Laboratory Lesson 2 Enzyme and Factors that Affects Enzyme Activity.pdf

Biochemistry for Medical Laboratory Science BACHELOR OF SCIENCE IN MEDICAL LABORATORY SCIENCE LECTURE/RECORDED VIDEO BASED Laboratory: Enzymes and Factors that affect  Enzyme and substrate concentrations enzyme activity...

Biochemistry for Medical Laboratory Science BACHELOR OF SCIENCE IN MEDICAL LABORATORY SCIENCE LECTURE/RECORDED VIDEO BASED Laboratory: Enzymes and Factors that affect  Enzyme and substrate concentrations enzyme activity CATALASE OBJECTIVES  To protect them from oxidative damage. 1. To isolate and determine the properties of  It will occur when cells or other catalase molecules in the body come into 2. To determine the effect of sulfides on the contact with oxidative catalase activity compounds. 3. To prove the presence of a proteins factor in enzymes4)To determine the effect of Materials temperature on enzyme  1 pc small potato with peel TOPIC OUTLINE  Filter paper 1 Theory  6% H2O2 2 Materials  Filter paper or cotton/gauze 3 Procedures  Guaiac solution 4 Nature of Enzyme and its extraction  Saliva 5 Factors that affect enzyme activity  Starch solution  Acidified iodine solution (0.001N) Theory  0.1N NaCl  0.05N HCl  Enzymes are complex organic substances  0.05N NaOH produced by an organism to alter the rate of  Blender biochemical reactions that take place in the cell.  Funnel  Cheesecloth  In line with metabolic processes which  Wooden splint provides energy for the maintenance life support system of an organism. NATURE OF ENZYME AND ITS EXTRACTION  Consist of degrading chemicals relatively with high potential energy to the PROCEDURES products with low potential energy. A. Preparation of Catalase Enzyme Examples of degrading chemicals of those enzyme:  Grate a potato, including the peel, into a fine  Conversion of glucose to carbon dioxide pulp (a blender may be used for this). Add and water 50mL of water in a beaker and allow the  The energy evolved is collected, mixture to stand  It is also stored. for 10minutes,  It is utilized by cell growth. swirling  Intended for reproduction and occasionally synthesis.  It is also included within the  Strain through cellular repair materials. cheesecloth and discard the  Show high degree of specificity residue. Filter again through  All enzymes have a protein component and filter paper. are affected by a number of physical and (NOTE: If filtering chemical factors that denature proteins through filter paper takes too long, use cotton or gauze).  Extremes of pH and temperature Use the filtrate for the following tests:  Presence of metal and non-metal ions  Presence of alkaloidal reagents TRANSCRIBED BY JEAN HERSHEY REYES 1 Biochemistry for Medical Laboratory Science BACHELOR OF SCIENCE IN MEDICAL LABORATORY SCIENCE LECTURE/RECORDED VIDEO BASED  Mix 5mL of the filtrate and 5mL of 6% B. EFFECT OF pH H2O2 in a test tube. Immediately insert a glowing wooden splint.  Label three test tubes and mix the substances indicated below:  There is a formation of bubbles, it is the oxygen upon exposure to hydrogen peroxide  It is indication that the catalase is present.  The oxygen gas is rising from the tube. We should test if the bubbles are really oxygen.  Catalase speeds up the decomposition of the  Mix well by shaking each test tube and hydrogen peroxide place in a water bath maintained at producing two harmless body temperature (37C). substances which is the water and the oxygen.  Recording the time at 3-minute intervals, test for the presence of starch using 0.001N iodine solution. B. Protein Nature of Enzymes  Record the time needed for the deep  To 5mL of the catalase extract, add 5mL blue color of starch with iodine to of 3M NaOH. fail to appear.  Mix the two solutions by tapping the tube against the palm of your hand.  From light orange brown original color.  Deep blue means that the  Add five drops of CuSO4 solution to the starch has been completely mixture. hydrolyzed to glucose.  Optimum pH for the  Purple Color- indicates that the proteins are enzymatic activity of salivary present in the extract. amylase ranges from 6-7  Salivary amylase is most active on 6.8 pH FACTORS THAT AFFECT ENZYME ACTIVITY  Our stomach has high level of A. PREPARATION OF ENZYME acidity which causes the salivary amylase to denature and change its  Extract 10-15mL of saliva from a shape. student donor and use it to test for  The salivary amylase does not the effects of pH and temperature function once it enters to the  Amylase stomach.  Our saliva contains special enzyme which is the salivary amylase. C. EFFECT OF TEMPERATURE  It helps with the digestion of  Label three test tubes; add 10mL of starch in our food. 1% starch suspension and 1mL of  Converts carbohydrates into 0.1N NaCl solution. sugars.  Immerse tube in a beaker of water at room temperature; tube 2 in a water TRANSCRIBED BY JEAN HERSHEY REYES 2 Biochemistry for Medical Laboratory Science BACHELOR OF SCIENCE IN MEDICAL LABORATORY SCIENCE LECTURE/RECORDED VIDEO BASED bath at 37C; and tube 3 in a boiling water bath.  Ranges from 32-37C  In low temperature (Tube 1), the amylase will break starch down slowly due to reduce kinetic energy.  Low temperature has possible to have blue color but the reaction is slow.  In high temperature (Tube 3), the amylase will break the starch slowly but not at all  Using a pipette, add exactly 2mL of saliva into each tube. Mix well and monitor in a manner similar to part II-B. Note the length of time it takes before the blue color with iodine no longer appears. TRANSCRIBED BY JEAN HERSHEY REYES 3