In this experiment concerning the activity of enzymes we tested the different effects of various concentrations, pH, Temperature, and Inhibitors over intervals of time. For the effects of concentration of enzyme extract, or peroxidase we mixed six of seven tubes to get three different concentrations of extract. By doing this we wanted to know whether the concentration would positively or negatively affect the activity of the enzyme, in which we predicted that the concentration would increase the activity. The three extract concentrations being 0.5ml from the mixing of tubes 2 and 3, 1.0ml from tube 4 and 5, and 2.00ml from 6 and 7, while the first tube of the seven that wasn’t mixed was the control containing zero extract. The control and the mixed tubes together made 8ml, and was poured into cubettes to be placed in the spectrometer at an absorbance of 500nm.
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.
We hypothesized that a medium pH buffer added to the hydrogen peroxide an peroxidase reaction would be the best condition for the enzyme activity due to it being the more neutral than the high, being basic, and low, being acidic, pH.
Figure 2 is a representation of the average saturation of each cuvette at a specific point time as a function. The y-axis shows the specific saturation points from figure 1, and the x-axis provides the different levels of pH. The pH scale provided on the x-axis ranges from 0 to 14, 0 being the most acidic and 14 being the most basic. The point chosen from figure 1 was the saturation levels of each cuvette at 110 seconds. The saturation point was chosen because in the previous graph at time 110 seconds the reactions of
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 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.
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
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.
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
will be working at the pH 7 the majority of the time and our bodies
HRP has been around for a long time and used in a wide variety of products such as food taste enhancer or spices to pharmaceutical products. Horseradish peroxidase is an important heme-containing enzyme that has been studied for more than a century but the first ever recorded observation of a reaction catalysed HRP is found in a note published by Louis Antoine Planche (Veitch, Nigel C 2004). HRP was first detected by Theorell in horseradish roots.Horseradish peroxidase has been known to degrade certain recalcitrant organic compounds such as phenol and substituted phenols, but for the first time it is shown that HRP is effective in degrading and precipitating industrially important azo dyes(Bhuniaet al. 2001).
The most frequently used heme peroxidase in the enzymatic oligomerization/polymerization of arylamines is isolated from the roots of horseradish (Armoracia rusticana) and belongs to the class III family of secretory plant peroxidases (Veitch, 2004). Similarly to all other heme peroxidases, horseradish peroxidase (HRP) has an iron(III) protoporphyrin IX prosthetic group located at the active site (Veitch, 2004), Fig. 1. The most abundant isoenzyme of HRP is HRP C (Veitch, 2004), Fig. 2. For HRP C the catalytic mechanism for the oxidation of arylamines (ArNH2) at the expense of hydrogen peroxide (H2O2) is the same, the so-called “peroxidase cycle” (Veitch, 2004), Fig. 3. Following the two-electron oxidation of the native Fe(III) enzyme in the
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.