Lab Report Peroxidase

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.

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.

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).

One of the best-studied peroxidases is horseradish peroxidase (HRP), which has a heme-iron co-factor. In most heme-peroxidases the iron atom in the active center undergoes a reversible change of its oxidation state. The reaction proceeds in three distinct steps. In first step, the resting state high-spin Fe(III) is present, which is oxidized by hydrogen peroxide to form an unstable intermediate called compound I (Co-I) with Fe(IV), releasing water in the process. Compound I is not a classical enzyme–substrate complex, but rather a reactive intermediate with a higher formal oxidation state (­5 compared with ­3 for the resting enzyme). Thus, compound I is capable of oxidizing a range of reducing substrates. This reactive intermediate oxidizes

5ml pipettes graduated in 0.1 ml units with suction devices or automatic pipetters and tips

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

An enzyme is a protein macromolecule which acts as a catalyst, an agent which speeds up reactions without being consumed by it. They are vital to life; cellular chemical reactions would not occur fast enough to support life, without the aid of enzymes. They do this by lowering the activation energy (EA), which is the energy that must be added to the reactants at the start of reactions, it has to be reached in order for the reaction to occur (Reece, Wasserman and Urry). There are hundreds of enzymes known, but not all cells contain the same ones, an example of this is catalase which will be the experimental enzyme in the lab.

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.

Examination of Temperature Change in Yeast Solution with the Addition of Hydrogen Peroxidase Dr. Shazia Ahmed Kushal Bhatt by April Wyatt (Biology 1111.03) Friday Sept 25, 2015   Introduction: Hydrogen peroxide is an oxidizing agent which affects melanin production within the body (the chemical which controls the color of human hair and the pigment of human skin). Hydrogen peroxide (in very small percentages) is often found in products such as hair bleach and teeth whitening kits because of this property. (World Health Organization, 1997) Hydrogen peroxide is considered a toxin by the U.S. National Library of Medicine (US National Library of Medicine, accessed 2015) and the Agency for Toxic Substances and Disease Registry.

Hypothesis: If the pH is around seven, then the rate of the reaction for the enzyme peroxidase will be the highest. This is because the optimal pH range for the enzyme peroxidase is around seven and at its optimal pH an enzyme functions the best.

Tables 2,3,4,5 and 6 show that as duration increased the absorbance also increased for each pH. The solution in the conical flask became darker (yellow) in time this is because the substrate, p-nitrophenyl phosphate was catalysed by acid phosphatase, releasing Nitrophenolate anion. It was the Nitrophenolate anion giving off the yellow colour; the presence of this feature increases the absorbance rate. The addition of sodium hydroxide distorted the shape of the enzyme making it no longer effective in its function.

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.

Enzymes are macromolecules that act as a catalyst, and it’s a chemical agent that accelerates the reaction without being consumed by the feedback or the results (Campbell and Reece, 2005). After the adjustment by the enzymes, the chemical movement through the pathways of metabolism will become awfully crowded because many chemical reactions are taking a long time (Campbell and Reece, 2005). There are two kinds of reactions in nature. The first one is Catabolic reaction and the second one is Anabolic reaction. Catabolic reactions are large molecules that are broken up into smaller molecules (Ahmed, 2013). Anabolic reactions are small molecules that join to make larger molecules, like polymerization (Ahmed, 2013). If you