Lab Report 1 Principles of Biology 1 (BIOL 100) Fall 2001 Gerard Chretien Living cells perform a multitude of chemical reactions very rapidly because of the participation of enzymes. Enzymes are biological catalysts, compounds that speed up a chemical reaction without being used up or altered in the reaction. The material with which the catalysts reacts, called the substrate, is modified during the reaction to form a new product. But because the enzyme itself emerges from the reaction unchanged and ready to bind with another substrate molecule, a small amount of enzyme can alter a relatively enormous amount of substrate.

This report will illustrate the enzymatic action of the enzyme, which is common in plants. To study this particular enzyme in a laboratory, the natural substrate catechol is oxidized by the removal of two hydrogen atoms. The substrates of the enzyme are catechol and oxygen. These sub states react with one another within the active site of the enzyme. The products formed by this reaction are has a brown color, you can see that the reaction has taken place. This is called the fruit browning reaction.

Benzo quinone inhibits the growth of microorganisms and prevents damaged fruit from rotting. In undamaged cells is stored in vesicles and does not interact with catechol. In the presence of the enzyme: Catechol+1/2 O 2  +H 2 OThe structure of the enzyme is mainly dependent on the active site and variable groups. Extreme temperatures or extreme p Hs can alter the structure of an enzyme. Enzymes function to lower the activation energy to break the bonds. They achieve this by putting stress and pressure on the bonds or creating a micro environment for the substrate.

A change in the temperature or a fluctuation in pH can alter the enzyme's structure. Anent temperature the alteration of the enzymes occurs when the temperature is very high and the enzyme denatures and is unable to perform the desired task. The temperature is so high that the active site of the enzyme changes and it is unable to bond with substrates. Part I: Effect of Temperature on Enzyme Activity (Invertase) The rate of an enzyme-catalyzed reaction will increase with temperature to maximum rate and then sharply decrease. The rate increases to about 40 degrees Celsius, at which point the structure of proteins is disrupted (denaturation), resulting in an abrupt loss of enzyme activity. PROCEDURE: 1.

Number 3 test tubes (1, 2, and 3) with a permanent marker. Add 4 ml of a sucrose solution to each test tube. 2. Place tube 1 in a beaker of ice water; place tube 2 in a beaker of water at room temperature, and tube 3 in a beaker of boiling water. Wait 10 minutes for the sucrose solution in the tubes to equilibrate with the temperature of the water in the beakers.

3. After 10 minutes, add 0. 5 ml of 25 percent invertase (an enzyme derived from yeast) solution to each tube (swirl the invertase solution before use). Cover each tube and invert it several times to mix the contents during the 5 minutes. 4. After 5 minutes add 1 ml of Benedict's solution to each test tube.

Place the 3 tubes at the same time in a boiling water for 5 minutes. 5. Remove all 3 tubes from the water bath at the same time and observe the amount of precipitate and the color of the Benedict's solution in the tube. Then I would record my observation on a data graph sheet and write a statement concerning the effects of temperature on enzyme activity. Test Tube # Color Results Part II: EFFECT OF pH ON ENZYME ACTIVITY (Catechol Oxidase) The active site of an enzyme is affected by the presence of ions that change the charges within the protein molecule. The most favorable pH for an enzyme is called the optimum pH, because it is at this pH that the enzyme is most active.

A complete loss of enzyme activity will occur at extremely low or high p Hs because of the breakdown (denaturation) of the protein structure. PROCEDURE: 1. Number 5 test tubes (4, 6, 7, 8, and 10) with a water-soluble pen. Put 3 ml of phosphate buffer (pH 4, pH 5, pH 6, pH 7, pH 8, and pH 10) in the appropriately numbered test tubes. 2.

Add 10 drops of potato extract to each tube (an extract of potato will be used as the source for the catechol oxidase). 3. Add 10 drops of 0. 5 percent catechol solution to each tube and shake vigorously. 4. Shake the tubes at 1-minute intervals.

Examine the tubes after 5 minutes. The rate of the catechol oxidase activity is proportional to the color intensity that develops in the tube (the darker the brown color, the greater the enzyme activity). Then I would record my observations on the data graph sheet. Effect of pH on Catechol Oxidase Activity Test Tube Color Explanation pH - 4 pH - 6 pH - 7 pH - 8 pH - 10 Part III: Effect of Substrate Concentration on Enzyme Activity (Catechol Oxidase) If the amount of an enzyme is kept constant, but the amount of substrate is gradually increased, the rate of the reaction will increase to a maximum rate.

The maximum rate is the point at which the entire available enzyme is involved in enzyme-substrate complexes. PROCEDURE: 1. Number 6 test tubes (1, 2, 3, 4, 5 and 6) with a water-soluble pen. Add deionized water (pH is about 6. 5) and a 0. 5 percent catechol solution to the tubes as given below: #1 - 5 ml of deionized water + 1 drop of catechol #2 -- 5 ml of deionized water + 6 drops of catechol #3 - 5 ml of deionized water + 9 drops of catechol #4 - 5 ml of deionized water + 14 drops of catechol #5 - 5 ml of deionized water + 20 drops of catechol #6 - 5 ml of deionized water + 25 drops of catechol 2.

Add additional deionized water to each tube to make the level of fluid equal in all 6 tubes. 3. Add 30 drops of the potato extract to each of the tubes and shake. 4. Shake each tube at 1-minute intervals. After 5 minutes, examine the tubes and record the results of the experiment in the table below.

The rate of the activity is proportional to the intensity of the color developing in the tube (the darker the brown color, the greater the enzyme activity). Then I would record my observations of the tubes on the data graph sheet. Effect of Substrate Concentration on Catechol Oxidase Activity Test Tube Color Explanation 1 (1 drop) 2 (6 drops) 3 (9 drops) 4 (14 drops) 5 (20 drops) 6 (25 drops) Part IV: Effect of Pectin on ApplesaucePectinases are actually a mixture of enzymes, which along with others such as cellulose, are widely used in the fruit juice industry where they are widely used to help extract, clarify and modify fruit juices. To formulate a hypothesis about the effect of pectinate on applesauce I would utilize the hypothetic o-deductive (H-D) approach, which allows me to ask questions and test answers. The method has five stages: 1.

Making observation. 2. Asking questions. 3.

Forming hypotheses, or tentative answers to the questions. 4. Making predictions based on the hypotheses. 5.

Testing the predictions by making additional observations or conducting experiments. The information gained may support or yield opposite results based on predictions being tested. My independent variable would be time and the dependent one would be the enzyme pectinate. I believe the key feature of my experimentation is the control of most factors so that the influence of a single factor can be seen clearly.

The setting would take place within a laboratory, equipped with various components such as tubes, microscope and other related equipment. I would try to investigate the physical properties of pectin such as molecular weight, molecular conformation and aggregation of pectin molecules in the solution. In regards to the pulpiness of the applesauce, I would use the pectin as a emulsifier and stabilizer. This procedure would reduce the pulp of the applesauce considerably without making the solution too watery.