Bot110-Enzymes

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Feb 20, 2024

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Bot 110 Name Kylie Tyler____________ Enzymes Biochemical reactions in living organisms require the help of a special group of proteins called ENZYMES. Enzymes are reusable proteins that greatly speed up biochemical reactions. Because of their ‘reusable’ role, enzymes are referred to as biological catalysts. Enzymes carry out very distinct tasks by lowering the energy needed for these tasks (reactions). They act on specific starting materials called SUBSTRATES. Each enzyme can only take action on a particular type of substrate. The enzyme’s role is to somehow change the substrate into a ‘useful’ biological material. Cells are constantly battling to maintain a constant environment. This includes such physical parameters as pH, salt balance and temperature. This homeostatic balance is of great importance to a living cell because its enzymes have very specific pH, salt and temperature requirements. If these parameters change too much, a cell’s enzymes will no longer function (are denatured) and the cell will die. It is the purpose of this exercise to observe enzyme activity from a variety of biological source and observe the effects of pH and temperature on enzyme activity Part 1: Demo of Bromelain Enzyme Certain types of enzymes (PROTEASES) carry out the job of breaking down proteins into amino acids. These can be broken down further and can then be used to build other useful biomolecules. The cells in a pineapple contain the protease, bromelain. Bromelain is also found in pineapple juice (not surprising), which is why it is often used in marinades for meat. The enzymatic activity of bromelain will tenderize the meat by breaking down some of the meat protein. Unfortunately, it is difficult to visualize bromelain activity on meat. For this reason, we will use gelatin as a protein source to observe the proteolytic effects of bromelain. When gelatin is heated up it is in a liquid state. As it cools, the gelatin proteins interact to take on a semi-solid form. This solidified JELLO is a demonstration of protein interaction. Your instructor will prepare 350mLs of JELLO according to the following procedure: Beaker 1: JELLO, Water and FRESH pineapple; Beaker 2: JELLO, Water and cooked pineapple; Beaker 3: JELLO and Water). These beakers will be chilled for about 4 hours. Observe each beaker and record your results. Check in the boxes the consistency observed: Liquid Semi-solid Solid Beaker 1 – Fresh Pineapple X Beaker 2 – Cooked Pineapple X Beaker 3 – Water Only (Control) X Part 2: Amylase Enzyme Activity Safety- please replace the cap of the IKI solution when done using it and put it back in the ziplock bag of lab chemicals. Please wash your hands when you are done. Clean up any spills with a paper towel and dispose in trash. Starches are important energy storage molecules in plants. They are complex carbohydrates built from monosaccharide subunits glucose and stored in specialized plastids called amyloplasts. Because starches can readily be broken down to form useful sugars like glucose. In animals, starches are stored in the liver as a molecule known as GLYCOGEN. AMYLASES are enzymes that can break the various bonds that hold starches and complex sugars (polysaccharides) together. This enzyme activity will result in the release of maltose, which can then be broken down further to glucose. When you digest foods, the process of digestion begins in
your mouth. Your saliva contains amylases that immediately begin to break down starches. In this exercise you will observe the activity of amylase that is in your saliva that is able to break down starches from plants. 1)Prepare a starch solution: Add 2g of starch (use all of ziplock bag) to 75ml of cold water 2) Prepare the following 2 test tubes: Label one tube S for saliva and one tube C for control. Using a 10mL graduated cylinder to measure, add 3mLs of starch solution to the two different tubes. 3) Collect at least 4mLs of saliva in a 100mL graduated cylinder. You can chew sugarless, colorless gum to help produce saliva. 3) Once the saliva has been collected, quickly add 3mLs of saliva to the tube labeled S (use dropper to do this) and gently mix. Add 3mls of water to the tube labeled C (both tubes should now contain 6mLs of liquid) 4) Place the tubes in a beaker filled with 60C water (Hot tap water, about as hot as is comfortable on the inside of your wrist). Let this incubate for 15min. 5) Add 6 drops of IODINE swirl the tube and record the color on your data sheet. Blue Black – Starch (no conversion) Orange-red – Maltose Yellow-clear – Glucose . Tube with Saliva Tube with control Color after 15min Conversion Completed. Part 3: Potato Peroxidase Activity Some biochemical reactions produce unwanted by products such as PEROXIDES. Peroxides are byproducts of normal cellular metabolism, but they can be damaging to cells. However, nearly all living cells have evolved enzymes called PEROXIDASES that can degrade the peroxides (H 2 O 2 ) into H 2 O and O 2 . This release of O 2 causes the vigorous bubbling seen when Hydrogen Peroxide is placed on an open wound. In this exercise you will observe the enzymatic activity of PEROXIDASE (in a potato) as it degrades Hydrogen Peroxide into H 2 O and O 2 . H 2 O 2 peroxidase H 2 O and O 2 1) Cut two 5mm (thin) slices from the center of Solanum tuberosum (a potato). The slices all must be the same. If the slices are too thick it will be hard to tell what is happening. BOILED UNBOILED 2) Boil one slice for 3 min. (its works to just place the potato in a coffee cup of water and microwave it for 2 min.) Then place it on a paper towel to drain and cool. 3) Place the 2 slices on a piece of paper towel and label the paper so you can tell one slice from the next. 1 2 4 3
4) Cut theUNBOILED potato slice into 4 sections. Each section will be used for a different treatment. Designate each section as #1, #2, #3 or #4. Make sure you can keep track of which section is which! 5) Add the following solutions (one at a time) to the sections on the UNBOILED potato: Let the solution sit for 60 seconds, and then add 1 drop of Hydrogen Peroxide (H 2 O 2 ) to the same place. Section #1) Add 1 drop of dish soap (basic pH) Section #2) Add 1 drop of soda or vinegar (acidic pH) Section #3) Add 1 drop of 10% Sodium Chloride solution (mix a teaspoon of salt in 1/2cup of water) Section #4) Add 1 drop of water, then H 2 O 2 . 6) Add 2 drops of H 2 O 2 to each section and then wait 5 - 10 seconds and then observe any activity (bubbles) 7) Record your result on the data table below, rating the amount of bubbling (activity) on a scale of 0- 5. O = not activity, 5 = extreme activity Peroxidase Activity: Potato Treatment Activity Observed (0-5) Boiled 3 min. Potato Dishsoap (Basic pH) Vinegar (Acidic pH) 10% NaCl - table salt Water (Control) Conclusions – 1) After seeing the Bromelain Enzyme activity experiment, explain why it might be better to use FRESH pineapple juice (instead of cooked) in a meat marinade. Fresh pineapple juice contains active bromelain enzymes, which can effectively tenderize meat by breaking down its proteins. Cooking pineapple juice can denature or deactivate these enzymes, reducing their effectiveness in tenderizing the meat. 2) Why would it be important for animals to have evolved an enzyme that quickly releases glucose sugar from starch in the mouth? The evolution of this enzyme benefits animals by supporting energy production, enhancing digestion efficiency, ensuring immediate nutrient access, and aiding survival in challenging environments. 3) What is the Iodine used for in the Amylase Activity experiment? What does it do? Iodine is used in the experiment to test for the presence of starch. Iodine reacts with starch to form a blue- black color, providing a visual indicator of the presence of starches. In the experiment, adding iodine to a solution containing both amylase (enzyme) and starch, the disappearance of the blue-black color indicates that the enzyme has catalyzed the breakdown of starch into simpler sugars, such as maltose. This color change helps in determining the activity of the amylase enzyme on the starch substrate. 4) What observation did you make to determine if potato Peroxidase was active in part 3 of this exercise? Why does this happen? I was able to observe a change in color of the substrate solution after adding hydrogen peroxide. The color change indicated that the enzyme was actively breaking down hydrogen peroxide into water and oxygen. This occurs because peroxidase catalyzes the reaction between hydrogen peroxide and a substrate, leading to the
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formation of a colored compound. Overall, the observation of the color change confirmed the activity of peroxidase in the potato extract. 5) Why is it important for potatoes (or humans) to have peroxidases in their cells? Peroxidases are crucial enzymes present in potatoes and humans, playing significant roles in biological processes. In potatoes, peroxidases aid in defense against pathogens, wound healing, and creating defensive compounds. In humans, they contribute to the immune response, thyroid hormone production, and detoxification. Overall, peroxidases are essential for maintaining cellular health and function in both organisms. Part 4: Extra Credit – Amyloplasts in Potato (2 points) Make a wet mount slide of potato. Use a butter knife to gently scrap the open surface of a potato to collect cells. Spread this material onto a clean microscope slide. Add a drop or two of the IKI starch indicator solution onto this sample and then cover with a cover slip. View this slide at 400X. Make a drawing (color this) of your observations. Then label the plasma membrane, cytoplasm, and amyloplasts (these will be a bluish purple color)

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