This lab will determine and analyze the effects of various hydrogen peroxide concentrations on the catalysis of the enzyme turnip peroxidase. The enzyme, peroxidase, is used very frequently in modern cell and molecular biology experiments as its function is to catalyze the conversion of hydrogen peroxide into water and oxygen. Substrate and enzyme binding are specific, meaning only certain enzymes will bind with certain substrates. The enzyme-substrate complex, where catalysis takes place, is formed when the active site of the enzyme binds with the substrate. After the chemical reaction is complete, the enzyme is unchanged, however, the substrate has been converted into new products (Hardwick and Pass 2018).
In this particular experiment, the substrate hydrogen peroxide will be tested in concentrations of 1.25mM, 2.5mM, 5mM, 7.5mM, 10mM, and 20mM along with a pH 6 buffer and guaiacol dye. However, a similar study, published in the Journal of Mathematic Chemistry, had been conducted by a team of researchers using peroxidase derived from horseradish as opposed to turnip (Bispo 2013; Bonafe 2013; Koblitz 2013; Silva 2013; Souza 2013).
Every chemical reaction needs a minimum amount of energy in order for it to occur, this is called the activation energy. Enzymes, as catalyzes, can lower the activation energy needed by interacting with the reactants but not being consumed by the reaction itself (Hardwick and Pass 2018). In the present experiment it is hypothesized that as the concentration of substrate increases, the rate of the reaction will decrease. Therefore, greater concentrations of hydrogen peroxide are hypothesized to take longer in the reaction than lower concentrations.
Materials and Methods:
Six test tubes, six cuvettes, samples of substrate hydrogen peroxide (stained with guaiacol) of the following concentrations: 1.25mM, 2.5mM, 5mM, 7.5mM, 10mM, 20mM, six mL of pH buffer, Kim wipes, spectrophotometer are needed to conduct this experiment.
Procedure is as follows. Six test tubes were labeled containing samples of different concentrations of hydrogen peroxide substrate from 1.25mM to 20mM. Each sample also contains guaiacol dye and 1mL of pH buffer. The first cuvette is wiped with a Kim wipe and with the tube the spectrometer is blanked at 500nm. 0.1mL of 10% turnip peroxidase is added to the cuvette. The tube is thereby placed into the spectrophotometer and concentration is read from 10 second time intervals beginning at 0 seconds and ending at 60. The spectrometer is blanked with each cuvette and previously mentioned steps are repeated for each of the six test samples. The data was recorded and placed onto graphs for appropriate analysis. The graph readings provide an explanation of the effects of substrate concentration on the catalysis of a reaction.
Upon completion of this experiment, the previously stated hypothesis was deemed to be correct. It was assumed that the higher the substrate concentration, the longer a chemical reaction would take. The Optimal substrate concentration was found throughout experimentation to be 7.5mM by examining Graph 3: Slopes of Substrate Concentrations. As slope represents the reaction rate, it can be determined that the highest slope, or the peak in the graph, can be interpreted as the optimal concentration for the experiment.
Turnip peroxidase, our enzyme, reacted with hydrogen peroxidase, our substrate at different concentrations in order to determine the optimum concentration. Excess amount of enzyme but not enough substrate indicates that the reaction will be limited by the substrate availability. Once you add more hydrogen peroxide to the solution, the reaction rate will increase as more substrate molecules can collide with the enzyme, forming more product. One study stated that the peak of the reaction would occur as the solution becomes saturated with the substrate molecules (Urry et Al. 2014).
Possible sources of error throughout this experiment consist primarily of human error but could also be caused by spectrophotometer malfunction. Of these errors, inexperience, incorrect blanking, and false or delayed readings are among the most likely to occur in this lab. Cuvettes, prior to blanking and readings may not have been completely cleaned of dirt, grime, and fingerprints using a Kimwipe may also affect results in this case. In conclusion, aside from errors, the initial hypothesis was deemed correct and the optimal substrate concentration was determined to be 7.5mM.
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