The researcher conducted this experiment in order to investigate how various factors effected enzymes. Those factors were the concentration of the enzyme, the temperature it had to work in, and the pH level of the solutions as well. All three were hypothesized to speed up the rate at which the enzyme acted. A series of several tests were carried out to find the answer to each problem. First, the scientist witnessed the enzyme diastase work in a natural environment, which was a room temperature starch solution.
Five trials involving enzyme and starch solution were carried out using increasing amounts of diastase to test the effect of concentration. Cold, room temperature, body temperature, and hot enzyme solutions were timed and observed to find out what effect temperature had on the reactions. To see how pH level effected enzyme reactions, the researcher used hydrochloric acid, sodium hydroxide, and distilled water to create solutions. The data recorded from the investigations were very useful in helping the scientist gain an understanding of how enzymes work and how some factors can either aid or hinder their processes.
This investigation was carried out in order for the researcher to gain an understanding of enzymes in how they work, and the relative speed at which they carry out their processes. Before it was conducted, the scientist had very limited knowledge of the proteins, so this was a good way to find out about them first hand. Gaining an understanding of enzymes is very important, because they are so significant to the health of living things, from plants, to animals, to human beings. Their role in body is actually so necessary, that without them, it would be impossible to live. Besides gaining an understanding of the function enzymes carry out in the body, this experiment was also carried out so that the function of the commercially available enzyme solutions could be observed.
Before starting the experiment, the researcher hypothesized that high concentrations of enzymes, exposure to high temperatures, and acidic pH levels would all increase the efficiency of enzyme activity. A large number of enzymes available would mean that more substances can be catalyzed at the same time. High temperatures speed up the rate that molecules move, so the enzymes would be able to reach more molecules at a time, and also speed up reaction. Also, acids have a low pH number and give off heat, so they may also speed up reaction time in the same way as temperature. The scientist also learned about the structure of enzymes, and how denaturation occurred. Such material is useful for scientists and biologists to know because they aid in the creation of drugs and medication that may help someone with health problems.
The smallest structures of enzymes are amino acids. They are the nitrogen-containing basic building blocks of proteins, so they are used in the synthesis of many of them. A total of 20 of them exist in nature, unique only by their R groups. Eight of them are essential amino acids, meaning that they are not created in the body, but they are necessary, so they must be consumed to be acquired. They carry out significant functions from providing aid in building cells, RNA, and DNA, tissue repair, antibody formation, and helping muscle activity (Kimpel, 2000). They may come together in a process called dehydration synthesis, which means a loss of water, but a combination of two amino acids. A peptide bond keeps them together, and they form a protein.
The essential amino acids, as identified by Christensen (2011), would be histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan and valine. They must be acquired through one’s diet, otherwise the body will break down other things like muscle to find reserves or substitutes. Even though the body cannot make them, plants must obviously be able to provide them. Alanine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, proline, serine and tyrosine are the non-essential amino acids that the body can produce naturally. Cysteine and glutamic acid may become essential when the body is stricken with serious illness. As a matter of fact, supplements of any of them can help cure some diseases or conditions.
For instance, scientists at the National Institute of Medical Sciences and Nutrition in Mexico City, Mexico found that when they provided arginine and citrulline to cardiology patients, they had a beneficial effect on their heart functioning. As previously stated, amino acids come together to form proteins. There are several different kinds of them in the body and in nature. On a molecular level, they are polymers of amino acids; therefore they may also be called polypeptides. They are complex molecules whose functions are determined by their shape. The smallest structure of a protein is the primary structure. Put simply, it is a sequence of amino acids joined together by peptide bonds in a long chain. (Clark, 2012). This specific sequence is unique to every protein, and no two are the same. Loss or removal of even one amino acid from the chain will affect the ability of the protein to function.
By appearance, it can be said that the primary structure resembles a ribbon. When the “ribbon” coils into an alpha helix, or folds into a beta-pleated shape, it is now in its secondary structure. In the alpha-helix shape, the amino acid chain is coiled in a way that it looks like a spring. The beta pleated sheet, they’re folded so that they lie next to each other, and head in opposite directions. Multiple hydrogen bonds are created in both shapes to keep them together. When those folds or coils fold over once more, it creates the tertiary structure of proteins. Now the overall shape of the chain in a ball of amino acids that are cleverly, and intricately twisted around each other. In this stage, the molecule is now three dimensional, and in models or diagrams, the primary and secondary structures may still be identified within this tertiary one.
This shape comes to be due to the non-covalent interactions between amino acids to help it hold its form. (Bowen, 2002). The quarternary structure is the last possible structure of the amino acid chain, which is a combination of at least 2 tertiary structures. From a biological point of view, proteins are both present in and acquired for the body. It is widely known that they are essential to good health. What people really need them for are the amino acids that they contain. According to the Division of Nutrition, Physical Activity, and Obesity in the Center for Disease Control and Prevention (2012), there are 3 categories of proteins: complete, incomplete, and complementary. A complete protein provides all of the essential amino acids previously discussed, and they may also sometimes be called the high quality proteins.
Good sources of complete proteins are any product that you can obtain from an animal, such as meat, eggs, cheese, and fish. The second best protein category would be an incomplete protein, that lack only one or more of the essential amino acids. A complementary protein is similar to an incomplete protein. It’s one that lacks some of the amino acids, but is usually combined another food that can provide the ones that are missing. One example is rice and beans; rice itself does not have enough of the essential amino acids to really benefit the body, but when consumed with beans, the intake of essential amino acids is increased to a healthy amount. Professionals recommend that 10-35% of daily intake should be protein.
Enzymes are the specific protein that investigated by the researcher. They are also known as catalysts, because they speed up molecular reactions. Their relationship with substrates is demonstrated in the “lock and key” model. In that model, the enzyme is the lock, and the substrate is the key that “unlocks” the reaction. The substrate can fit perfectly into the active site on the enzyme, and that process is called competitive inhibition. Substrates are made to fit specifically into one enzyme shape. In allosteric inhibition, an allosteric inhibitor molecule moves into its regulatory site on the enzyme after the substrate has changed the active site’s shape. Diastase is the specific enzyme the researcher worked with in the experiment. It plays an important role in the digestive system, but is also known to breakdown starch into glucose molecules.
Denaturation is the unraveling of a protein’s structure, and since that trait is responsible for their proper functioning, a change means a useless enzyme that can’t do its job. In the study, the researcher tested how the concentration of an enzyme, the temperature it was at, and the pH level it was exposed to effected the overall reaction. It made sense to hypothesize that the higher the concentration of enzyme that exists in a solution, then the faster the reaction between enzyme and substrate will happen. Enzymes can only bind with one substrate at a time, so if there are excess proteins open and awaiting a substrate, then more of them will be catalyzed at the same time, and it would speed up the reaction. High temperatures also have this effect. In warmer solutions, molecules are moving rapidly, and bumping into each other often; therefore, in a solution with enzyme, the reaction will happen faster than in normal conditions because substrates move quickly into their active sites.
As for pH levels, the farther away it is from a neutral 7, the slower the reaction will take place. Increasingly acidic or basic solutions are the opposite of highly concentrated amounts of enzyme. The two extremes in pH can cause the denaturation of the structure of the enzyme, and re-shape it so that it doesn’t perform its job at all, or properly. The more of the enzymes that become unraveled, then the less will be in a good enough condition to carry out the reactions. Biuret’s solution is one chemical used in the experiment to detect the presence of the gelatin protein, before the enzyme protease was tested. This solution is a combination of sodium hydroxide and copper sulfate. When exposed to a protein, it turns purple in color, and changes to pink in the short chains of amino acids. It’s the copper ions that are present in the solution that cause this reaction. (Cara Lea Council-Garcia, 2002).
In the investigation, protease was added to a solution with Biuret’s already present in it, to see how it affected the gelatin protein. Protease, also known as papain, is a commercially available enzyme that is not naturally present in the body, but may be exposed to from certain bacteria. Reason being is that it causes severe conditions and illnesses in humans, because it hydrolyzes the peptide bonds found in proteins. When amino acids come together in dehydration synthesis, they form those peptide bonds to stay together at the cost of water, so when the papain comes in and adds the water again, the protein breaks down and malfunctions.
Proteins are essential to the body, so such an enzyme should be avoided as well as possible. The reason that the researcher used the series of tests to answer the hypothesis made is because proteins themselves are a very complex molecule to understand. The five activities carried out each gave the scientist new information on enzymes that greatly helped to comprehend their functions in the body, and their necessity to life.
One of the labs demonstrated how temperature affected enzyme activity, and another helped to explain what effect pH would have on the proteins. Another one of the activities showed how enzyme concentration affects the speed of reactions. Data from those trials combined with information gained from the several others helped the scientist find a specific answer to the hypothesis that was created. It was quickly learned how those factors affected the work of enzymes.
Materials and Method
To conduct this experiment, the researcher gathered certain materials in the laboratory. A total of 50 milliliters (mL) of diastase solution and distilled water were gathered, along with 40mL of starch solution. Another 10mL of gelatin and papain solutions were also necessary. The researcher used 2 glucose test strips, 3 medicine cups, 3 plastic pipets, 1 spot plate, 4 test tubes, and a stopwatch. Special substances included Lugol’s starch indicator solution, enzyme solution, dilute hydrochloric acid, dilute sodium hydroxide solution, and 30 mL of Biuret solution. Safety materials included an apron, protective gloves, and goggles, as the starch indicator solution is poisonous and skin contact is dangerous.
The first part of the experiment required the scientist to label 3 medicine cups and plastic pipets by the following: (1) enzyme solution, (2) distilled water, and (3) starch solution. Ten milliliters of each were poured into their respective medicine cup. Using the second pipet, the researcher added 5 drops of distilled water to each of the two wells on the spot plate. Using the third pipet, 2 drops of starch solution were added to the wells containing distilled water. The first pipet was used to add a single drop of enzyme solution to one well only, keeping the other as a control. After 3 minutes, the scientist dipped separate glucose test strips into each well, waited 10 seconds to observe a color change, and recorded the observation.
Then, a single drop of the starch indicator solution was added to the two wells, and another color change was observed and recorded. For the second part, the researcher used the same pipets and medicine cups. Using the first pipet, a single drop of enzyme solution was placed into each of the 12 wells on the spot plate. Using the second pipet, four drops of distilled water were put into each well. As for the third pipet, a single drop of starch solution was added to the wells next. The scientist then quickly placed a single drop of starch indicator solution to the first well on the spot plate and started the stopwatch for the “time 0” sample. After 30 seconds, a drop of the indicator was also added to the second well, and the stopwatch was restarted. Single drops of indicator were continuosly added to each well as 30-second intervals until there were blue-black color changes, and recorded the data in table #2 as trial #1. Following that, the spot plate was thoroughly rinsed and dried.
The same steps were followed using 2 drops of enzyme, 3 drops of distilled water, and 1 drop of starch solution in the spot plate wells. They were stopped for the same reason as before, which was an observation of no blue-black color change, and recorded as trial #2. After rinsing the spot plate again, the steps were repeated with 3 drops of enzyme, 2 drops of distilled water, and 1 drop of starch solution in each well, and the times were recorded as trial #3. For trial #4, again, the same steps were repeated, this time using 4 drops of enzyme, 1 drop of distilled water, and 1 drop of starch solution. The scientist used 5 drops of enzyme, 1 drop of starch solution, and no distilled water for trial #5. The third activity was conducted by first placing two drops of starch solution into each of the four corner wells on the spot plate.
The materials were then organized so that the next step could be completed rapidly. A cold enzyme solution was placed into one of the 4 corner wells, a room temperature enzyme solution was placed into the next, a body temperature enzyme solution was placed in the third, and a boiling enzyme solution was placed in the fourth. Five drops of enzyme solution were immediately added to each of the four corner wells, and the stopwatch was started. After 60 seconds, a single drop of starch indicator solution was added to each well, then the color changes were observed and recorded in the third data table.
For the fourth activity, 3 drops of distilled water were placed into each of three wells on the spot plate, and 2 drops of enzyme solution to the same 3 wells. One drop of hydrochloric acid was added to one of the three wells, one drop of sodium hydroxide was placed into the second, and one drop of distilled water in the third. Two drops of starch solution were added to all three welss, and timing was started immediately.
After 2 minutes, one drop of starch indicator solution was put in each well, and the final color change was recorded. The researcher obtained 10mL of warm gelatin solution, and added 8-10 drops of it into each of two wells on the plate. An equal amount of biuret solution was added to each of the two wells on the spot plate, to look for a purple color change. To one of the wells, a few drops of protease were placed in it, and the solution was observed for a few minutes to see the purple color disappear. Observations were recorded in the fifth data table.
The results showed that the enzymes break down certain substances into their smaller compounds, and that certain conditions such as the concentration of the enzyme, its temperature, and the pH level it’s exposed to have adverse effects on the reactions. Enzyme activity was first indicated in the first activity, when the solution that had enzyme added to it yielded a (……) test result for glucose, and a (….) result for starch. They indicated that the starch molecules were broken down into glucose. It was also observed that higher concentrations of enzyme take a longer time to do their job than lower concentrations, because the starch indicator solution stopped yielding blue-black colored results so soon.
Diastase enzyme also showed that it works best in the room and body temperature extremes. The cold and hot solutions yielded positive results for the presence of starch, so the enzyme didn’t work. The researcher also found that acidic and basic solutions inhibit enzyme reaction, because the hydrochloric acid and the sodium hydroxide yielded a positive test for starch as well. Papain, or protease, breaks down the protein gelatin into amino acids, but it did not do so in the experiment. The solution remained purple in color, so no breaking down of gelatin was observed, because it should have turned pink.