Enzyme 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

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 type of peroxidase is used is called turnip peroxidase. Turnip peroxidase is made up of Guaiacol and hydrogen peroxide. The reactants to the product are turnip peroxidase or called tertraguaiacol and water. The color of the react is brown. In the experiment was conducted there were baseline experiment, temperature, pH, 10X substrate, Inhibitor, and half the amount of enzyme.

The same solution of 0.5 ml BSA was then added from test tube 1 to the test tube 2 after being properly mixed, and from test tube 2 the solution was being added to test tube 3, and so forth all the way up to test tube 5, with the same exact procedure. From the last tube, we then disposed the 0.5 ml solution. After above procedures, we now labeled another test tube “blank”; 0.5 ml blank distilled water was purred into the tube with the serial dilution of 1:10. We also had a tube C labeled “unknown” with the same 0.5 ml of solution. And after adding 5ml of Coomassie Blue to each tube (1-5) and to the blank, the result of absorbance was read at 595 nm.

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.

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.

1. We measured 2 mL of diluted hydrogen peroxide (the substrate), 1 mL of guaiacol (the product indicator), and 1 mL of neutral buffer (pH 7) with a syringe and disposed it into tubes 1, 2 , 4, 9, 11, and 12.

The first experiment begun by filling a 600-ml beaker, almost to the top, with water. Next, a 10-ml graduated cylinder was filled to the top with water. Once water was added to the beaker and graduated cylinder, a thumb was placed over the top of the graduated cylinder. This would ensure that no water was let out and no bubbles were let into the graduated cylinder. Next, it was turned upside down and fully submerged into the beaker. Then, a U-shaped glass tube was attained. The short end of the glass tube was placed into the beaker with the tip inside of the graduated cylinder. Next, a 50-ml Erlenmeyer flask was received. After, 10-ml of substrate concentration and 10-ml of catalase/buffer solution were placed into the flask. A rubber stopper was then placed on the opening of the flask. After adding these, the flask was held at the neck and spun softly

Peroxidase is an enzyme found in potatoes that catalyzes the breakdown of hydrogen peroxide, H2O2, into O2 gas and water. We examined the different pH environments that can affect the enzyme activity during the breakdown of H2O2. In order to do this, we added different levels of pH, low, medium, and high, into different test tubes with the enzyme and H2O2, and we then inverted the tube. The amount of O2 gas produced was then measured and recorded. The result was that the higher pH produced more gas, followed by medium pH, then low pH. The enzymes were more active in the pH of about 10. It increased

After the substrate solution was added, five drops of the enzyme were quickly placed in tubes 3, 4 and 5. There were no drops of enzyme added in tubes 1 and 2 and in tube 6 ten drops were added. Once the enzyme solution has been added the tubes were then left to incubate for ten minutes and after five drops of DNSA solution were added to tubes 1 to 6. The tubes were then placed in a hot block at 80-90oC for five minutes. They were then taken out after the five minute period and using a 5 ml pipette, 5 ml of distilled water were added to the 6 tubes and mixed by inversion. Once everything was complete the 6 tubes were then taken to the Milton Roy Company Spectronic 21 and the absorbance of each tube was tested.

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.

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 name of the enzyme used in this lab is called peroxidase. “In plants the enzyme is called peroxidase. A related enzyme named catalase performs the same function in animals” (Pearce, 2017). These enzymes breakdown hydrogen peroxide (which is a by – product of metabolism).

Incorporation of assay controls included setting up a spectrophotomer and running the chart recorder with a full-scale deflection before the start of the assay. The set recorder had a corresponding value of 1 for the change in the absorbance. Therefore, prior testing was done to observe whether a change occurred in the readings. This helped to indicate that the results were valid, as they could have been affected by a fault during the setting up of the spectrophotometer. On the other hand this was considered as one of the controls for the experiment. Nevertheless, a new cuvette had to be used for each assay.

waxed and then rehydrated through descending graded ethanol series down to distilled water. To block the endogenous peroxidase, the rehydrated sections were treated with 6% hydrogen peroxide for 10 min. For epitope retrieval, sections were microwaved in citrate buffer, pH 6 for a total 20 min. Non-specific staining had been blocked by superblock (UV block) for 10 minute.