Bio lab Report

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BIOL-150

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Biology

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Feb 20, 2024

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Jack Kallstrom 10/30/22 Effect of Different NaCl Concentrations on Rutabaga Peroxidase Enzyme Activity Introduction: All plants and animals contain enzymes. These enzymes serve many different functions, including breaking down of toxic material. Peroxidase is an enzyme that is very effective at breaking down toxic waste produced from aerobic metabolism (Storer et al., 2022). When peroxidase is mixed with H 2 O 2 , it produces a reaction that breaks these substances down. This reaction can be seen more clearly through the formula: 2H 2 O 2 + Peroxidase → H 2 O 2 + O 2 (gas). The oxygen gas produced is what we use to measure the rate of reaction. We do this by using an indicator, guaiacol, which when reacting with the oxygen forms tetra-guaiacol. We then perform an enzyme assay using a spectrophotometer to measure absorbance rates. In this experiment though, on top of the standard conditions, I will be testing the effects of salt on the enzymatic activity of peroxidase. The peroxidase we will be using is from the rutabaga plant, where peroxidase is found in abundance. I will be measuring three different table salt (NaCl) concentrations: 0.5%, 1%, and 2% and their effect on the rate of reaction of the peroxidase enzyme. I will perform this by setting up 4 tubes. I will have a standard conditions tube, then 3 experimental tubes each with a different salt concentration in them. All tubes will be set at 5 pH and 22 degrees Celsius. This will allow me to isolate the effect the different salt concentrations have on the peroxidase enzyme. Salt concentration could have a major effect on the enzyme reaction rate (Cummings, 2005). If the salt concentration is too high, then the enzyme site will be blocked by ions from the NaCl (Husytee, 1987) which would inhibit the reaction with the H2O2 from taking place. Obviously, if the reaction can’t take place or is lowered, then the rate of enzyme activity will be lower as well. As stated above, the main goal of this experiment is to measure the effects of different salt concentrations on peroxidase enzyme activity. Based on the available scientific research, my hypothesis is that the salt will lower enzyme activity, and thus the rate of reaction in the experiments. If this is the case, enzyme activity will decrease as salt concentration increases. Materials and Methods: A standard curve needed to be set for the experiment to have something to compare the experimental values to. This was done by making six different test tubes with varying combinations of water and 150 mg/mL Tetra-guaiacol solution in them to bring the total volume to 5 mL. After the solutions were mixed in their respective tubes, they were allowed to sit at room temperature for 5 minutes before being put in a spectrophotometer to measure the absorbance at 470 nm. These absorbance figures allowed for the standard curve formula to be found.

For the experiment, four tubes of Guaiacol substrate tubes were prepared with 7.0 mL water, 0.3 mL 0.1% H2O2, and 0.2 mL guaiacol for a total volume of 7.5 mL. Four salt and peroxidase enzyme tubes were also prepared as well. One tube did not have salt in it, as that was the standard condition, while the other three tubes had an increasing amount of salt concentration in them, going in order, 0%, 0.5%, 1%, and 2%. The salt solution that was used to make the different concentrations was 5% NaCl solution. All four enzyme tubes had 2mL of peroxidase enzyme in them and a differing amount of water and salt concentration that added up to 7.5 mL. After being allowed to sit for around 15 minutes to incubate at room temperature, one substrate tube was combined with one enzyme tube and placed in the spectrophotometer. The absorbance value at 470nm was measured every 30 seconds for three and a half minutes. The absorbance values were then plugged into the standard curve equation to find the concentration of tetra-guaiacol. This process was repeated for all three experimental tubes as well. Table 1, Standard Curve of Tetra-Guaiacol Concentrations with Water Dilutions: Tube Water Tetra-Guaiacol Solution Tetra-guaiacol concentration in tube 1 5 mL 0 mL (0 mL / 5 mL) 150 mg/mL = 0 mg/mL 2 4 mL 1 mL (1 mL / 5 mL) 150 mg/mL = 30 mg/mL 3 3 mL 2 mL (2 mL / 5 mL) 150 mg/mL = 60 mg/mL 4 2 mL 3 mL (3 mL / 5 mL) 150 mg/mL = 90 mg/mL 5 1 mL 4 mL (4 mL / 5 mL) 150 mg/mL = 120 mg/mL 6 0 mL 5 mL (5 mL / 5 mL) 150 mg/mL = 150 mg/mL These tubes were prepared to make the standard curve. Each tube had a total of 5 mL in them. They consisted of a combination of water and the brownish tetra-guaiacol solution. Six tubes were prepared, each ranging from 5 mL of water to 0 mL of water combined with 0 mL of Tetra- Guaiacol to 5mL. To find the concentration of tetra-guaiacol was then calculated by dividing the concentration of tetra-guaiacol in the tube by the volume of water then multiplying it with the concentration of the tetra-guaiacol being used. Table 2, Guaiacol Substrate Tubes: 7.0 mL Water 0.3 mL 0.1% H2O2 0.2 mL Guaiacol Total Volume = 7.5 mL This table shows the preparation of the substrate tubes for the experiment. Four substrate tubes were prepared, all with identical conditions within the tube. This was not an experimental variable so what was in the substrate tubes did not need to be changed. The hydrogen peroxide and guaiacol, when mixed with the peroxidase enzyme to form the tetra-guaiacol. This will then

be measured through the absorbance and converted to the tetra-guaiacol concentration in the solutions to see the effect the salt had on the peroxidase enzymes. Table 3, Peroxidase and Salt experimental Tubes: Peroxidase Volume Water Volume Experimental Variable volume Ending Volume Final Variable Concentration Standard Condition 2.0 mL 5.5 mL 0 mL 7.5 mL 0 Experimental Condition #1 2.0 mL 4.75 mL .75 mL 7.5 mL 0.5% Experimental Condition #2 2.0 mL 3.5 mL 1.5 mL 7.5 mL 1% Experimental Condition #3 2.0 mL 2.5 mL 3.0 mL 7.5 mL 2% Final Concentration = exp var total / total volume x stock concentration The table above shows how the experimental tubes were prepared. Like with the substrate tubes, each of the four tubes had a final total volume of 7.5 mL. The amount of peroxidase in each tube was kept constant. The final variable concentration shows the concentration of salt in each tube compared to the water and peroxidase. The experimental variable volume shows the real amount of 5% NaCl that was put into the experimental tubes. Results : All tests were carried out the same manner, with the substrate and enzyme tubes being prepared separate then being combined right before being put into the spectrophotometer for the readings. The only thing that was different was the salt concentrations in each of the enzyme tubes. The results of each reading from the spectrophotometer shows the potential effect that the different salt concentrations had on the peroxidase activity. As seen in the tables and graphs below, the salt concentrations of 0.5%, 1%, and 2% did not appear to have a substantial impact on the peroxidase enzyme activity, as all the salt concentrations had higher slopes and concentrations than the standard curve. All the experimental conditions and tests were similar in the regression statistics that they produced. All conditions tested had high R 2 values, with the lowest being 98.66%. The three salt concentrations all had similar slopes as well, although they were all significantly different compared to the standard curve. Table 4 : Standard Curve for peroxidase assay at standard conditions Tube Tetra-guaiacol, mg/mL Absorbance at 470nm 1 0 mg/mL 0.000 2 30 mg/mL 0.120 3 60 mg/mL 0.231

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