Enzymology Lab

The Effects of Substrate Concentration on Enzyme Activity Wyatt Sinclair Biology 1310 Eric Pass November 8th, 2018 The Effects of Substrate Concentration on Enzyme Activity 2 ABSTRACT In this lab an experiment was conducted to determine the effect substrate concentration had on enzyme activity of turnip peroxidase. A standard BSA curve was also created to test the concentration of an unknown sample of bovine serum albumin. The tests were conducted using a spectrophotometer to measure the light absorbance of varying samples. From that measurement we were able to determine the reaction rate for each subsequent level of substrate concentration. For the bovine serum albumin, tests were conducted to create a standard BSA table and graph. From that graph a best fit line was created which was used to find the unknown concentration. Based on the data collected the optimum substrate concentration was found to be around 20 mM per 0.1ml of 10% turnip peroxidase. The unknown BSA concentration was found to be 0.293mg/ml. INTRODUCTION Enzymes are one of the most important things in our cells. They are critical for speeding up chemical reactions, making DNA, building up large molecules, controlling cell growth, and keeping cell life under control (James Roland 2018). These crucial parts of our cells were discovered as early as 1835 by Swedish chemist Jon Jakob Berzelius, but it wasn’t until 1926 when James B. Summer acquired the first ever enzyme in pure form (Worthington Chemical Corporation 2018). There are lots of things that can affect the performance of a particular enzyme. temperature, PH, substrate concentration, and enzyme concentration to name a few. For our experiment we tested the effect varying substrate concentrations had on enzymatic activity. A substrate is the reactant on which an enzyme works (Cain, Durnford, and others 2015). In order to test how substrates effected our enzyme (turnip peroxidase), we took multiple samples of varying substrate concentration, mixed them with our enzyme and placed them in a spectrophotometer. The spectrophotometer measures concentration by analyzing the brightness of light when it is shone through our subject. Spectrophotometers have been used since 1940 when they were invented by Arnold J. Beckman. This was a huge invention which took weeks’ worth of analysis with only around 25% accuracy to mere minutes of analysis with a 99.99% accuracy (Christy P 2017). I had hypothesized that because for other experiments the substrate concentration was constant at 5mM, this would be the optimum substrate concentration for a 100 uL volume of enzyme. MATERIALS AND METHODS A BSA standard curve was created and used to determine the concentration of an unknown sample. To do this, five samples were taken and put into a spectrophotometer to get the absorbance value of each sample. It should be noted that the spectrophotometer was set at 540 nm wavelength. The data was recorded into a table and then Microsoft excel was used to plot a graph. From that graph a line of best fit was made. Using the formula found, the unknown sample was found. Experiment 2 then began. Six test tubes were labelled, and 1 ml of PH six buffer was added to each tube. 1ml of substrate concentration was added to each tube. Substrate concentrations varied from 1.25mM to 20mM. The spectrophotometer was set to 500 nm and as quickly as possible, 0.1 ml of 100 ul 10% turnip peroxidase was added to a sample and it was placed in the spectrophotometer. An initial absorbance reading was recorded and after that a The Effects of Substrate Concentration on Enzyme Activity 3 recording was made every ten seconds for 60 seconds. This process was repeated for each different sample and the spectrophotometer was blanked before each test. After all the data was recorded it was put onto a graph via Microsoft excel. The graphs were then analyzed to determine which substrate concentration was the most effective. RESULTS Figure 1. BSA Concentration vs Absorbance Figure 1 shows the relationship between the increase in BSA concentration and how it affected our absorbance. The trend appeared to show that as BSA concentration increased, so did the absorbance level. Tube # H2O (mL) BSA Stock (mL) 2 mg/mL Biuret Reagent (mL) Absorbance at 540 nm BSA Concentration (mg/mL) 1 0.9 0.1 1 0.248 0.1 2 0.8 0.2 1 0.259 0.2 3 0.6 0.4 1 0.280 0.4 4 0.3 0.7 1 0.343 0.7 5 0.1 0.9 1 0.408 0.9 The Effects of Substrate Concentration on Enzyme Activity 4 Table 1. BSA Concentration vs Absorbance table Table 1 shows how the data was arranged while the experiment was happening. As with Figure 1, the table shows the increase in absorbance as the BSA concentration increases. Figure 2. Change in Absorbance over Time Figure 2 shows the change in absorbance at 500nm for the enzymatic activity at differing substrate levels. The general trend showed that as the reaction proceeded over the first minute, the higher concentration samples had higher absorbance. Figure 3. Substrate Concentration vs Reaction Rate. The Effects of Substrate Concentration on Enzyme Activity 5 Figure 3 shows how the reaction rate increased with each different substrate concentration sample. The overall trend shows a non-linear increase in reaction rate. The graph resembles a root function, indicating that there will be an optimum substrate concentration near 20mM. DISCUSSION Looking first at our BSA standard curve (figure 1), we can observe that the overall trend was that an increase in BSA resulted in an increase in absorbance. These results seem in line with other experiments of similar fashion (Man, 2012). Furthermore, these results seem to make sense when we think about the context of the experiment. As more BSA stock is added, less light will be able to pass through the sample, leading to an increase in absorbance. Now turning out focus to the substrate experiment, our trend results are concurrent with the general scientific understanding for how substrate concentration would affect enzymatic activity (Cain, Durnford, and others 2015). As substrate level increases, the amount of collision between the enzyme and substrate would increase too. This would cause a continued increase in reaction rate until the enzyme becomes saturated. When an enzyme becomes saturated, it is working at its maximum reaction rate. This is why our figure 3 looks like a root function and appears to become flat after a certain concentration. My hypothesis was incorrect. The data from our experiment seems to indicate that the optimum substrate concentration was more like 20mM. REFRENCES 1.Christy P. The History of Spectrophotometry. Sciencing [Online]. From: https://sciencing.com/history-spectrophotometry. Accessed 2018 November 4. 1.Unknown. Introduction to Enzymes. Worthington Biochemistry [Online]. From: http://www.worthington-biochem.com/introbiochem. Accessed 2018 November 5. 1.Biggers A. Roland J. Why Are Enzymes Important. Healthlie [Online]. From https://www.healthline.com/health/why-are-enzymes-important. Accessed 2018 November 5. 1.Cain. Durnford. Chapter 8. . Biology, Second Canadian Edition. 2015. P154-174. 1.Pass, E.W. and Hardwick, T. 2018. Biology 1310 Lab Manual – Fall 2018. Charlottetown: UPEI. p81-106.