Testing for the Activity of a Mitochondrial Enzyme
BIOL: 1411: 0A09
Jordyn Kuehl
October 3, 2017
Partners: Lexi Zocher, Steve Coutteau
I. Question & Hypothesis
In experiment I and II we attempted to take cell fractions of cauliflower, created through a series of differential centrifugations, and ultimately determine which cell fraction contained the greatest number of mitochondria. The Citric Acid Enzyme succinate dehydrogenase (SDH) is a biochemical marker that allows us to indirectly asses the presence of mitochondria. SDH is an enzyme found in the inner mitochondrial membrane and is responsible for catalyzing the oxidation of succinate into fumarate. The mitochondria can be isolated through differential centrifugation and then treated
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We were only able to take 3 readings (at 0 min, 7 min, and 14 min) of the test tubes (E1-E4). The greatest change in absorbance was obtained for test tube E4 (Supernatant 3) at 1.8 nm. This tells us that supernatant 3 contained the largest number of mitochondria relative to the other cell fractions. E3 (pellet 3), contained the lowest absorbance reading, indicating that it had the lowest number of mitochondria present. This was shown experimentally when the solution of E3 (Pellet 3) remained the initial shade of blue, and did not become lighter over time. Without the SDH enzymes found in the inner mitochondrial membrane the DCIP does not get reduced, therefore there is no color change in the solution. The dependent variable of this experiment was the change in absorbance at 600 nm, as the differing number of mitochondria in the cell fractions is what causes the change in absorbance. The independent variable was the number of mitochondria in the cell fractions, as the mitochondria levels were manipulated, the values of reduction of DCIP varied. The control was the solution E1 (None), which contained no cauliflower cell fraction. This provided data that guaranteed a solution without any mitochondria present, that tubes E2-E4 could be compared to.
Figure 3. Effect of Temperature on Enzyme Activity Over Time. This experiment involved taking four identical test tubes and performing
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Generally, when the temperature is greater the decrease or change in absorbance is greater. The highest readings are for the solutions carried out at 3.7 C and 65.0 C. This is to be expected as they are the two highest temperatures used in experiment III. Although the solution carried out at 65.0 C was expected to have a greater change in absorbance than the solution carried out at 37.0 C, this could be due to the enzymes denaturing in the higher temperature of 65.0 C. The two lowest reading were also identical despite their significant temperature
Understanding the activity of enzymes in different muscle types can aid greatly in obtaining more information about other processes such as metabolism of the tissues (Anderson et al., 2012). There are many different methods in order to achieve this information based in two different major categories but the most convenient method is one called continuous assay. This process includes the use of a spectrophotometer to continuously monitor the assay. This method allows for an easy way to calculate the initial rate of reaction as one can establish time points easily. In the lab performed the continuous method was used in order to determine the measurement of the activity enzyme succinate dehydrogenase (SDH). SDH is most commonly found in mitochondria. It is present in many different muscle tissues including the heart, red, and white tissue. In the following lab the enzyme was tested and measured in these three muscle types in the Oncorhynchus mykiss in order to determine which type contained the highest and the lowest activity. This enzyme is involved in multiple processes such as involved in both the Krebs cycle and the electron transport chain (ETC) (Smith, 2014). Its role in
The first hypothesis was unsupported as Tube 4 (positive control) had the most succinate (.2mL) but ended with the lowest % transmittance out of the three sample we were examining (Tubes 2-4). We believe this discrepancy to be due to the variability of the mitochondrial solution. If the Tube 4 mitochondrial suspension had less concentrated enzyme than the solution for Tube 3 and Tube 2 then Tube 4 would be biologically incapable of matching their rate of reduction. It's worth mentioning that Tube 2 and 3 had a %T change of 23.1% and 23.2% respectively while Tube 4 exhibited a change of only 20.4%; further supporting the idea that Tube 4, despite its increase in succinate concentration, wa lacking in other enzymes/proteins necessary for the
To test the effect of temperature, place 4, clean test tubes in a test tube rack and label them “T 0-5,” “T 20-25,” “T 30-35,” and, “T 50-55.” Add 3 mL of 3% H2O2 and 3 mL of water to each test tube. To measure enzyme activity at 0-5°C, prepare a water bath with a temperature range of 0-5°C in a 600 mL beaker. Place Test Tube T 0-5 in the water bath and record the temperature in Table 4. Add 2 drops of the enzyme solution to the test tube, and connect the free end of the plastic tubing to the connector in the stopper. Click the “Collect” button to collect data, and
At 46°C, there is no rate of enzyme activity. The graph presents a direct relationship between the temperature and rate of enzyme activity. The line of best fits shows that as temperature increases, the rate of enzyme activity decreases. Conclusion: If the enzyme is placed in higher temperatures than its optimal temperature, then the rate of enzyme activity will decrease or possibly denature.
At the end of three minutes the absorbance level was only 0.234. The hot temperature seems to have slowed the enzyme’s ability. The other group’s ice bath reading started at a low 1.326 it then grew rapidly over thirty seconds to the 2.4 range that the room temperature was also at. The cold temperature seemed to slow the enzymatic activity. The difference between the cold and room temperatures can be observed in graph 1
In Experiment One, the data signifies that the velocity of the enzyme increased as the concentration of the substrate increased. However, the data from the second experiment draws inconclusive. The qualitative observations of the color change were a clear sign to start the experiment over again due to possible contamination, and that was not done. This could possibly be a reason why the data shown in the effects of temperature is so skewed. While temperature should rapidly increase the rate of enzyme activity until a certain point at which the enzyme denatures, the data shows denaturation of all enzymes besides Tubes 3 and 4, the color changing solutions in both parts of Experiment Two.
Four independent variables of specific temperatures will be used: 0° C, 23° C, 37° C and 55° C. Will enzyme activity be positively or negatively affected by increasing temperature? Also, is there a temperature at which the enzyme will start to denature or destroy its tertiary structure and unravel, compromising its ability to function? The hypothesis is that if temperature is increased, then the enzyme activity will also increase, producing more oxygen. However, at too high a temperature, the enzyme will denature and not be able to produce oxygen.
Further research alludes to the fact that this may be the first study which demonstrates increased mitochondrial function as caused by the bacterium’s polysaccharide outer capsule. Researchers are in the process of synthesizing drugs that could potentially protect the mitochondria and greatly diminish cell death, as well as bacterial replication by blocking the effects of the outer capsule. This particular group intends on advancing their study of drugs to mice and to observe its
Temperature has a negative and positive effect on enzymes. As the temperature increases from 0 to 40 degrees (See Fig 2) the movement of the enzyme and substrate quicken and will bind more often.But, as the temperature increase from 40 degrees the enzyme and substrate slow and cannot bind as quick and therefore at 63 degrees production stops.
The purpose of this lab experiment was to determine the relationship between temperature and the rate of enzymatic activity in yeast cells. In the lab, the temperature was the independent variable. The temperatures consisted of 6°C, 24°C, 34°C, 46°C. The dependent variable in the experiment was the rate of enzymatic activity in yeast cells. The temperatures were tested by using a LabQuest and pressure probe that tested pressure inside the plastic test tube. LabQuest graphed the data and created a line of best fit that was used to determine the slope of the graph. The slope of the graph represents the rate of enzymatic activity. The slope was found for each temperature in 2 different trials. Then, the rate of enzyme activity (kPa/sec) for each temperature in the 2 trials were averaged. These averages were used to develop a graph that shows the relationship between temperature and the rate of enzyme activity. According to the results of the experiment, as the temperature increases, the rate of enzymatic activity decreases. Each enzyme has its own optimal temperature in which it can function efficiently.
To find the effect of temperature on the activity of an enzyme, the experiment deals with the steps as follows. First, 3 mL if pH 7 phosphate buffer was used to fill three different test tubes that were labeled 10, 24, and 50. These three test tubes were set in three different temperature settings. The first test tube was placed in an ice-water bath for ten minutes until it reached a temperature of 2° C or less. The second tube’s temperature setting was at room temperature until a temperature of 21°C was reached. The third tube was placed in a beaker of warm-water until the contents of the beaker reached a temperature setting of 60° C. There were four more test tubes that were included in the procedure. Two of the test tubes contained potato juice were one was put in ice and the other was placed in warm-water. The other two test tubes contained catechol. One test tube was put in ice and the other in warm water. After
The biochemical process at work is the cause of mitochondrion, a membrane-bound organelle found in the cytoplasm of
To determine how different temperatures and how they can directly affect the outcome of enzyme activity, we will need to look at the actual temperatures used in the study as well as the specific type of enzyme used. In remembering that enzymes; Proteins speed up the rates of chemical reactions in the types of living organisms used in the lab experiment this report is based upon. With our groups’ hypotheses, including prediction: The determination factor being can a higher temperature decrease the enzyme activity? In this study we as a lab group needed to be able to form conclusions with knowing at what temperatures did we see the results?
The purpose of this experiement is to determine the effect of temperature on the rate of enzyme
A team of researchers from Boston University, supervised by Thomas Tien, studied the mitochondrial function