Introduction: E + S > ES > E + P Enzymes are proteins which act as a catalyst in biochemical reactions(affect the rate of achemical reaction). The substrate binds to the active site of the enzyme. Any deformation of the active site will affect the activity of the enzyme, these are some ways that enzyme action may be affected because of them: 1- Salt concentration: If it is close to zero or very high the enzyme will precipitate . 2- PH: Neutral range is found to be the best for most enzymes, they can be denatured by higher or lower levels.
3- Temperature: Chemical reactions can speed up by raising the temperature although there is an optimum temperature of enzyme catalyzed reactions . 4- Activators: Increase rate of the reaction. 5- Inhibitors: Decrease rate of the reaction. Enzyme catalysis ( it is an experiment to calculate the rate of the reaction and how much substrate disappears over time in an enzymatic reation. ) The hypothesis: If the conversion of hydrogen peroxide to water and oxygen by the enzyme catalase is observed , the amount of oxygen generated can be measured and the rate of the enzyme catalyzed reaction can be calculated.
The primary reaction catalyzed by catalase is the decomposition of H2O2 to form water and oxygen. 2H2O2 > 2H2O+O2(gas) Catalase + Hydrogen Peroxide –> Complex –> Catalase + Water + Oxygen The materials: * 1. 5% H2O2 , H2SO4 , at least 400 ml of catalase and 2% KMNO4. * Distilled water * Pipette (for adding water) * At least 4 syringes to avoid contamination * At least 8 beakers to perform titrations (for the reaction mixture, the chemicals and distilled water) General Procedure.
In this experiment the disappearance of the substrate, H2O2, is measured as follows: 1. A purified catalase extract is mixed with substrate (H2O2) in a beaker. The enzyme catalyzes the conversion of H2O2 to H2O and O2 (gas). 2. Before all the H2O2 is converted to H2O and O2 , the reaction is stopped by adding sulfuric acid (H2SO4). The sulfuric acid lowers the pH, denatures the enzyme, and thereby stops the enzyme’s catalytic activity. 3. After the reaction is stopped, the amount of substrate (H2O2) remaining in the beaker is measured. To measure this quantity, potassium permanganate is used.
Potassium permanganate (KMnO4), in the presence of H2O2 and H2SO4 reacts as follows: 5 H2O2 + 2 KMnO4 + 3 H2SO4 -> K2SO4 + 2 MnSO4 + 8 H2O + 5 O2 Note that H2O2 is a reactant for this reaction. Once all the H2O2 has reacted, any more KMnO4 added will be in excess and will not be decomposed. The addition of excess KMnO4 causes the solution to have a permanent pink or brown color. Therefore, the amount of H2O2 remaining is determined by adding KMnO4, until the whole mixture stays a faint pink or brown, permanently. Add no more KMnO4 after this point. The Blank Assay..
To determine the amount of H2O2 initially present in a 1. 5% solution, one needs to perform all the steps of the procedure without adding catalase to the reaction mixture. This amount is known as the baseline and is an index of the initial concentration of H2O2 in solution. In any series of experiments, a baseline should be established first. Procedure for Establishing blank 1. Put 10 mL of 1. 5% H2O2 into a clean beaker. 2. Add 1 mL of H2O (instead of enzyme solution). 3. Add 10 mL of H2SO4 (1. 0 M) Use Extreme care in Handling Acids. 4. Mix well by swirling. 5. Use a new syringe to remove a 5 mL sample of this mixture.
Place this 5 mL sample in another beaker for “titration”, and assay for the amount of H2O2 as follows: Place the beaker containing the sample over white paper. Use a burette to add potassium permanganate a drop at a time to the solution until apersistent pink or brown color is obtained. Remember to gently swirl the solution after adding each drop An Enzyme-catalyzed Rate of H2O2 Decomposition: Procedure: 1. Label 7 clean cups as follows: 10 seconds, 30 seconds, 60 seconds, 90 seconds, 120 seconds, 180 seconds, 360 seconds. 2. Fill a beaker with approximately 80 mL of H2SO4. Be careful with H2SO4!.
If you spill it on your skin, flush with LOTS of water immediately! (This is a dilute sulfuric acid, but it will still burn if you don’t get it off. ) Get a 10 mL syringe and reserve it for sulfuric acid. (One group member should be in charge of sulfuric acid. ) 3. Get another beaker and put approximately 10 mL of catalase into it. Take it back to your desk and keep it on ice! (This is very important—catalase disintegrates quickly. ) Get a 1 mL syringe and reserve it for catalase. 4. Get approximately 80 mL of H2O2 in a plastic cup, and then put 10mL in each of the labeled plastic cups. 5. Get a stopwatch and figure out how to use it!
6. Put 1 mL of catalase in the “10 second” cup. Swirl gently and constantly for 10 seconds. At 10 seconds, add 10 mL of H2SO4 , swirl to mix (“alphabet time”), and set the cup aside. 7. Put 1 mL of catalase in the “30 second” cup. Swirl gently for 30 seconds. At the end of 30 seconds, add 10 mL of H2SO4, swirl to mix and set the cup aside. 8. Repeat #7 for each of the remaining cups, varying the amount of time at which you stop the reaction with H2SO4 . When you have completed the reaction in each of the cups, remove a 5 ml sample and determine the amount of H2O2 present by running a titration Results:
Blank: final reading of syringe(0 ml) – initial of burette (6 ml)= 6ml KMNO4 added to blank. The amount of KNO4 used is proportional to the amount of H2O2 present in the blank. The average for baseline is 5. 4375. KMNO4 (ml)| 60 sec. | 90 sec. | blank| 6 ml | 6 ml| final reading| 1. 2 ml| 1. 1 ml| initial reading| 5. 8 ml| 5. 8 ml| amount of kmno4 consumed (final- initial)| 4. 6 ml| 4. 7 ml| amount of h2o2 used (blank – amount of kmno4 used)| 1. 4 ml| 1. 3 ml| time (seconds)| H202 decomposition (ml)| 10| 1. 4| 30| 0. 4| 60| 0. 5| 90| 0. 5| 120| 2. 5| 180| -0. 1| 360| 2. 3| Enzyme catalyzed H202 decomposition Time (seconds).
H202 decomposition (ml) Reference : http://www. scribd. com/doc/82892303/AP-Lab-2-Enzyme-Catalysis-Lab http://www1. hudson. k12. ma. us/teachers/jsnyder/files/Lab02. pdf http://davisbiology. com/Documents/LAB%202%20Enzyme%20Catalse. pdf http://www. ptbeach. com/cms/lib02/NJ01000839/.
Centricity/Domain/113/ap%20biology%20Labs/Investigation%2013%20Enzyme%20Lab. pdf http://giannou. weebly. com/uploads/5/6/4/8/5648347/lab_2. pdf http://www. chem. qmul. ac. uk/iubmb/enzyme/EC1/11/1/6. html http://www. judeo-christian-co-op. com/catalase. htm http://www. ncbi. nlm. nih. gov/pubmed/8676564 http://www. gopetsamerica. com/dog-health/hypocatalasia. aspx.