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AP BIOLOGY ENZYME LAB

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Chelsey Puffer

on 22 October 2012

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Transcript of AP BIOLOGY ENZYME LAB

BROMELAIN Found in stems and juice of pineapples or tablets What we did: 1. Experiment with Catalase to measure different factors of the enzyme's activity
2. Based off of what we learned, we made up our own experiment using bromelain Catalase catalyzes this reaction I. The Catalase Experiment 2H2O2-->2 H2O + O2 or Substrate→ Products In this lab, we analyzed the enzyme catalase. Catalase / cat·a·lase / (kat´ah-lās) Catalase is found in high concentrations in the liver. Livers work to detoxify substances that would cause to the body. Catalase is very useful in this process as it decomposes a dangerous substance, hydrogen peroxide, into harmless oxygen and water. In this lab, the rate of enzyme activity was determined by measuring the volume of O2 over the time it took to be produced. The first test served as the control, which we ran three times. The control is meant to act as a standard from which to compare all other results that have changed due to a changed variable. The first test merely showed the reaction rate of catalase at 26º, a pH of 7, a substrate concentration of 3.0%, and no ion concentration. Without knowing how quickly catalase decomposed hydrogen peroxide under these conditions, we would have no basis on which to compare how altering the conditions made the rate change. Control (Test #1) Part A ---------> Data Findings (Control) In this experiment, we saw how different substrate concentration effected the rate of enzyme activity. We tested the rate of enzyme activity in 0%, .3%, 1.5%, and 3.0% substrate. The 3.0% was the control we did in part A. Part E
Substrate Concentration Average of Controls 3.0% In the control experiment, there was no oxygen produced because there was no reaction taking place. There was no substrate for the enzyme to react with, therefore no reaction and production of product. Equation to Determine the Rate:
Slope = ∆y∆x = 100−22.6150−30 = 0.65 ml/sec .3% The 0.30% solution experiment shows that the reaction started off slowly with a rate of change of 0.167mLO2/sec. It increased slightly to 0.200mLO2/sec but then decreased until leveling off at 0.033mLO2/sec. 1.5% The 1.50% solution of substrate has a very similar rate to the .30% solution. The rate starts off slowly at a rate of 0.167mLO2/sec and increases slightly to 0.200mLO2/sec. The rate stayed in that general area and took a little while longer to level off at .033mLO2/sec. The Time Course of Enzyme Activity 3.00% The 3.00% solution started off at a higher rate of 0.467mLO2/sec. This reaction continues to increase. The rates of change slowly start to level off, but it is still reacting and reaches a rate of 0.100 at 270 seconds. Compared to the other reaction, this is later. The 0.30% and 1.50% reactions have this rate around 120 and 150 seconds, and then begin to level off. Part F
Effect of Ionic Concentration on Enzyme activity Part B The Effect of Enzyme Concentration
on Enzyme Activity In this experiment, we saw how using different ionic concentrations effected the rate of Enzyme activity. We used 10%, 2%, and 0% of NaCl. The 0% NaCl is the control for this experiment. Here we repeated the experiment from Part A, but used 75%, 50%, and 25% of the original enzyme solution. This allowed us to see the effect of the enzyme concentration on the activity. Part D
Effect of pH on Enzyme Activity Calculations:
Rate of reaction with 0.75 enzyme concentration
Slope = ∆y∆x = 90−20150−30 = 0.58 mL/sec
Rate of reaction with 0.50 enzyme concentration
Slope = ∆y∆x = 95−12300−30 = 0.31 mL/sec
Rate of reaction with 0.25 enzyme concentration
Slope = ∆y∆x = 47−5300−30 = 0.16 mL/sec For this experiment we changed the pH of the catalase by adding hydrogen peroxide. We then tested the rate of enzyme activity of these different pH values. We used pH values of 4, 7, and 10. Concentration and Rate The solution with the pH of 7 has a more gradual rate that starts off slowly, increases slightly them levels off around zero. The solution with the pH of 10 starts off with a faster rate than starts to level off at the end of the timed period. The solution with a pH of 4 has a very high reaction rate, it doesn’t even begin to level off at the end of the time trial. Based on other data, the reaction should begin to level off somewhere shortly after 300 seconds. 1. The reaction was slower as the enzyme concentration decreased. This makes sense because catalase works by interlocking with hydrogen peroxide in the active site, breaking its bonds, changing it into its products, and then returning to its original shape. When there is a higher concentration of enzyme, this means that more enzymes are able to decompose more H2O2 molecules at once, and thus creating a faster reaction. The amount of substrate did not change, so fewer enzymes could not do as much decomposition as more enzymes together could. Applying the Findings 2. The reason for the leveling off in the enzyme concentration graph has to do with the substrate acting as a limiting reagent. The enzyme could theoretically produce the same amount of product at a constant rate, but there was a limited amount of substrate to decompose. Once the catalase changed the bulk of the H202 into its products, it no longer had substrate left to change. With an infinite amount of substrate, the rate of enzyme activity would not level off in the manner it does in the graph. Part C The Effect of Temperature on Enzyme Activity II: Our Experiment Using Bromelain In this section, we repeated Part A again, but used 3 different temperatures: 5 degrees C, 37 degrees C, and 100 degrees C (boiled catalase). This allowed us to see the effect of different temperatures on enzyme activity. Slope Calculations:
Rate of reaction at 26ºC
Slope = ∆y∆x = 99−15240−30 = 0.40 mL/sec
Rate of reaction at 5ºC
Slope = ∆y∆x = 87−11300−30 = 0.28 mL/sec
Rate of reaction at 37ºCSlope = ∆y∆x = 103−30150−30 = 0.61 mL/sec
Rate of reaction at 100ºSlope = ∆y∆x = 0−0300−30 = 0 mL/sec Applying the Findings 1. The optima is 37º. Higher temperature means that there is faster movement among the molecules, which means more collisions. When catalase collides more frequently with the substrate, catalase decomposes it at a higher rate. On the other hand, when temperature is low, the enzyme and substrate do not come into contact as often and the rate is slower. Generally, a higher temperature means a higher rate, but at particularly high temperatures, the enzyme is denatured and ceases to function at all. Its form unraveled and its form was what enabled the decomposition in the first place. This is why we see the sharp decline to zero after the optima in the idealized graph above. 2. Boiling catalase causes it to gain too much kinetic energy. The tertiary and secondary structures are altered and it loses its precise form for decomposing hydrogen peroxide. In the case of catalase, this denaturing is irreversible. 3. The temperature optima for human enzymes is 37ºC. The solution with 10%NACL starts off a slightly slower rate, but begins to level off. The solution with the 2%NACL solution increases very high rate but slows down shortly thereafter. After some trial and error, we were able to get quantifiable data from our lab. This quantifiable data actually backed up our original thoughts, plans, and position. After removing the cups of jell-o and pineapple/bromelain solution (and our control of water), we discovered that the cups held jell-o like substances of different consistencies. The water was completely formed, with 0 grams of liquid. If held upside down, nothing would come out. This was our control, so these findings make sense, and tell us that the jell-o and method we were using to chill and form the jell-o were good and acceptable to use. The canned juice mixture was fairly formed, but had some liquid visible in the jell-o. When we poured this through the small sieve, we were able to measure the amount of liquid to be .95 grams. We believe that this is because the canned juice would contain less bromelain as it has gone through processing and the pineapple has been altered before entering the can, thus potentially losing some of the bromelain. Less bromelain would mean more jell-o formation, which would be backed up by our findings. Next, we examined the hot fresh juice. This seemed to have even more liquid in and around the formed jell-o. We strained it using the same method and found the remaining liquid to be 6.25 grams. This is a fair amount more, but the solid was still a much greater percent of the mixture as a whole. We believe that this is because the heat denatured some of the bromelain when we boiled the fresh juice before entering it into the mixture. The fresh juice would have more bromelain than the canned juice to begin with (seeing as the pineapple is in its natural state), but the high heat denatured some. This left the hot fresh juice with more bromelain than the canned, but not enough was denatured to stop most formation of jell-o. Finally, we checked the fresh juice. The fresh juice was entirely liquid, no solid or semi-solid had begun to form. The liquid was 15.71 grams. This means that the fresh juice has the most amount of bromelain, that none had been denatured and so it was able to prevent the gelatin from acting and the jell-o from forming. This not only backs up our original position on the interaction between the bromelain enzyme (pineapple) and gelatin substrate (jell-o), it further assured us of our other findings in their correctness. Bromelain, when not denatured by processing or heat, acts upon gelatin and prevents its formation. Our lab was a success in the end. Supplies needed: canned pineapple juice, 1 package of sugar free jell-o, water, and fresh pineapple juice made from squeezing chunks of fresh pineapple into a beaker or glass. Additional supplies include a mass scale, plastic pipets, ice, a bucket for the ice, timer, and four small containers with lids. Procedure:
1. Gathered all supplies. This included canned pineapple juice, fresh pineapple juice, pipets, containers, a scale, a sieve, and a stove (or bunsen burner).
2. Heat some of the fresh pineapple juice to boiling point.
3. Take 10 ml of each mixture and put into a sealable small container.
4. Heat 1 cup of water to boiling point.
5. Mix one packet of sugar-free jell-o powder in.
6. Take 10 ml of jell-o for each mixture, so each is now 20 ml of mixed substance.
7. Promptly put into a bucket with ice layered below and above the containers.
8. Time for 18 minutes.
9. Observe every five minutes from outside container.
10. After 18 minutes, remove and put substance through sieve.
11. Take the liquid that is drained into the small “boat” and place on scale.
12. Record mass.
13. Evaluate.
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