Lab discussion
- 1. Biochemistry Activity 3A Enzymes Group 3 – BMLS-2A
- 2. To be able to know the properties of an enzyme To be able to test for the presence of an enzyme To be able to test the specificity of an enzyme Objectives
- 3. Procedures Preparation for Potato Extract: 1. Peel and grate a potato into 100 mL distilled water. 2. Let it stand for 10 minutes. Stir occasionally. 3. After 10 minutes, strain through cheese cloth. 4. Filter the extract using filter paper. Use the extract in the following tests.
- 4. Procedures Biuret Test 1. To 1 mL of extract, add 2 mL of 10% NaOH Solution. 2. Mix thoroughly and add 2-3 drops of 1% Copper Sulfate solution. Observe the appearance for violet color.
- 5. Result: Blue (negative) Expected result: Violet (positive) Results and Observations
- 6. Procedures Test for Catalase Activity 1. To 5mL of extract, mix it with 1 mL of 3% H2O2. 2. Observe whether the gas evolve supports combustion, by way of holding a splinter over the mouth of the test tube. 3. Add 1 mL of 0.5% Benzidine. Note the formation of the blue to green coloration.
- 7. Result: Brown (negative) Expected Result: Blue to green coloration Results and Observations
- 8. Procedures Preparation of a dilute solution of Salivary Amylase 1. Rinse mouth several times with water. 2. Collect 1 mL of saliva. 3. Prepare a 1:8 solution of saliva by adding 0.25 mL of saliva to 20 mL of distilled water. Use prepared test in the following tests.
- 9. Procedures Test for Specificity of Enzyme Action 1. Prepare two test tubes, each containing 2 mL of 0.02M Phosphate buffer pH 6.7 and 1 mL of 0.9% Sodium Chloride solution. 2. Test tube no. 1: Add 1 mL cooked starch and 1 mL of salivary amylase solution. 3. Test tube no. 2: Add 1 mL 1% glycogen solution and 1 mL of salivary amylase solution. 4. Stand for 15 minutes at room temperature. 5. Stir the mixture in both test tubes. Place one drop of each solution in separate evaporating dish or spot plate. 6. Add a drop of Iodine solution. Observe the color produced. 7. Repeat the test at 5 minutes intervals for an hour. Tabulate results.
- 10. Result: Results and Observations Time (minutes) Test Tube No. 1 Changes Test Tube No. 2 Changes 5 positive As time passes, the color turns lighter. Negative No change. 10 Positive Negative 15 Positive Negative 20 Positive Negative 25 Positive Negative 30 Positive Negative 35 Positive Negative 40 Positive Negative 45 Positive Negative 50 Positive Negative 55 Positive Negative 60 Positive Negative
- 11. 1. Principle involved in the use of Biuret Test. Biuret Test is based on the ability Cu (II) ions to form a violet-colored chelate complex with peptide bonds (-CONH-groups) in alkaline conditions. 2. How will you prove the presence of catalase? Show chemical equation. A rapid appearance of sustained gas bubbles is an indication of the presence of a catalase. 2 H2O2 2 H2O + O2 3. How will you prove the specific action of salivary amylase? Enzymes are normally very specific , catalyzing at most only a few types of chemical reactions. The enzyme amylase acts on the substrate starch catalyzing its break down into simple sugars. Amylase will not catalyze the break down of the complex sugar sucrose into simple sugars. Indicators can be used to test for the presence of these reactions like iodine solution in the iodine test, amylase in the starch (straight chain) forms helices where iodine molecules assemble and forming blue-black color. While amylopectin (branched portion) in glycogen forms much shorter helices and iodine are unable to assemble and only forming orange/yellow hue. Analysis and Conclusion
- 12. Biochemistry Activity 3B Factors Affecting Enzyme Action Group 3 – BMLS-2A
- 13. Describe the effect of temperature and pH in enzyme action. Explain how changes in pH and temperature affect enzyme action. Objectives
- 14. Procedures Effect of Temperature 1. Prepare three test tubes and label them respectively. 2. Test tube no. 1: 5 mL 1% cooked starch solution + 1mL saliva. Keep in water bath at 40ºC. 3. Test tube no. 2: 5 mL 1% cooked starch solution + 1 mL saliva. Keep in water bath at 60ºC. 4. Test tube no. 3: 5 mL 1% cooked starch solution + 1 mL saliva. Keep in water bath at 10ºC. 5. At 15 minutes interval, take a drop of the reaction mixture from each test tube, stir the mixture first before taking a drop, and test with iodine solution. Perform the iodine test for a period of an hour. At 60ºC At 10ºC At 40ºC
- 15. Result Results and Observations Time (minutes) Test tube no.1 @ 40ºC Test tube no. 2 @ 60ºC Test tube no. 3 @ 10ºC 15 White surrounded by black ring Blue black or something dark in color Light yellow with visible black ring formation in center Orange or something light in color Yellowish black *after 6 minutes – violet Blue black or something dark in color 30 Light yellow surrounded by black ring Blue black or something dark in color White with visible black ring formation in the middle Orange or something light in color Yellowish black *after 40 minutes – black Blue black or something dark in color 45 yellow surrounded by black ring Blue black or something dark in color Light yellow with visible scattered black particles Orange or something light in color (darker) yellowish black *after 10 mins. – Light Violet Blue black or something dark in color 60 Dark yellow Blue black or something dark in color violet Orange or something light in color Dark yellow Blue black or something dark in color At 40ºC At 60ºC At 10ºC
- 16. Procedures Effect of pH 1. Prepare four test tubes containing equal amounts of egg white. Place the test tubes in hot water bath. When the egg white is coagulated, cool n=and mark the height of the solidified egg white. 2. Test tube no. 1: 5 mL 2% pepsin + 10 drops 0.4% HCl 3. Test tube no. 2: 5 mL 2% pepsin + 10 drops of 0.4% Na2CO3 4. Test tube no. 3: 5 mL 2% pancreatin + 10 drops of 0.4% HCl 5. Test tube no. 4: 5 mL pancreatin + 10 drops 0.4% Na2CO3 6. Keep the test tubes in a beaker of water controlled at 40ºC for one hour. 7. Use a pH paper to determine specific pH of each test tube. 8. Determine the extent of digestion in each test tube by noting the amount of protein dissolved or disintegrated. 9. Perform Biuret Test of 1 mL of supernatant liquid from each test tube. 10. Compare the colors obtained with 0.5% peptone standard.
- 17. Result Results and Observations Test tube pH Color Expect ed color Result No 1. pepsin + HCl Acidic Purple Purple + No. 2 pepsin + Na2CO3 Basis Purple Purple + No. 3 pancreatin + HCl Acidic Purple Purple + No. 4 pancreatin + Na2CO3 Basic Purple Purple +
- 18. 1. Explain the mechanism of enzyme action at varying temperature. Give the principle involved. Being a protein, an enzyme is denatured at high temperature and becomes inactive. Initially as the temperature increases, the rate of enzymatic reaction also increases but it has a limitation. When the temperature affects the molecular structure of the enzyme it gets inactivated. 2. How does different pH affect enzyme action? Explain your results using this principle. Discuss Isoelectric pH enzyme action. Enzymes are picky with pH levels, as they are with everything else. They have an optimal level at which they work the best, and anything above or below that level, their activity begins to slow down until they shut down all together. Analysis and Conclusion