Peroxidase Enzyme Lab Report

In the blank cuvettes, the 2 mL of pH levels 2,5,7, and 10 were added first, followed by the 1mL of peroxidase. However our experimental cuvettes contained both hydrogen peroxide and guaiacol. Before the cuvettes were placed in the spectrophotometer we added 0.1 mL of H202, followed by 0.02 mL of guaiacol these two substances were added last immediately before they were placed in the spectrophotometer. Para film was placed over the cuvette opening, and the assay solution was shaken and placed into the spectrophotometer where its saturation level was then tested. We recorded the saturation level of the solution every 15 seconds for 3 minutes. This process was repeated two times for each pH level for a total of two trials.

The baseline experiment is the control group. If the time increases over a period a time then turnip hydrogen peroxidase will increase absorbance.

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.

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).

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

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.

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.

will be working at the pH 7 the majority of the time and our bodies

Enzymes are catalysts that function to speed up reactions; for example, the enzyme sucrose speeds up the hydrolysis of sucrose, which breaks down into glucose and fructose. They speed up reactions but are not consumed by the reaction that is taking place. The most important of the enzyme is the shape as it determines which type of reaction the enzyme speeds up. Enzymes work by passing/lowering and energy barrier and in doing so; they need to bind to substrates via the active. Once they do, the reaction speeds up so much more quickly than it would without the enzyme. Coenzymes and cofactors aid the enzyme when it comes to binding with the substrate. They change the shape of the active site so the substrate can bind properly and perform its function.

Enzymes are biological catalysts, which accelerate the speed of chemical reactions in the body without being used up or changed in the process. Animals and plants contain enzymes which help break down fats, carbohydrates and proteins into smaller molecules the cells can use to get energy and carry out the processes that allow the plant or animal to survive. Without enzymes, most physiological processes would not take place. Hundreds of different types of enzymes are present in plant and animal cells and each is very specific in its function.

“Enzymes are proteins that have catalytic functions” [1], “that speed up or slow down reactions”[2], “indispensable to maintenance and activity of life”[1]. They are each very specific, and will only work when a particular substrate fits in their active site. An active site is “a region on the surface of an enzyme where the substrate binds, and where the reaction occurs”[2].

There are thousands of chemical reactions that occur in an organism that make life possible. Most of these chemical reactions occur too slowly on their own. Enzymes are protein catalysts that speed up chemical reactions in a cell. Catalysts are not changed by the reactions they control, and are not used up during the reaction. Enzymes therefore, can be used over and over again. Enzymes are large complex proteins made by the cell and allow chemical reactions to take place at the temperature of the cell. These catalysts are needed in only very small amounts because a single enzyme molecule can complete the same reaction thousands of times in one minute.

Enzymes are biological catalysts, which speed up the rate of reaction without being used up during the reaction, which take place in living organisms. They do this by lowering the activation energy. The activation energy is the energy needed to start the reaction.

Based on the data, the absorbance when the pH was seven was the highest. It was the lowest in an acidic environment at a pH of three, but slightly higher than the acidic in the basic environment at a pH of eleven. The rate of the reaction could be measured through the absorbance. When hydrogen peroxide breaks down, it produces oxygen gas which can react with the guaiacol to form tetraguaiacol. The solution turns brown and the darker it is, the more oxygen is produced and the greater the absorbance. At the pH of seven, the solution was the darkest, meaning the reaction proceeded quickly and the rate was higher. The reason that peroxidase functioned the best at around the pH of seven is because that is the optimal pH in cells for the enzyme. Enzymes work best at their optimal conditions. They are sensitive to their environment and tiny changes such as changes in pH can cause them to stop functioning. The shape of the enzyme or the active site can be changed so it will not attach to the substrate and become inactive. One

The experiment that the class worked on was about peroxidase. Peroxidase is part of the enzyme group that presents most living organisms (Ahmed, 2013). Peroxidase interferes with the removal of hydrogen peroxide (Ahmed, 2013). Hydrogen peroxide is a toxic product that have normal metabolism before it causes any cell damages (Ahmed, 2013). Peroxidase has two substrate and both of them must present a reaction (Ahmed, 2013). One of the two substrate is H2O2 and other one just depends on the organism or the cell type (Ahmed, 2013). The substrate that the class uses is turnip extract. In the class there were five experiments to do but the class were assigned into groups and each group were going to do two experiment. The names of the experiments are: Baseline, Temperature, and pH.