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AP Biology Lab Factors Influencing Enzyme Activity

Biology Lab
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on 22 October 2012

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Transcript of AP Biology Lab Factors Influencing Enzyme Activity

AP Biology Factors Influencing
Enzyme Activity Lab By: Chase, Brian, Walter, and Nick The General Questions: 1. Balanced Equation: 2 H2O2-->2 H2O + O2 2. Enzyme: Catalase 3. Catalase is found in highest concentration
within the liver in the human body. Catalase breaks down H202 into water and oxygen. It allows the liver to filter such toxins from the blood formed by a byproduct of metabolic processes.
4.) The rate was determined by taking an accurate reading of the amount of oxygen being produced over a period of 30 seconds. We applied a designated amount of H202 with 1 ml of yeast. As the oxygen level rose within the submerged graduated cylinder we took an accurate measurement. What is Catalase? Catalase is an enzyme found in all organisms (mainly the liver for humans) that catalyzes the decomposition of hydrogen peroxide to water and oxygen. Part A: The Time Course of Enzyme Activity Time (30 second intervals) mL O2 Time Course of Enzyme Activity Water Temperature: 24 degrees Celsius Rate: .5 ml/sec , Possible Factor: Didn't put yeast back to get heated/mixed Rate: (30, 9) (60, 19) -> (19-9)/(60-30) -> 10/30 -> 1/3 -> .33 ml/sec Rate: .36 ml/sec Initial Rate: .345 ml/sec or .35 ml/sec Starting Time(s): 0 sec.
Starting ml O2 : 0 ml Part B: Enzyme Concentration .75 ml of Catalase Solution .50 ml of Catalase Solution .25 ml of Catalase Solution Rate: .30 ml/sec Rate: .23 ml/sec Rate: .10 ml/sec ml
02 Time (30 second intervals) Effect of Enzyme Concentration Part B Questions
Q1.) Summarize the effect of concentration on enzyme activity.
1.) As the concentration of enzyme decreased, so did the amount of oxygen that was being produced.
As the process takes place, the yeast concentration reacts with the hydrogen peroxide and produces oxygen. With each new trail, less amounts of yeast were added into the reaction. This, in turn, resulted in less amounts of oxygen to be measured. If compared to the control, we see that a high amount of oxygen was produced with a full 1 ml of yeast. with the three trials of .75, .5, and .25 ml of yeast. lower readings of oxygen was recorded
Q2.) What would cause the rate of enzyme activity to level off on the enzyme concentration graph?
2.)If an enzyme reaction reaches a maximum effecienc,y in which concentration and enzyme are at an optimum production level, the reaction would plateau. When put into a graph, the reaction would slowly increase before reaching the climax and then level off. Part C: Temperature Time (30 second intervals) Effect of Temperature on Catalase Starting Time(s): 0 sec.
Starting ml O2 : 0 ml Part D: Effect of pH on Catalase The Experiment Purpose: The purpose of this experiment is to investigate the effect of different environmental variables on the enzyme Catalase. The Apparatus: Enzyme Utilized: Catalase Substrate: H202 Product Measured: O2 Products of Substrate:
H20 + 02 Factors: Time, Enzyme
Concentration,
Temperature, pH, Ionic
Concentration, Substrate
Concentration Boiling Catalase to 100 degrees Celsius 5 degree Celsius water temperature 37 degree Celsius water temperature Rate: 0 ml/sec Rate: .2 ml/sec Rate: .5 ml/sec Starting Time(s): 0 sec.
Starting ml O2 : 0 ml ml
O2 Sample Calculation: (150,89) (180,104) -> (104-89)/(180-150) -> 15/30 -> .5 ml/sec Sample Calculation: (30,2) (9,60) -> (9-2)/(60-30) -> 7/30 -> .23 ml/sec Effect of pH Time (30 second intervals) mL
O2 pH of 4, 5 ml of H202 with 4 pH buffer pH of 10, 5 ml of H202 with 10 pH buffer Rate: .067 ml/sec Rate: .1 ml/sec Sample Calculation: (210,31) (240,33) -> (33-31)/(240-210) -> 2/30 -> 1/15 -> .067 ml/sec Starting Time(s): 0 sec.
Starting ml O2 : 0 ml Part C Questions
Q1.) Summarize the effect of temperature on enzyme activity
1.) in regards to temperature, the reaction is very sensitive to changes in heat or lack of. The optima temperature for enzyme reactions to take place is at 37 degrees Celsius. When brought to the extreme of 100 degrees Celsius, the enzyme denatures and produces zero amounts of oxygen. When placed in the other extreme of 5 degrees Celsius, the production of oxygen is slower than that of warmer temperatures. Finally when tested at 37 degrees, the reaction produces oxygen at such an excessive rate that it peaks the scale at the 3 minute mark with a rate of 8 ml per second.
Q2.) Explain the specific effect of boiling the enzyme
2.) When the enzyme is brought to a boil, the enzyme itself becomes denatured. When placed into the reaction with yeast, no oxygen is produced, thus no measurements of change and rate can be taken.
Q3.) What is the temperature optima foR human enzymes?
3.) 37 degrees Celsius Part E: Effect of Substrate Concentration 10 mL of Distilled Water .3 % , 3mL H202 + 7 mL Distilled Water Rate: 0 ml/sec 5 ml of H202 + 5 ml water Rate: .1 ml/sec Rate: .2 ml/sec Starting Time(s): 0 sec.
Starting ml O2 : 0 ml Part F: Effect of Ionic Concentration Time (30 second intervals) mL
O2 Effect of Ionic Concentration 10% of NaCl 2% of NaCl Control: 5 ml water + 5 ml H202 Rate: .1 ml/sec Rate: .12 ml/sec Rate: .1 ml/sec Starting Time(s): 0 sec.
Starting ml O2 : 0 ml Sample Calculation: ( 120,17) (150,20) -> (20-17)/(150-120) -> 3/30 -> 1/10 -> .1 ml/sec Sample Calculation: (90,22) (120,28) -> (22-28)/(120-90) -> 6/30 -> 3/15 -> 1/5 -> .2 ml/sec Part D:
Q1.) Summarize the effect of pH on enzyme activity.
1.) The catalase produces oxygen faster in pH 10 rather than in pH 4. This is due to the fact that pH 10 is closer to the catalases optima environment when compared to that of pH 4. If you look at the control (pH 7), oxygen is produced a lot faster than either pH 10 or pH 4. This suggests that the catalase works best in an environment with a pH a little over
7.
Q3.) What is the pH optima for catalase?
3.) 7
Q4.) Do all human enzymes function within the same optimum pH range like they do with temperature?
4.) Not all human enzymes function within the same optimum pH range. Different pH levels are needed in order to carry out different functions of the body. These functions take place in different parts of the body, therefore, requiring different pH levels. The mouth has a pH level of 6.5 or higher which helps in the breakdown of starch whereas the stomach has a pH of around 3 in order to break down all of the foods. Part E Questions:
Q1.) Summarize the effect of substrate concentration on enzyme activity.
1.) The higher the substrate concentration is, the faster the reaction is supposed to take place. The reaction increases with the amount of substrate until the enzyme saturation has been reached. In our lab, we proved this to be true, as you can see from our charts.Although the we yielded correct results, they have the capability of being compromised depending on whether or not the solution is either too acidic or too basic.
Q2.) What would cause the rate of enzyme activity to level off on the substrate concentration graph?
2.) The enzyme reaching its point of saturation would cause the rate of enzyme activity to level off on the substrate concentration graph. Part F Questions:
Q1.) Summarize the effect of ion concentration on enzyme activity
1.) Ion concentration decreases the frequency of enzyme-catalyzed reaction. This is because the ions denature the enzymes. The ions bond to the r-groups and disrupt the structure of the enzymes as old r-group bonds are broken and new bonds are made. Personal Enzyme Catalysis Experiment Purpose: The purpose of this lab is to investigate the effect of an environmental variable on the catalytic function of an enzyme Enzyme Used: Protease Rate of Change Graph Substrate Concentration Time (30 second Intervals) ml
O2 Rate of Change for Control ml
O2 Time (30 second intervals) Rate of Change for Enzyme Concentration ml
O2 Time (30 second intervals) Rate of Change for Temperature Change ml
O2 Time (30 second intervals) Time (30 second intervals) ml
O2 Rate of Change for pH Barrier Rate of Change for Substrate Concentration ml
O2 Time (30 second intervals) Where is Protease found: Protease can be found within household items such as detergents or meat tenderizers. Hypothesis: The group believes that the Protease found in Bromelain will be able to dissolve jello to a liquid form (from a solid) in a certain amount of time. The group will be controlling water temperature as its environmental variable. The group predicts that the higher the water temperature, the faster protease will be able to dissolve the jello. Materials Used: Jello (cherry flavored), Bromelain, 2 Graduated cylinder, 10 ml graduated cylinder, test tubes, test tube holder, stirring rod, spoon, ring stand, hot plate, water, pestle and mortar Instructions:
1.) Buy a package of jello along with Bromelain pills.
2.) Use a cup of jello and place it into a graduated cylinder, record the volume of the graduated cylinder as well as the mass of the gelatin. Use water displacement if needed to measure the amount of gelatin within the cylinder.
3.) While measuring the volume of the jello, use a pestle and mortar with the bromelain pills to grind them into a fine powder, use only one pill per trial unless otherwise specified.
4.) Pour the now grinded tablet into 20 ml of water, mix thoroughly with a stirring rod and pour into a test tube.
5.) Time for 30 second intervals for 5 minutes and pour the mixture of water and protease into the graduated cylinder, record volume difference (if any).
6.) Repeat this procedure using differing factors such as water temperature and amount of protease used, record all results, calculate rate. Time (30 second intervals) Effect of Protease on Jello Protease Experiment Rate of Change of Protease Experiment Time (30 second intervals) Conclusion: In Conclusion, our experiment failed horribly. The group was not able to measure the rate in which protease dissolved jello back into a liquid. The group hypothesized that by mixing the Bromelain pills with water and pouring into a gelatin at varying temperatures, the protease within the Bromelain would break down the gelatin causing it to become a liquid. The Bromelain pills did not dissolve the gelatin into a liquid causing the experiment to not be successful.
Many factors could have lead to the reason why the experiment failed. First, the type of jello used may have caused a problem. Store bought jello may be less dense then homemade which could cause it too not dissolve as well in protease. Another problem the group noticed was a precipitate that formed when the Protease powder mixed with the water. When mixed, a thick, murky brown precipitate formed, creating a dense liquid that needed to be stirred. Perhaps using water with the Bromelain pills produced an error in testing. Inaccurate readings of volume may also have played a critical role in the failure of the experiment. Getting a solid into a graduated cylinder was no easy task and required using water displacement to approximate the measurements. There is also a chance that 500-1000 Mg of protease was not a sufficient amount to dissolve the gelatin. It may be required that a larger amount such as 2000-3000 mg of protease be even close to begin breaking down the gelatin.
Many things could have been done different within this experiment. A more precise reading of results would produce a more accurate reading of the measurements. Using a different provider of protease may have lead to stronger concentrations of it rather then using 500 mg of Bromelain. Using a different provider of protease may also result in not forming a precipitate with water.
Through our experiment we found that proteaSe has no effect on gelatin and does not break it down. This conclusion can likely be challenged and proven wrong thorough further testing. Suggested multiple tests are in order to provide any new data. Data and Observations: Data:
-Weight of Gelatin: 96 grams
- Water Temperature Control: 26 degrees Celsius
-Density of Gelatin: Density = mass/volume -> 96 grams/ 85 ml -> Density = 1.13 g/ml
-Constant: 20 ml of 26 degree Celsius water with 500 mg crushed protease tablets Observations:
-Hard to get jello into graduated cylinder, had to use water displacement to find accurate volume
-Water Temperature fluctuating slightly
-Thick precipitate forms when Bromelain powder comes into contact with water
-No real dissolving of jello when powder+water is added
-Attempted to use double amount of Protease (no effect), Higher water temperature (no change) Initial ml of Jello: 30 ml
Initial time: 0 seconds Water Temperature 37 degrees Celsius Control, 20 ml of water, 500 mg Protease 1000 mg Protease Rate: 0 Rate: 0 Rate: 0 5.) Our control for this experiment was too take 1 ml of yeast (catalase) solution and 20 ml of H202. This gave a clear base line of measurements to record over the 30 second intervals. Other processes required less amounts of yeast therefore our control gave us a limit to how our reactions should undertake. Time (30 second intervals) mL
O2 Effect of Substrate Concentration
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