Enzyme Assay Lab Report

The research and observations of this lab primarily focused on the enzyme activity of the enzyme Peroxidase. Peroxidase is a large protein and is composed of more than three hundred amino acids. The enzyme was selected as it is easy to experiment with and effectively showcases the effects of varying independent variables, such as pH and temperature. Peroxidase catalyzes the decomposition reaction of the chemical Hydrogen Peroxide ( H2 O2 ) into water and an electron donating molecule, which stands for R in the written chemical equation. ( The equation is displayed below:

Horseradish peroxidase Type 1 was used in this laboratory experiment, it was an enzyme that helped catalyze the oxidative coupling of vanillin to produce divanillin. The role of the enzyme is to increase the rate of the overall chemical reaction to reduce reaction time, therefore making the reaction process faster. The Horseradish peroxidase Type 1 achieved this by decreasing the activation energy required for a chemical to react, thus allowing the reaction to process through a lower activation energy, which increases the reaction rate and makes the reaction faster.

3) Adding less enzyme caused the reaction to proceed more slowly than when more enzyme was utilized.

In this experiment, the naturally occurring peroxidase is extracted from homogenized turnip (Brassica rapa) pulp (Coleman 2016). Its role in the environment is to remove toxic hydrogen peroxide during metabolic processes where oxygen is used (Coleman 2016). The goal of this experiment is to evaluate the change of absorbency of turnip peroxidase within a metabolic reaction utilizing oxygen. Any change noted is indicative of the peroxidase removing hydrogen peroxide. Within this experiment, the extract will be prepared, the amount of enzyme will be standardized, and the effect of changing the optimal conditions will be observed. If the enzyme concentration is increased then the rate of the reaction decrease. If the pH of solutions used is increased

Figure one depicts the reaction rate of peroxidase enzyme over time. The y-axis shows the absorbance of the assay solutions, and the x-axis depicts time it took for the reaction to occur in seconds.

The experiments were performed in the science lab 1.226 at the University of Texas Rio Grande Valley, Edinburg on October 2, 2017. The experiments were performed in a two-day process due to lack of time. Instructions were given by our TA on where to find the substances (guaiacol under the fume hood, turnip extract, peroxide, and distilled water were placed on our lab tables in dropper bottles, along with the spectrophotometer) and were told to get started. In activity 1 we will be testing 3 concentrations of an enzyme (0.5 ml, 1.0 ml, and 2.0 ml of turnip extract). To quantify the rate of reaction in turnips, guaiacol will be used as the color reagent. Guaiacol is oxidized when it encounters peroxide, allowing light at 470 nm to be absorbed and allowing us to measure the absorbance. In the first activity from experiment day 1, three test tubes were obtained and two clean cuvettes from our lab TA, and placed in a test tube rack on our lab tables. We used one of the test tubes to make the control, another to make the substrate and the last one to make the enzyme. We did this process 3 times to test the effects of the low enzyme concentration, medium enzyme concentration, and high enzyme concentration on the enzyme reaction rate. For the low enzyme concentration, on the control test tube we added 1.0 ml of guaiacol, 0.5 ml turnip extract, 0 ml of peroxide and 8.5 ml of distilled water, getting a total volume of 10 ml in the test tube. For the low enzyme concentration, on the

The effectiveness of peroxidase was measured in a varying pH environment. The environment’s pH can range from   1-14, 7 being neutral, 7-1 being more and more acidic towards 1, and 8-14 being more and more basic towards 14 (Raven, 2011).

Turnips and horse radish roots are rich source of this enzyme. In this experiment, we would carry out a reaction between hydrogen peroxide and guaiacol which is colorless dye, using peroxidase as a catalyst, to produce water and an oxidized form of guaiacol which is brown. The formation of brown color would serve as an indicator that the breakdown of Hydrogen Peroxide took place. The enzyme activity would be directly proportional to the brown color intensity. The color intensity would be measured using a spectrophotometer and standardized to find the corresponding concentration for each absorbance unit.

2. We measured 1 mL of turnip peroxidase (the enzyme) and 3 mL of neutral buffer (pH corresponding to the test tube number i.e. pH 5 in test tube 5) with a syringe and disposed it into tubes 3, 5, 6, 7, 8, and 10

The purpose of this experiment is to learn the effects of a certain enzyme (Peroxidase) concentration, to figure out the temperature and pH effects on Peroxidase activity and the effect of an inhibitor. The procedure includes using pH5, H202, Enzyme Extract, and Guaiacol and calibrating a spectrophotometer to determine the effect of enzyme concentration. As the experiment continues, the same reagents are used with the spectrophotometer to determine the temperature and pH effects on Peroxidase activity. Lastly, to determine the effect of an inhibitor on Peroxidase, an inhibitor is added to the extract. It was found that an increase in enzyme concentration also caused an increase in the reaction rate. The reaction rate of peroxidase increases at 40oC. Peroxidase performed the best under pH5 and declined as it became more basic. The inhibitor (Hydroxy-lamine) caused a decline in the reaction rate. The significance of this experiment is to find the optimal living conditions for Peroxidase. This enzyme is vital because it gets rid of hydrogen peroxide, which is toxic to living environments.

The purpose of this laboratory experiment is to explore the effects of pH has on a reaction rate. The reaction studied was the breakdown of hydrogen peroxide catalyzed by enzyme peroxidase. Peroxidase is a large protein containing heme co factors in its active site. Four trials were ran at pH levels of 3, 5, 7, and 9. I hypothesized that the reaction would run very quickly at a pH 7, since that is the normal condition of cells where peroxidase is found.

Hypothesis: If the concentration of the substrate is increased, then the rate of enzyme activity will decrease. This is because as the concentration of the substrate increases, there is an increasing amount of occupied active sites at any given moment. This will cause a decrease in the rate of enzyme activity as substrate-active site collisions are increasingly slowed down thus bringing down the rate of enzyme activity.

On the other experiment with 1.0 mL of Peroxidase, the solution rapidly change in color ranging from two to five in the first two minutes in the test and stop at seven at four minutes. This concentration test tells us that with more enzymes in the place, the chemical reaction will occur faster and more

The hypothesis is as the substrate concentration has an increase so will the reaction of velocity if the amount of enzyme is kept constant.

In the experiment we used Turnip, Hydrogen Peroxide, Distilled Water, and Guaiacol as my substances. On the first activity, Effect of Enzyme concentration of Reaction Rate for low enzyme concentration, we tested three concentrations of the turnip extract, and hydrogen peroxide. For the Turnip Extract I used 0.5 ml, 1.0 ml, and 2.0 ml. For hydrogen peroxide we used 0.1 ml, 0.2 ml, and 0.4 ml. We used a control to see the standard, and used a control for each enzyme concentration used. The control contains turnip extract and the color reagent, Guaiacol. We prepared my substrate tubes separately from the enzyme tubes. My substrate tube