All cells in a living organism require energy to drive various cellular activities such as transport of molecules and movement among others. The rate of cellular respiration may be affected by the substrate concentration, pH level and enzyme concentration among other factors. This experiment aimed to study the rate of cellular respiration in isolated mitochondria under different conditions. It was hypothesized that if the concentration of the substrate is increased then there will be an increase in the rate of cellular respiration. Mitochondrial suspension was used as the source of succinate that was used as the substrate. The level of DPIP reduced by the electron from succinate was used to measure the rate of cellular respiration. The rate was highest in Tube 3 that had the highest succinate concentration. The results obtained supported the hypothesis that as the concentration of the substrate is increased there is an increase in the rate of cellular respiration.
All cells in a living organism require energy to drive various cellular activities such as transport of molecules and movement among others. The energy used in the cells is in the form of ATP and provides the energy that is needed in a form that the cell can utilize for the various cellular activities. The formation of ATP in the cells is usually done using the organic molecules that are available in the form of food (Lehninger, Nelson and Cox 481). In situations where oxygen is absent, the formation of ATP follows the fermentation process. The fermentation process also occurs in yeast cells and the process is referred to as alcoholic fermentation. The alcoholic fermentation, which is also referred to as ethanol fermentation, utilizes various sugars such as fructose, glucose, sucrose among others. The sugars are converted into ATP and thereby releasing ethanol and carbon dioxide as the waste products. The total number of ATP molecules produced in this process is two (Seeley, Stephens and Tate 87).
In the presence of oxygen, the cells utilize the aerobic cellular respiration to transfer the energy contained in food to the molecules of ATP (Campbell and Farrell). The aerobic cellular respiration consists of several processes that make up a complex series of reactions. The reactions start with glycolysis, which takes place in the cytoplasm producing pyruvate molecules as the end products and two molecules of ATP (Pramanik 45). The produced pyruvate then enters into the mitochondria to enter into the next process of respiration which is the Krebs cycle. The Krebs cycle produces carbon dioxide, hydrogen ions and electrons. The electrons together with the hydrogen ions are passed to NAD+ and FAD electron carriers (Campbell and Farrell 568). These carriers take the electrons to the last process of aerobic respiration known as the electron transport chain. In this process, the electrons are transferred to oxygen molecules as the final electron acceptor and consequently producing ATP molecules. In the Krebs cycle, one of the steps involves the conversion of succinate to fumarate. The reaction is a reduction oxidation reaction where succinate loses its electrons and hydrogen ions to FAD to form fumarate and FADH2 (Lehninger, Nelson and Cox 601).
This experiment aimed to study the rate of cellular respiration in isolated mitochondria under different conditions. This was achieved by replacing FAD with DPIP, which results in a color change, to enable in mentoring the rate of respiration.
Hypothesis and Prediction
It was hypothesized that if the concentration of the substrate is increased, then, the rate of cellular respiration will increase. It is predicted that there will be an increase in the rate of cellular respiration when the concentration of the substrate is increased.
Materials and Methods
In the experiment, the materials used were 4 cuvettes, DPIP solution, Buffer, mitochondrial suspension, and succinate, Parafilm square, spectrophotometer, wax pencil, and Kimwipe.
Four cuvettes were obtained and labeled 1, 2, 3 and B. Tubes 1, 2 and 3 were prepared by measuring DPIP, buffer, mitochondrial suspension and added to the tubes using a pipette as shown in Table 1 below.
The top of each cuvette was covered with a parafilm square. A thumb was placed over the cuvette and gently inverted to mix the content. The cuvettes were allowed to stand for several minutes while preparing the blank, cuvette B and calibrating the spectrophotometer. To cuvette B, 4.6 mL of the buffer, 0.3 mL of mitochondria suspension and 0.1 mL succinate were added and the cuvette covered with a with a parafilm square. A thumb was placed over the cuvette and gently inverted to mix the content. The outside of the tube was wiped with a kimwipe. The cuvette B was used to calibrate the spectrometer. Succinate was added to cuvette 2 and 3 as specified in Table 1 and the cuvettes inverted to mix the content. Starting with Tube 1, the outside of the cuvettes was wiped and inserted into the spectrophotometer, and the readings noted. The absorbance was recorded at 600 nm at time 0, and the reading continued at an interval of 5 minutes for 30 minutes. The steps were repeated for Tubes 2 and 3.
The absorbance of the three tubes at an interval of 5 minutes was recorded in Table 1 below. In all the tubes, absorbance reduced as time progressed with the absorbance of Tube 3 recording the lowest absorbance after 30 minutes and Tube 1 the highest.
Using the results obtained, a graph of absorbance against time was plotted for the three tubes as shown in Figure 1 below.
Figure 1: Absorbance of DPIP reading change over time. The absorbance readings were taken at a 5-minute time interval after succinate was added.
The experiment aimed to study the rate of cellular respiration in isolated mitochondria under different conditions. It was thus predicted that the rate of cellular respiration will be a high when the concentration of the substrate is increased. In the experiment, effect of different substrate concentrations on the rate of cellular respiration was determined using succinate as the substrate. The reaction mixture also included di-chlorophenol-indophenol or DPIP which acted as the electron acceptor to take in the electrons produced from the succinate. When DPIP is reduced, its color changes from blue to colorless. This change in color was used to determine the amount of DPIP that was yet to be reduced. As the DPIP was reduced the intensity of the blue color was reduced, and thus the absorbance reading was less where reduction process was high as a result of high succinate concentration. A buffer was used to prevent changes, in pH level in the reaction solution since changes in pH, also affect the rate of a reaction.
In the three tubes, Tube 1 acted as the control since no succinate was added. The transmission rate was increased in a rapid rate in Tube 3 as the tube had the highest level of succinate. This caused more electrons to be released which reduced the DPIP to its reduced form. The reduction of the DPIP caused a reduction in the blue color allowing more light to be transmitted. Succinate is added last in the reaction since it is the substrate in the reaction and its addition would initiate the reaction. The results are in agreement with a previous study that reported an increase in rate of cellular respiration as the substrate concentration was increased (Dickey 6-8).
The results obtained from the experiment supported the hypothesis that rate at which cellular respiration occurs increases as the concentration of the substrate is increased. The rate was highest in Tube 3 that had the highest succinate concentration. Other independent variables that may be investigated are pH level, temperature and the enzyme concentration.
Campbell, Mary and Shawn Farrell. Biochemistry. Stamford: Cengage Learning, 2011. Print.
Dickey, Jean. Laboratory investigations for biology. San Francisco: Benjamin/Cummings Pub. Co., 2003. Print.
Lehninger, A. L., D. L. Nelson and M. M. Cox. Lehninger principles of biochemistry. 4th. New York: WH Freeman, 2008. Print.
Pramanik, D. Principles of Physiology. Kolkata: Academic Publishers, 2007. Print.
Seeley, R., T. D. Stephens and P. Tate. Anatomy and Physiology. 6th. New York: The McGraw Hill Companies, 2004. Print.