Abstract
Substrate concentration influences the rate of enzyme activity in biological reactions. This is attributed to the high impact between enzyme and substrate particles, which augments the tempo of reactions. This report focused on the effect of substrate concentration on succinate dehydrogenase during cellular respiration. It was observed that the rate of enzymatic reaction rose with the increase in substrate concentration. These were the expected results of the study. The absorbance of solutions containing enzyme-substrate complexes at varying levels of the substrate was established by spectrophotometry. Plotting time on the x-axis against absorbance on the y-axis resolved the actual connection between substrate concentration and the rate of reaction. This paper concluded that the activity of succinate dehydrogenase increased with the increase in substrate concentration.
Introduction
Energy is a vital requirement for all living organisms because it drives all metabolic processes that are necessary to sustain life. Living organisms spend energy as ATP (Adenosine Triphosphate) molecules created from the oxidation of glucose through cellular respiration. This process takes place in the mitochondria of living cells. The breakdown of glucose molecules involves two processes depending on the availability of oxygen. In the presence of sufficient oxygen, aerobic respiration takes place and the cell breaks glucose producing 38 molecules of ATP, carbon dioxide gas, and water. On the other hand, when oxygen is absent the cell breaks down glucose to ethanol and carbon dioxide in plants, and lactic acid in animals (anaerobic respiration). Anaerobic respiration generates only two molecules of ATP.
Cellular respiration takes place in two steps, which are glycolysis (in the cytoplasm) and the Tricarboxylic Acid Cycle or the Kreb’s Cycle (in the mitochondria). Kreb’s cycle is a progression of eight phases of oxidation-reduction reactions with precise enzymes catalyzing each step. Carbon dioxide, hydrogen, and electrons are emitted when a substance is converted from one state to another. The electrons and hydrogen ions are conveyed to two carrier molecules, NAD+ (Nicotinamide Adenine Dinucleotide) and FAD (Flavin Adenine Dinucleotide). These carrier molecules cart the electrons to the electron transport chain where ATP molecules are produced as the electrons move to oxygen, the last electron acceptor. Several factors influence the rate of enzymatic activity including temperature, pH, enzyme concentration, substrate concentration, and enzyme inhibitors. Strittmatter (612) indicates that several techniques establish various enzyme behaviors. These include “electron paramagnetic resonance spectroscopy, fluorescence enhancement or quenching, and near-ultraviolet absorption measurements” (Strittmatter 612). He further attributes fast mixing techniques to the successful application of kinetic investigation in reaction intermediates (612).
This study looked into the pace of cellular respiration in extracellular mitochondria in diverse conditions using “mitochondrial suspensions from pulverized lima beans.” It specifically determined the effect of change in the amount of succinate on the rate of cellular respiration. It emphasized the transformation of succinate to fumarate in Kreb’s cycle. This reaction was catalyzed by the enzyme succinate dehydrogenase and involved reduction and oxidation reactions. The study utilized an electron acceptor dichlorophenol indophenol (DPIP) to capture hydrogen ions and electrons freed from succinate. The original color of oxidized DPIP was blue, but it turned colorless when it was reduced by hydrogen ions. Therefore, the rate of color change by spectrophotometry was used to measure the rate of respiration. DPIP absorbed less light as it changed from blue to colorless. Therefore, more light went through the solution.
The study hypothesis was that a boost in substrate concentration enhanced the degree of cellular respiration.
Therefore, the study envisaged that escalating substrate concentration raised the speed of cellular respiration.
Materials and Methods
Four cuvettes were obtained and labelled 1, 2, 3, and B. The first step involved the addition of 4.4 ml of buffer, 0.3 ml of mitochondrial suspension, and 0.3 ml of DPIP to tube 1. To tube the second tube, 4.4 ml of buffer, 0.3 ml of mitochondrial suspension, 0.3 ml of DPIP, and 0.1 ml of succinate were added. To the third tube, 4.4 ml of buffer, 0.3 ml of mitochondrial suspension, 0.3 ml of DPIP, and 0.2 ml of succinate were added. The upper region of each cuvette was covered with a parafilm square and upturned lightly to blend the contents. These cuvettes were allowed to stand for a few minutes as the blank cuvette B was prepared. To tube B, 4.6 ml of buffer, 0.3 ml of mitochondrial suspension, and 0.1 ml of succinate were added. A kimwipe was then used to wipe outside the tube.
The spectrometer was calibrated by selecting “calibrate” then “spectrometer” from the “experiment” menu of the spectrometer. The substrate (succinate) was put afterwards into tubes 2 and 3 and the tubes upturned to mix up the substances. The outer surface of tube 1 was wiped and inserted into the spectrometer and the time recorded. The green arrow on the spectrometer was selected to get the readings, waiting a couple of minutes to view the results. The red “stop” icon was then pressed after viewing the results. Absorbance at 600 nm was then recorded against time in a table. These steps were repeated to obtain the absorbance values for tubes 2 and 3 at different times. The tabulated values were then used to plot a graph of time (x-axis) against absorbance (y-axis).
Results
Table 1: Table of absorbance values for each tube at various time intervals. The values were obtained by measuring the absorbance of tubes 1, 2, and 3 at five-minute intervals. The three tubes contained varying amounts of the substrate succinate. Tube 1 contained no succinate, tube 2 contained 0.1 ml of succinate, and tube 3 contained 0.2 ml of succinate.
Discussion
The experimental design for the study involved the use of similar tubes with identical total volumes of substances. It was noted that the total volume per tube was 5.0 ml. However, the actual components of the tubes were put in varying proportions. For example, for tube 1 no succinate was added. For the second tube, 0.1 ml of succinate was added, but the amount of buffer was reduced by 0.1 ml. The buffer reduction catered for the additional 0.1 ml of the substrate, which maintained the total volume in the tube at 5.0 ml. For tube 3, 0.2 ml of succinate was added to 4.2 ml of buffer and mitochondrial suspension. A 0.2 ml reduction in the amount of buffer took care of maintaining the final volume at 5.0 ml. However, the amount of mitochondrial suspension was kept constant at 0.3 ml in all the tubes demonstrating that the quantity of the enzyme was constant throughout the experiment. DPIP was also kept constant at 0.3 ml in all the tubes. What the study varied was the quantity of succinate in the tubes, which signified the variation of substrate concentration in the enzyme catalysed reactions. The role of the buffer was to provide a constant pH for all the reactions so that a change in pH did not interfere with the results. Tube B contained 4.6 ml of buffer, 0.3 ml of mitochondrial suspension, and 0.1 ml of succinate but without the DPIP. The role of this tube was to act as the control by calibrating the spectrometer.
Transmittance increased rapidly in tube 3 because it contained the highest amount of succinate. Low absorbance values signified high transmittance rates because absorbance was contrariwise proportional to the amount of light put out. The blue DPIP color absorbed more amount of light (transmitted less light) than the colorless solution. Therefore, the rate of increase in transmittance (equivalent to a decrease in absorbance) signified an increase in the rate of reaction. Tube 1 recorded the least transmittance (highest absorbance) because it did not contain succinate (there was very little substrate). Tube 2, on the other hand, displayed moderate absorbance and transmittance values. That was because it contained moderate quantities of the substrate.
Succinate was added last to the tubes to prevent the enzyme-substrate reaction from starting before completion of spectrometer calibration.
The experimental results verified the hypothesis. The results indicated that indeed an increase in substrate concentration increased the rate of the reaction. That was because the transmittance values increased with the increase in substrate concentration thereby representing a high reaction rate. A reduction in substrate concentration yielded low transmittance values indicative of the low reaction rates. According to Strittmatter increasing the substrate concentration increased the number of enzyme active sites that were bound to substrates, hence increasing the rate of activity (126). In reference to previous studies by Wong and Hanes, Strittmatter indicated that substrates also functioned as enzyme modifiers (126). A separate study by Swenson and Betts also revealed that the amount of aerobic respiration in yeasts was dependent on the quantity of glucose substrate (387). Yeast cells incubated with glucose exhausted the content of amino acids in the medium after some time. The probable reason for the depletion of amino acids was that the yeast cells converted the amino acids to protein in the presence of energy from aerobic respiration of yeasts (Swenson and Betts 387). Withdrawal of glucose from the medium increased the quantities of amino acids in the resultant medium.
The procedure could also be used to probe other independent variables such as temperature and enzyme concentration. The effect of temperature on enzyme reactions could also be established in the same way by measuring the absorbance (or transmittance) at different temperatures of the reactants. In addition, a similar setup could also be utilized in the determination of the consequence of enzyme concentration on enzyme-catalyzed reactions. To ascertain the effect of enzyme concentration, the absorbance (or transmittance) at different enzyme concentrations could be measured. The setup could maintain the quantity of substrate in the tubes and vary the quantities of enzyme.
The study concluded that an increase in substrate concentration produced a subsequent increase in the rate of enzyme reactions.
Works Cited
Strittmatter, Philipp. “Dehydrogenases and Flavoproteins.” Annual Reviews of Biochemistry. 35.1966(1966):309-334. Web.
Swenson, Paul A. & Robert F. Betts “Aerobic Fermentation and the Depletion of the Amino Acid Pool in Yeast Cells.”Journal of Cell Physiology. 3.46(1963): 387-403. Web.