Bio lab Report

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

4 90 mg/mL 0.345 5 120 mg/mL 0.476 6 150 mg/mL 0.569 Absorbance 0.002524 +0.003835 (concentration) R 2 99.88% Slope 0.003835 T-value of Slope 55.02 SE Coef 0.000066 p-value 0.0001 The standard curve represents the peroxidase assay at standard conditions. No salt was added to this solution. As shown in the table, the equation for the standard curve is Abs = 0.002524 +0.003835 (concentration). This will be used to calculate the tetra-guaiacol concentrations in the other tests. The R 2 value for this was 99.88%, meaning the data fits the regression model very well and could be repeated. The high R 2 value ensures that we have set everything up correctly, including using good pipetting techniques, to get our standard curve. Table 5: Rate of hydrolysis of H 2 O 2 in Standard and Experimental Conditions through Tetra- Guaiacol Concentration (mg/mL). Standard Curve equation used to convert absorbance ratings from spectrophotometer to Tetra-Guaiacol concentration as time increases. Time (seconds) Standard Condition, mg/mL 0.5% Salt Concentration, mg/mL 1% Salt Concentration, mg/mL 2% Salt Concentration, mg/mL 0 14.99 22.55 35.07 35.33 30 38.19 58.53 80.70 74.44 60 58.01 89.82 120.33 108.08 90 76.00 118.50 155.27 138.89 120 91.65 144.06 188.65 167.29 150 105.71 168.05 217.65 193.87 180 116.68 189.69 245.23 217.59 210 130.76 208.99 270.23 239.76 R 2 98.66% 99.04% 99.07% 99.19% Rate of Reaction 0.5403 0.8814 1.1099 0.9654 t-value 21.05 9.64 12.95 11.97 SE Coef 0.0257 0.0354 0.0440 0.0355 p-value 0.0001 0.003 0.001 0.001 Shown in the table is the rate of hydrolysis of all the conditions along with their regression statistics. All three salt concentrations ended up with a higher concentration of tetra-guaiacol and rate of reaction than the standard condition. All three of the salt concentrations have a similar slope, with the 1% concentration having the highest slope of any of the conditions

tested. All four trials have a very high R 2 rate, meaning that there wasn’t much statistical error and they all fit the regression line well. Table 6: Statistical Comparison of reaction rates in enzyme assays Standard Condition Standard Rate (Slope) Experimental Condition Experimental Rate (Slope) t-value F(x) No Salt 0.5403 0.5% Salt 0.8814 9.64 0.003 No Salt 0.5403 1% Salt 1.1099 12.95 0.001 No Salt 0.5403 2% Salt 0.9654 11.97 0.002 The table above shows the experimental conditions being compared to the standard condition on account of t-value and F(x). All the experimental conditions had a calculated p-value of less than 0.05, so their F(x) is significantly different, meaning there is a discernible difference between them and the standard condition. This was calculated by taking the absolute value of the larger slope minus the smaller slope then dividing it by the larger SE coefficient. Figure 1: The standard curve of tetra-guaiacol concentrations at 470 nm. Regression stats include Abs = 0.002524 +0.003835 (concentration), R 2 = 99.88%, T-value of slope = 55.02, SE Coef = 0.000066, and p-value = 0.0001 Above is the graph for the standard curve of the experiment. The slope and line of regression for the tetra-guaiacol concentration compared to the absorbance is shown. It has a positive,

that they were significantly different than the standard condition. This shows that the salt concentrations used did not slow down the rate of reaction of the peroxidase enzyme. This was a very unexpected result considering the pre-experimental research seemed to suggest that the addition of salt would denature the enzyme and slow down activity. One explanation that can be offered is that the salt concentration wasn’t high enough to impact. This was seen in an experiment where they performed basically the same experiment. In this experiment, they used salt concentrations going up in 5% increments from 0-15% and still found no impact on the rate of reaction. Since I had much lower salt concentrations than that, it could just show that there wasn’t enough salt to affect the enzyme (Ukessays). It could have also been human error, through wrongful mixing or an error when preparing the tubes. Since all three salt concentrations are higher though and not just one outlier, it is unlikely that human error was involved regarding a mixing up of materials. Sources: Storer, James D., De Jong, Peter J., Sanchini, Paula (2022). Cell and Molecular Biology Laboratory: An Introduction to Molecular Biology Techniques and Statistical Analysis. Student Manual. pp. 66 Campbell, Neil., Jane Reece (2005). Biology, 7th ed. Beth Wilbur. Benjamin Cummings, Publishing Menlo Park, California. pp. 150-157. Huystee, R. B (1987). Some molecular aspects of plant peroxidase biosynthetic studies. The Journal of Plant Physiology. 38: 205-219. UKEssays. (November 2018). Salt Concentration Effect on Reaction Rates. Retrieved from https://www.ukessays.com/essays/biology/salt-concentration-on-turnip-peroxidase-activity- biology-essay.php?vref=1