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Clemson University *

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Chemistry

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Feb 20, 2024

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Cellular Respiration Lab Report By: Jacob Patete Introduction Cellular respiration is one of the most important mechanics for any organism to obtain the energy to function. The main goal of cellular respiration is to break down glucose and produce ATP for the body; this is what this report will explain. The experiment involves the use of enzymes that break down the glucose inside the mitochondrial membrane and testing it at different pHs to see the reactions. This is because enzymes will denature when the conditions are not ideal like the pH is higher or lower than normal. This experiment is an important study because we use cellular respiration every second of our lives for energy and if one part of that system fails then w e can’t function. This experiment's hypothesis would be the pH level used on the succinate. So, if the optimum pH for succinate is the one where it will denature the least, then that pH will have the highest percent transmittance rate. Since enzymes need conditions to be just right with the solution being too acidic or basic, I predict that the optimum pH will be somewhere between the pH of 6 and 8 as this is the neutral zone. The main purpose of this experiment is to research how enzymes will react when exposed to different variables so we can use apply that data to the functions of complex organisms. Methods and Materials The steps to this experiment are simple to carry out, but a little time-consuming when running through it. For this lab, the required materials are 6 cuvettes , pH’s of 2, 5,7,9, and 11, succinate, pH indicator solution, test tubes, mitochondria suspension, a timer, DPIP, and a spectrophotometer. The first step for the lab is to create the five pH solutions from the materials to have a different range of acidic to basic with a neutral by first starting with a 4.2 mL pH buffer solution, then add 0.3 mL DDIP, and finally 0.3 mL of mitochondrial suspension. Once the pH, DPIP, and mitochondrial suspension are measured out add 0.2 mL of succinate to the five tubes except the blank to record the enzyme reaction values. Then acquire 6 cuvettes and add one of each of the pH solutions into them with the sixth only having only the solvent in it to be used as a blank for the spectrophotometer. Once all the solutions are prepared seal the cuvettes and place the blank into the spectrophotometer making sure that it reads 100%. This is where the timer comes in to record the percent transmittance rate every five minutes for 30 minutes starting at 0. Record everything on a table for the time and percent transmittance rate as the enzymes mix with the solution. After the experiment when all the data is recorded look at the pHs of each cuvette and report which is the most optimum.
Results (Graph 1) The results from the experiment should tell how the enzymes inside the mitochondrial membrane reacted when placed at different pH. The main trends in the graph when recording the different times show that tubes 2, 4, and 5 show a constant line with most of the transmittance percentages being the same with the enzymes denaturing as indicated from the low rate. The biggest results from the experiment were tubes 1 and 3 with the rate being total opposites for both. Tube 1 had a pH of 2 making it the most acidic of the experiment. While reading the graph the transmittance percent rate was the lowest out of all the tubes and as time went the rate almost decreased to zero indicating that the enzymes were denatured to the point where it became dysfunctional. Tube 3 was at a pH of 7 making it neutral for the enzymes. When starting at the zero-minute mark it had the highest transmittance percent rate of all the tubes and as the time when on every five minutes the rate increased quickly doubling the rate of reactions when the experiment ended at the thirty-minute mark. Discussion y = -0.1693x + 7.1536 y = -0.0443x + 9.9643 y = 0.6671x + 17.736 y = -0.0464x + 10.168 y = -0.0179x + 10.382 0 5 10 15 20 25 30 35 40 45 0 5 10 15 20 25 30 35 Transmittance Percentage Time (Min) Cellular Respiration optimum pH Tube 1 pH 2 Tube 2 pH 5 Tube 3 pH 7 Tube 4 pH 9 Tube 5 pH 11 Linear (Tube 1 pH 2) Linear (Tube 2 pH 5) Linear (Tube 3 pH 7) Linear (Tube 4 pH 9) Linear (Tube 5 pH 11)
The results from the experiment tell how the succinate reacts under the different pH’s and which one of those is the most optimum for the enzymes inside the mitochondrial membrane to function. The hypothesis from the experiment is supported by the graph reinforcing the statement that enzymes denature in high acid or base solutions. The transmittance rate from tube 3 has doubled from the start and is the highest out of all others indicating that the optimum pH for the enzyme is 7. This is supported by the sources about the effect of pH on enzymatic reactions with acids disrupting the hydrogen bonding formed between the side chains with their H+ with tube 2 being a clear indicator of this scenario. Bases also seem to do this, but not as extreme as acids with tubes 4 and 5 having some transmittance. This experiment ultimately shows that enzymes thrive when conditions are perfect for them, and the slightest change can cause them to stop functioning. There could be some changes to the experiment to improve it would be more to experiment with other dyes than just DDIP to get more results. Another improvement would be to do every test tube individually without time constraints to focus more on the percentage. This experiment has shown that enzymes are the main driving force of bodily functions and that a change in anything like pH or temperature can lead to dysfunction. We use enzymes every day like peptides and amino acids, so we must study how enzymes denture so our bodies can function properly. Works Cited Kishore, Devesh, et al. “Thermal, Chemical and Ph Induced Denaturation of a Multimeric β - Galactosidase Reveals Multiple Unfolding Pathways.” PloS One , Public Library of Science, 2012, https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3503960/. “Che 120 - Introduction to Organic Chemistry - Textbook: Chapter 9 - Proteins and Enzymes.” LibGuides, https://guides.hostos.cuny.edu/che120/chapter9. “Effect of Ph on Enzymatic Reaction.” Effect of PH on Enzymatic Reaction - Creative Enzymes, https://www.creative-enzymes.com/resource/effect-of-ph-on-enzymatic- reaction_51.html. Libretexts. “10.7: The Effect of Ph on Enzyme Kinetics.” Chemistry LibreTexts, Libretexts, 1 Sept. 2020, https://chem.libretexts.org/Bookshelves/Physical_and_Theoretical_Chemistry_Textbook
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