BMS 100 Enzyme Lab

Background
In a chemical reaction, one or more reactants are transformed into one or more products. The sum of all chemical reactions that occur in an organism is referred to as metabolism, a term that is based on the Greek word for "change."
In 1897, Hans and Eduard Buchner inadvertently used yeast extracts to ferment (change) sugar into alcohol in the absence of living yeast cells. The term "enzyme" was used to describe the substance(s) in yeast extract that brought about the fermentation. It was not until 1926, however, that the first enzyme was obtained in pure form.
An enzyme is a protein that catalyzes, or speeds up, a specific chemical reaction. Enzymes are essential to sustain life because, without them, most chemical reactions (metabolism) in cells would occur too slowly, or would lead to different products.

Substrate (reactant) attaches to active site of enzyme, enzyme facilitates chemical reaction, products are released, and enzyme is ready to repeat the cycle with a fresh substrate molecule.
Animations: How Enzymes Work | Amylase and Starch Digestion

The substrate must "fit" into the active site of the enzyme.

Peroxidase: A Model Enzyme
Free radicals are highly reactive molecules with unpaired electrons. A superoxide radical -- an oxygen molecule with an extra, unpaired electron -- is one type of reactive oxygen species and can be accidentally generated during energy metabolism or by exposure to UV and certain other types of radiation. Capable of causing oxidative stress, reactive oxygen species have been implicated in cell death, aging, and many disease processes. Reactive oxygen species are also generated in white blood cells where they are used to destroy bacteria.
Antioxidants are molecules that scavenge free radicals, thereby protecting the body from oxidative stress. Some antioxidants convert superoxide to hydrogen peroxide, which is itself potentially toxic. Vitamins C and E and other molecules present primarily in fruits and vegetables are also able to temporarily "quench" free radicals.
Peroxidase, also known as catalase, is an enzyme that is present in most cells but particularly active in the liver. It converts hydrogen peroxide to water and oxygen (see formula on handout) and is responsible for the "fizzing" that occurs when hydrogen peroxide is poured on a wound.




PART ONE – Effect of Shape of Substrate Molecule on Enzyme Activity

Rennin (chymosin) is a protease (protein-digesting enzyme) synthesized by cells in the stomach wall. Its role in digestion is to coagulate casein (milk protein) in the stomach, a process of considerable importance in a very young animal. If casein were not coagulated, it would rapidly flow through the stomach and miss the initial digestion of its proteins. Rennin efficiently converts liquid milk to a semisolid, allowing it to be retained in the stomach for a longer period of time.
    Procedure (please use the larger test tubes)
  1. Put approximately 3 ml. of soybean "milk" into a large test tube. Label the tube with S for soy milk.
  2. Put approximately 3 ml. of cow's milk into another large test tube. Label the tube C for cow's milk.
  3. Place the tubes into the warm water bath.
  4. Let the tubes stand for 5 minutes to allow the contents to come to bath temperature. While they are warming you may begin Part Two of today's experiment.
  5. After 5 minutes, add 1 ml. of the enzyme solution to each tube. Be sure to hold the tubes vertically so that the drops fall into the milk and not onto the insides of the tubes. Shake the tubes to mix the contents, then place them back into the bath and let them stand for 10 more minutes.
  6. After the 10 additional minutes, look at the contents of the tubes and indicate on your individual response form whether the milk was solidified (+) or not (-).
  7. Cleanup: Pour contents of tubes into sink (if not solidified). Do not attempt to remove solidified milk from tubes. Put all tubes back in racks.

PART TWO – Effect of pH on Peroxidase Activity
Do not eat, drink, or touch your eyes during this experiment.
    Procedure
  • Prepare the “liver homogenate:”
    1. put 50 ml. of cold water into a beaker or graduated cylinder
    2. use a “weigh boat,” scale, and instruments to obtain a 3 gram (approx.) piece of liver
    3. put all of the liver and pour a small amount of the water out of the beaker and into a mortar
    4. use the pestle to homogenize the liver
    5. pour the remainder of the water into the mortar; the resulting mixture is the “liver homogenate”
  • Put approx. 15 ml. of hydrogen peroxide into a beaker or test tube.
  • Use a large pipette and pump to transfer 2 ml. of hydrogen peroxide into each of five (5) small clean test tubes. Label them as follows:
    • Tube #1 (acid)
    • Tube #2 (base)
    • Tube #3 (neutral)
    • Tube #4 (neutral)
    • Tube #5 (neutral)
  • To the acid tube (#1), add a few drops of HCl, stir with a wooden stick, and touch the stick to a small piece of pH paper placed on paper towel. Add acid until the pH is approximately 3 or lower.
  • To the base tube (#2), add NaOH until the pH is approximately 10 or higher.
  • For the neutral tubes (#3-5), measure the pH of the hydrogen peroxide.
    • If the pH is between 6 and 8, proceed to the next step.
    • If the pH is not between 6 and 8, please inform the instructor.
  • Use a small plastic disposable pipette to add 1 ml. of liver homogenate to each of the five tubes.
  • For tubes #1–3, agitate the tube and measure the maximum height to which the foam rises above the top of the liquid (not the bottom of the tube). If the foam flows over the top, record as "overflow" for that tube. Record the heights on your individual response form.
  • Agitate Tubes #4 and #5 and place them in a warm water bath to incubate.
  • Periodically remove Tubes #4 & #5 from the water bath and agitate them.
    • If new bubbles or foam form in either tube, place both tubes back into the bath.
    • If no new bubbles or foam form in either tube, proceed to the following steps:
    1. Add another 1 ml. of liver homogenate to Tube #4. Use a wooden stick to thoroughly mix the contents of the tube. Does the reaction resume with fresh liver homogenate? (Record answer on Response Form.)
    2. Use a large glass pipette to add 2 ml. of fresh hydrogen peroxide to Tube #5. Use a wooden stick to thoroughly mix the contents of the tube. Does the reaction resume with fresh hydrogen peroxide? (Record answer on Response Form.)
    When finished, please rinse glassware, mortars & pestles with running water and wash your hands thoroughly.
    Stay in the lab area for the review and quiz.