Friday, November 15, 2013

Cellular Respiration Lab

Purpose: The purpose of this lab was to determine how much carbon dioxide gas was produced during cellular respiration for each different type of seed. We used both germinated and non-germinated seeds at room temperature and at cold temperatures. Our independent variable was the germinated versus non-germinated, and cold temperatures versus room temperature. The dependent variable was the amount of carbon dioxide produced. We used glass beads as the control group. 

Introduction: Cellular respiration is the catabolic pathway that breaks down glucose into carbon dioxide, water, and energy. It has three stages-glycolysis, the citric acid cycle, and oxidative phosphorylation. During glycolysis, glucose and other sugar molecules are broken down into pyruvate. Pyruvate then loses a molecule of carbon dioxide and attaches to a coenzyme A, and becomes Acetyl CoA. Acetyl CoA then goes through the citric acid cycle where more carbon dioxide is released and Acetyl CoA attaches to an existing 4-carbon molecule called oxaloacetate to form citrate. The next steps of the cycle decompose citrate back to oxaloacetate so that the process can start over with another Acetyl CoA molecule. While the citrate was being decomposed, it produced NADH and FADH2, which are electron carriers. They then carry electrons to the electron transport chain, which uses the energy given off by transporting the electrons to power the proton-motive force. The protons that are pumped into the intermembrane space then flow through a transport protein called ATP synthase. The flow of H+ ions through ATP synthase powers the synthysis of ATP. Cells use this ATP to carry out different functions, and it is necessary to life. Germination is when a seed has been dormant for a period of time, but when its need of water, oxygen,and sunlight are met, the seed begins to grow and undergo cellular respiration. Non-germinated seeds, however, will not. 

Methods: First off, we collected the germinated corn seeds at room temperature and the non-germinated corn seeds by getting 25 of each type. We connected the CO2 gas sensor to the lab quest so we could collected our data. We then took the room temperature and stated it in a data table. Then, we had to dry the germinated seeds at room temperature and then put them in the respiration chamber and put the CO2 gas sensor in it. We let it sit for about one minute then started collecting data for 10 minutes. A graph of carbon dioxide vs. time is then showed and we had to figure out the slope of each trial. We took out the sensor and put the germinated seeds in ice cold water and let it sit for about 20-25 minutes, while we set up and started the trial of the non-germinated seeds. We did the same process as we did before for the room temperature germinated seeds. After the non-germinated seeds were done, we dried off the germinated seeds that were in the ice water and put them in the respiration chamber, and went through the same process a 3rd time. The next day, we used glass beads and used the CO2 sensor and the respiration chamber to figure out our control group, and lastly we used the lab quest to graph all of the trials so we could compare them. 

Below are the respiration rates of germinated corn at room temperature (23 degrees Celsius) and cooled (15 degrees Celsius). Then, we provided the respiration rate of glass beads as a control group, as they would undergo no respiration. 

Graphs and Charts
Below is the graph of our first trial, where germinated seeds were used at room temperature. The rate of respiration is .680 ppm/s.
Then, we have our second trial using no germinated seeds. As you can see, though the graph looks similar, the scale is much smaller. The rate of respiration is only .071 ppm/s.
For the third trial, the cold germinating seeds were used. The rate of respiration was higher than that of the room temperature, as the slope was .699 ppm/s.
Our last trial was the glass beads. Again, the scale is much smaller than the others, and the slope is much smaller than it appears. Our control group's rate came out to .029 ppm/s.
Finally, we put each graph on the same screen. The cold germinated seeds a re on top, followed by the room temperature germinated seeds, the no germinated seeds, and the glass beads.
There is one product of cellular respiration that we measured in this lab: CO2. Since rates of CO2 production changed depending on each trial, we were able to deduce that cellular respiration was occurring. As we can see through our measurements, the germinating seeds had much higher rates of cell respiration. This is because they have begun to use cellular respiration with the available amounts of water and oxygen in order to carry out cellular functions and growth. The non-germinated seeds have not met their requirements of water and oxygen, and will not be able to undergo cellular respiration like the germinated do.  Because of this, their rate is much lower, and represents that of the glass beads. Since glass beads are not living, they will not release any CO2. Temperature also affected our results. We thought that cooling the seeds would decrease their rate of respiration, due to slowing down cell functions. However, this was not the case. The rate for the cooled seeds was higher than the rate for the room temperature seeds. This could be because we did not keep the cool seeds at the same temperature during the six minute testing process. If the seeds started to warm up, their rates of respiration would increase more dramatically than seeds remaining at the same temperature. To amend this in the future, we could put the respiration chamber in a cool environment and then take measurements. 

Conclusion: The different types of seeds all had different amounts of carbon dioxide produced during cellular respiration depending on whether or not they were germinated and whether the germinated seeds were cold or at room temperature. For the room temperature seeds, corns slope was .68 and radish was .61, so those two outcomes were very similar. Peas rate of respirations slope was .32. For the rate of respiration at cold temperature, corn and radish both had .70 for their slope which shows this similarity in their cellular respiration rates and the peas had a slope of .97. Then for the non-germinated seeds the corn and peas were actually more similar, the corn was at .07 and the peas were .05 and the radish was .20. The rate of cellular respiration for the glass beads, which was our control group was different, for all 3 of our lab groups, but close in the number. For group 1, which was our group, our slope for glass beads was .03, for group 2 it was .00 and for group 3 it was -.02. Due to the results, we know that the glass beads should all be around zero. However, it is difficult to say which type of seed is the most efficient, as both the radish and pea seeds have high numbers for different trials.

Monday, November 4, 2013

Enzyme Lab

The purpose for experiment 2A was to determine how the enzyme and substrate acted in the reaction. The enzyme in this experiment was the catalase from yeast, and the substrate was the hydrogen peroxide. We added the catalase to the hydrogen peroxide, and oxygen and water were released in the form of bubbles.

In experiment 2C, the purpose was to find out the rate of uncatalyzed hydrogen peroxide decomposition. We let a cup of hydrogen peroxide sit out overnight, and the next day tested it to see how much of the solution was left, and how much of it had spontaneously reacted to form water and oxygen gas.

For experiment 2D, the purpose was to find the effect of an enzyme on the rate of hydrogen peroxide decomposition. To accomplish this, we measured how much subrate was disappearing over time. We did this by titrating the solution with potassium permanganate to determine whether leaving the catalase in the hydrogen peroxide longer would increase, decrease, or not effect the rate of the decomposition of hydrogen peroxide. 

Below is the equation of the spontaneous reaction of hydrogen peroxide into water and oxygen gas. Using catalase speeds up this reaction.

An enzyme is a macromolecule that speeds up a reaction without being consumed. A substrate is the reactant that the enzyme acts on. When you bond them together, it becoms an enzyme-substrate complex. Hydrogen peroxide is made of two hydrogen molecules and two oxygen molecule. When an enzyme, catalase, is added to the hydrogen peroxide, it releases water and oxygen. The rate of reaction means how quickly the reaction happens. Using hydrogen peroxide and potassium permanganate, we established a baseline, or the calculation that served as a comparison for all later tests. 

First off, we had to do a baseline for the uncatalzyed rate of hydrogen peroxide decomposition by putting a small amount quantity of it into a beaker, letting it sit overnight uncovered at room temperature. Then we put 1 mL of water into it and 10 mL of sulfuric acid . We mixed it and took out 5 mL to titrate and find the baseline. We then found the base line for an enzyme-catalyzed rate of hydrogen peroxide, doing the same steps as before if it had been a day or more since the practice baseline we did the day before the lab. Next, we tried to determine the course of an enzymatic reaction, by measuring the amount of substrate that disappeared. So we determined the reaction rate after 10, 30, 60, 90, 120,180 and 360 seconds. We put 10 mL of 1.5% hydrogen peroxide in a clean beaker and add 1mL of catalase and swirl for 10 seconds each time. Two of us would time it and stir the catalase/H2O2 solution and then add the 10 mL of sulfuric acid to stop the reaction, then The third would take 5 mL out of each solution then titrate it with potassium permanganate until it was pink or brown. Then we recorded it in a data table to compare the amount of hydrogen peroxide used in each test and the baseline.

Titrating to test hydrogen peroxide concentration

Before yeast

Getting catalase from yeast

Sulfuric Acid Being Inserted to end reactions on time.

Below is the measurement for the baseline. 3.9 mL of potassium permanganate was necessary to react with 5 mL of hydrogen peroxide.
Now, we can see the baseline compared to other tests. For example: When catalase reacted with hydrogen peroxide for ten seconds, 2.6 mL of KMnO4 was necessary to react with the remaining H2O2, whereas 3.9 mL was used in the baseline.
Graphs and Charts
  Here is the graph of the amount of hydrogen peroxide used in the enzyme- fueled reaction for each amount of time.
  If we look at the data for the timed trials, two patterns are clear. First, as the amount of time catalase and H2O2 react increases, the amount of potassium permanganate needed to filtrate the sample decreases. Second, as the amount of time increases, the amount of H2O2 used increases. (This increase can also be seen in the graph showing hydrogen peroxide decomposition.). These results go along with our predictions. Since catalase is the enzyme we are using, on our H2O2 substrate, as time goes on we expect more reactions to occur. As these reactions continue, hydrogen peroxide is used. The more time allotted for the enzyme and substrate to react, the more product is produced and substrate is used up. It is for this reason that we see the increase in the amount of H2O2 used. As more hydrogen peroxide is broken down by catalase, less is left to react with potassium permanganate  in the titration. Therefore, it makes sense for the amount of KMnO4 needed to decrease as time intervals increase. To stop the reactions, sulfuric acid is used. This works because the ph of sulfuric acid is very far from the optimum ph of catalase. The enzyme begins to denature in the harsh environment, and reactions cease to occur between the H2O2 and catalase. The results went along with our knowledge of the characteristics of enzymes on all fronts.

One conclusion that we came up with was that if we boiled the catalase solution, the proteins in the enzymes would be denatured and no reaction would have occurred. Then, for the time samples as the amount of time increased, the amount of potassium permanganate decreased because the yeast, which was the catalase solution, was allowed to react with the hydrogen peroxide for a longer period of time. For the same reason,  the amount of hydrogen peroxide used increased because of the amount of reactions that was going on. In short, as time goes on, more reactions between enzymes and their substrates can occur.