percentage. Then the four test tubes that contained the enzyme were placed in the Spectrophotometer and their values were compared with the blank test tube. The transmission for each of the four variable tubes was averaged to obtain an average for each temperature value. Finally a graph was made using the given data. The data obtained in the experiment was then compared with the best fit line of the graph of the given data, and the rate of enzyme activity for each temperature was calculated.
Using the calculated data, a new graph was made with temperature and rate and the X and Y axis, to show visually the effect of temperature of enzyme activity. Used in this experiment were 6 large beakers, for the heating and cooling of the temperature baths. Also used were a few small beakers to hold the sucrose solution, the buffer, and the Invertase. To hold the 4 variable solutions and the one control for each temp value, 30 regular test tubes were used. To heat the large beakers two electrically powered burners were employed. To write on the test tubes the groups used wax pencils, and finally to accurately measure amounts of each solution syringes were used.