Absorbance Experiment With Turnip Peroxidase

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

And finally into test tube 3, I pipetted 1.0 ml turnip extract and 4.0 ml of water. The contents of test tube 1 was poured into a spectrometer tube and labeled it “B” for blank. “B” tube was now inserted it into the spectrometer. An adjustment to the control knob was made to zero the absorbance reading on the spectrometer since one cannot continue the experiment if the spectrometer is not zeroed. A combination of two people and a stop watch was now needed to not only record the time of the reaction, but to mix the reagents in a precise and accurate manner. As my partner recorded the time, I quickly poured tube 3 into tube 2. I then poured tube 2 into the experiment spectrometer tube labeled “E” and inserted it into the spectrometer. A partner then recorded the absorbance reading for every 20 seconds for a total of 120 seconds. After the experiment, a brown color in the tube should be observed to indicate the reaction was carried out. Using sterile techniques, any excess liquid left was disposed

The baseline is a control and shows the amount of decomposed hydrogen peroxide in the initial sample.

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

Introduction:Enzymes are made up of proteins which are produced within living cells and act as catalysts which speed up chemical reactions. They are made up of long chains of amino acids containing carbon, hydrogen, oxygen and nitrogen. Enzymes are structured to be

If temperature of the water(enzyme environment) is increased to 35°C, then the enzyme activity will

There were three test tubes in which the experiment was held. A relatively equal sized portion of raw potato (this contained the enzyme [a biological catalyst] hydrogen peroxidase) was placed in each tube. Then, enough water to cover the potato was added. Proceeding this, each of the test tubes were assigned a temperature; cold, room temperature or warm (this was written on the tag so that they were not confused). The test tube destinated ‘cold’ was placed in a ice bath for five minutes. At the same time, the ‘hot’ test tube was placed in a hot water bath for five minutes. Meanwhile, the room temperature test tube sat at room temperature for five minutes. When the five minutes were over, the test tubes were returned to the rack (so that they were able to be observed). Then, the test tubes were allowed to sit at room temperature for five more minutes. Once that period of time was over, 2 ml of hydrogen peroxide (the substrate) was added to each tube.

Figure one depicts the reaction rate of peroxidase enzyme over time. The y-axis shows the absorbance of the assay solutions, and the x-axis depicts time it took for the reaction to occur in seconds.

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.

Enzymes are defined as catalysts that speed up chemical reactions but remain the same themselves. The shape of an enzyme enables it to receive one type of molecule and that specific molecule will fit into the enzyme’s shape. Where a substance fits into an enzyme is called the active site and the substance that fits into the active site is called a substrate. Several factors affect enzymes and the rate of their reactions. Temperature, pH, enzyme concentration, substrate concentration, and the presence of any inhibitors or activators can all affect enzymes. Temperature can affect enzymes because if the temperature gets too high, it can cause the enzyme to denature. pH can affect an enzyme by changing the shape of the enzyme or the charge properties of the substrate so that either the substrate cannot bind to the active site or it cannot undergo catalysis. Every enzyme has an ideal pH that it will strive in. Increasing substrate concentration increases the rate of reaction because more substrate molecules will be interacting and colliding with enzyme molecules, so more product will be formed. Inhibitors can affect enzymes and the rate of their reactions by either slowing down or stopping catalysis. The three types of inhibitors include competitive, non-competitive, and substrate inhibition.

The first part of the experiment measured the effects temperature has on the enzyme activity. Our hypothesis for part one is that peroxidase activity is affected by temperature. One can predict that if the temperature of the environment around the enzyme increases, then the enzyme activity will increase. With that being said, there is an optimal temperature for peroxidase, so at some point the peroxidase will decrease in activity once it exceeds it’s optimal temperature. The second part of the experiment measured the effects of inhibition and how it influenced enzyme activity. A hypothesis for part two would be that peroxidase activity is affected by an addition of an inhibitor. If hydroxylamine is added to the reaction mixture, then the breakdown of hydrogen peroxide will decrease. Since hydroxylamine is an inhibitor, one can predict that the rate of peroxidase activity will decrease and the hydrogen peroxide concentration in the mixture will be much

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 initial experiment was a success. As our treatment group spent more and more time under the lights, the absorbance rate continues to decrease toward zero. Once our 30 minutes were up, the absorbance rate in each tube was significantly lower than at the start of our experiment. In contrast the two control groups did significantly lower the absorbance. Each control lacked one of the vital aspects of photosynthesis, one being light, and the other being chloroplast. Neither of the control groups (Control 1 or 2) showed any signs of photosynthesis. Control 1 was exposed to light, but contained no photosynthetic organelles thus the absorbance throughout the 30 minutes varied minimally, mostly staying stagnant. Control two which contained chloroplast but was not exposed to any light failed to lower the absorbance at all and in fact increased the absorbance over the 30 minutes. However, the treatment group contained both and ultimately performed photosynthesis as we expect therefore, confirming our assumption that chloroplast were the organelles required for photosynthesis in plants and that light is required to perform said photosynthesis. The treatment group, containing both the chloroplast and being exposed to light provided evidence that photosynthesis was taking place as the absorbance lowered at each 10-minute interval. Having a less absorbance would be desired because as DCIP became reduced we would expect the solution to become more and more clear, thus less

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.

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