Hydrogen Peroxidase Lab Report

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

The preparation for the experiment started by gathering the solutions of enzyme Peroxidase, substrate hydrogen peroxide, the indicator guaiacol and distilled water. Two small spectrometer tubes and three large test tubes with numbered labels. In addition, one test tube rack, one pipet pump and a box of kimwipes were also gathered. Before the experiment, the spectrometer must be set up to use by flipping the power switch to on. Following, the machine was warmed up for 10 minutes and the filter lever was moved to the left. In addition, I set the wavelength to 500 nm with the wavelength control knob. Before the experiment, I had to create the blank solution by pipetting 0.1 ml of guaiacol, 1.0 ml of turnip extract and 8.9 ml water into tube #1. Following the creation of the blank, a control 2% solution was created.

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

The data in proves that our hypothesis was correct. When we increased the temperature to 35°C, the the enzyme activity increased because kinetic energy increased, increasing the collisions between the substrate and the enzyme, and thus creating a higher chance of reaction. When we increased the temperature to 45°C, the enzyme activity decreased as the enzyme became denatured,because the atoms in the enzyme had enough energy to overcome the hydrogen bonds between the R groups that give the enzyme its shape From our data, we could conclude that the optimal temperature of turnip peroxidase is around 35°C and around 45°C, it will start to denature.

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

In the first part of the enzyme lab, we mixed a substrate and an indicator with an enzyme. There was also a neutral buffer in each of the chemical mixtures. The neutral buffer regulated the pH to around 7. We got a color palette and once we mixed each together, we observed and saw a change in the color of the substance. The darker and more brown the substance got, the more oxygen produced by the reaction. Our results showed that amount of oxygen produced increased about 10% a minute until it sort of equilibrated at 4 minutes and didn’t change to the fifth minute mark. If we were to change anything we did in the experiment, we would make our comparisons to the chart more precise. Overall we thought it

The time in the water bath was also controlled to ensure that the enzymes were left to react for the same amount of time, making the experiment

The null hypothesis for the first experiment was that substrate concentration would have no effect on the reaction rate. It was hypothesized that the reaction rate would increase with rising substrate concentrations, until all active sites were bound. The null hypothesis for the second experiment was that temperature would not have an effect on reaction rates. It was hypothesized that until the enzyme is denatured, as temperature increased, so would the reaction rate.

In both experiments, there was one environment that the enzyme worked best it. The graphs show that in the salt experiment 4% salt concentration was the optimum environment for the enzyme, and pH 6 buffer solution was the optimum pH environment for the enzyme. In the other environments the enzyme did not work as fast, therefore not creating as much product. The hypothesis was that the enzyme would work the most efficiently in the most neutral environment. The data does support this hypothesis; therefore we would accept the hypothesis. The neutral state for pH is pH 7, so the enzyme would be most active at exactly pH 7. With pH 6 buffer solution, the enzyme is working, but after pH 7, the enzyme begins to denature at becomes less effective. For the salt experiment, the hypothesis was that the enzyme would be more productive in the solution that had the least salt concentration. As the salt concentration increase, the enzyme would be being to denature. In this experiment, the hypothesis was incorrect, so it had to be

How do various factors, pH, enzyme concentration, ionic concentration, substrate concentration, and temperature, effect turnip peroxidase absorbance/ transmittance rate? The initial experiment was testing the effects of pH buffers (3,5,7,9,and 11). If the pH buffers of 3, 5, 7, 9, and 11 are tested on the turnip peroxidase, the enzyme will function best, and the reaction rate will be the fastest, under conditions of a neutral pH because the cytoplasm of most cells has a pH of about seven and usually extreme pH’s in an enzymes environment usually denature the enzyme, restricting it from catalyzing the reaction. First, a baseline was tested to establish a standard control for a reaction to reference to when testing other factors, to

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 total change in temperature of the baseline experiment was 1.9 degrees Celsius and the total change in temperature for the 2x substrate experiment was 4.7 degrees Celsius. The reaction was positive and exothermic, however was much greater than twice the magnitude of the baseline experiment reaction. The expectation was that because there was twice the substrate to be consumed by the hydrogen peroxidase that it would result in approximately twice the magnitude of energy released in heat. The basis of the hypothesis was correct that there would be an overall increase in the magnitude of reaction but there was no way to tell the exact rate of reaction so the hypothesis can neither be confirmed nor denied with the experiments conducted.

Enzymes are made up of thousands of amino acids that are linked together, to form a specific shape in order to create a chemical reaction. Functions of an enzyme includes increasing the speed of a reactions, they also act with one reactant to produce products and they regulate high to low and low to high enzyme activity. Peroxidase are enzymes whose primary function is to break down hydrogen peroxide (H2O2). Isoenzymes are enzyme group that catalyzes the same reaction but receive different products.

What are the effects of different temperatures on the decomposition of Hydrogen Peroxide with the Enzyme Catalase. Different enzymes work best at different optimal temperatures, and with this experiment we hope to discover which temperatures are unideal for Catalase. The substrate is H2O2 which binds to the active site of the enzyme Catalase. The reaction in question is as written below:

Hydrogen peroxide is a toxic byproduct of cellular functions. To maintain hydrogen peroxide levels the catalase enzyme deconstructs hydrogen peroxide and reconstructs the reactants into oxygen gas and water. The catalase enzyme is found inside cells of most plants and animals. Regulating the levels of hydrogen peroxide is crucial in homeostasis and analyzing it’s optimal conditions for performance is just as important. To understand the optimal environment for this enzyme, they are put into different environments based off protein activity (enzymes are proteins). Catalase samples will be put into different hydrogen peroxide environments based off pH and temperature. The more active the enzyme, the more oxygen and water it will produce. Enzyme activity can be seen through the release of oxygen in the hydrogen peroxide. Since oxygen cannot be accurately measured, the data will consist of the longevity of the reaction in different environments. If the pH is higher than 7, then the reaction rate will increase due to the ample amount of hydrogen ions in the hydrogen peroxide. However the pH level cannot be higher than 10 or else there will be too many hydrogen atoms in the peroxide for the enzyme to be able to deconstruct them. If the temperature is increased, then the reaction rate will increase due to the ample amount of energy and movement in the hydrogen peroxide and enzyme.