Effects of Sodium Chloride on the activity of Polyphenol Oxidase located on potato extract.
Abstract
Polyphenol Oxidase (PPO) is an enzyme that catalyze the oxidation of phenols. This particular enzyme is located in a lot of fruits and vegetables such as potato. This enzyme when exposed to oxygen, oxidizes and it is the reason why fruits gets brown after they are peeled. In this experiment we will used potato extract that contain PPO and will look for the affect of Sodium Chloride in the activity of this particular enzyme.
Introduction
Polyphenol Oxidase (PPO) is a major enzyme present in fruits and vegetables. It is responsible for the browning of vegetables and fruits that contains this particular enzyme. When exposed to air, PPO
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It shows that absorbance increases as time passes but also it shows that the more substrate present the more absorbance. We can clearly see that the line of the 6th test-tubes (more concentrated one) is way higher than the one with no substrate. All of the test-tubes data have each a linear-correlation (R2 >95%) with a slope more and more positive as the concentration of substrate increase. The only data that can be a little less precise is the data of the first test tube (trend line with 0mL substrate). Some issue with the spectrophotometer may have altered the results and thus this particular trend-line may be not correct (all the data from test tube 1 should be the same for all 15 minutes as no substrate as been added). This particular graph shows us that substrate concentration plays a role in enzyme activity as we already know and that the absorbance continued to increases after 15 minutes. With this particular data we were able to construct a graph of Substrate concentration versus Enzyme activity (slope) and we compared our graph with the data of a control group that did not added any NaCl in the solutions. This graph (figure 2) is one of the key to answer our hypothesis. In fact, with this particular graph we can definitely see that Sodium Chloride is an inhibitor of the enzyme PPO. The curve of the experimental data (green) is clearly below the control group curve, which means that the enzymatic activity slows down in presence of NaCl. But, we also want to know if this particular inhibitor is a non-competitive or competitive one. The third graph (figure 3) is a lineweaver-burk plot and will represent the inverse graph of the figure 2 curves. This particular graph will convert the original data that was curves into linear correlation so that we are able to made some assumption on the characteristics of the
Data from Table 1. confirms the theory that as the concentration of glucose increases so will the absorbance of the solution when examined with the glucose oxidase/horseradish peroxidase assay. Glucose within the context of this assay is determined by the amount of ferricyanide, determined by absornace, which is produced in a one to one ratio.1 Furthermore when examining the glucose standards, a linear calibration curve was able to be produced (shown as Figure 1). Noted the R2 value of the y = 1.808x - 0.0125 trend line is 0.9958, which is statistically considered linear. From this calibration curve the absorbance values of unknowns samples can be compared, and the correlated glucose concentration can then be approximated.
In the lab, Inhibiting the Action of Catechol Oxidase we had to investigate what type of enzyme inhibition occurs when an inhibitor is added. Catechol oxidase is an enzyme in plants that creates benzoquinone.Benzoquinone is a substance that is toxic to bacteria. It is brown and is the reason fruit turns brown. Now, there are two types of inhibitors, the competitive inhibitor and non-competitive inhibitor. For an enzyme reaction to occur a substrate has to bind or fit into the active site of the enzyme. In competitive inhibition there is a substrate and an inhibitor present, both compete to bind to the active site. If the competitive inhibitor binds to the active site it stops the reaction. A noncompetitive inhibitor binds to another region
The results of this experiment showed a specific pattern. As the temperature increased, the absorbance recorded by the spectrophotometer increased indicating that the activity of peroxidase enzyme has increased.At 4C the absorbance was low indicating a low peroxidase activity or reaction rate. At 23C the absorbance increased indicating an increase in peroxidase activity. At 32C the absorbance reached its maximum indicating that peroxidase activity reached its highest value and so 32 C could be considered as the optimum temperature of peroxidase enzyme. Yet as the temperature increased up to 60C, the absorbance decreased greatly indicating that peroxidase activity has decreased. This happened because at low temperature such as 4 C the kinetic energy of both enzyme and substrate molecules was low so they moved very slowly, collided less frequently and formed less enzyme-substrate complexes and so little or no products. Yet, at 23 C, as the temperature increased, enzyme and substrate molecules
Catalase is a common enzyme that is produced in all living organisms. All living organisms are made up of cells and within the cells, enzymes function to increase the rate of chemical reactions. Enzymes function to create the same reactions using a lower amount of energy. The reactions of catalase play an important role to life, for example, it breaks down hydrogen peroxide into oxygen and water. Our group developed an experiment to test the rate of reaction of catalase in whole carrots and pinto beans with various concentrations of hydrogen peroxide. Almost all enzymes are proteins and proteins are made up of amino acids. The areas within an enzyme speed up the chemical reactions which are known as the active sites, and are also where the
This evidence alone suggests that higher increases in substrate concentration causes smaller and smaller increases in enzyme activity. As substrate concentration increases further, some substrate molecules may have to wait for an active site to become empty as they are already occupied with a substrate molecule. So, the rate of the reaction starts to level off resulting in a plateau in the graphs. This means that the reaction is already working at its maximum rate, and will continue working at that rate until all substrates are broken down. The only way the reaction rate would increase, is if more enzyme was added to the solution. This confirms that increases in substrate concentration above the optimum does not lead to greater enzyme activity. Therefore, the rate of reaction is in proportion to the substrate
Catecholase is an enzyme formed by catechol and oxygen used to interlock oxygen at relative settings, and it is present in plants and crustaceans (Sanyal et. al, 2014). For example, in most fruits and vegetables, the bruised or exposed area of the pant becomes brown due to the reaction of catechol becoming oxidized and oxygen becoming reduced by gaining hydrogen to form water, which then creates a chain that is is the structural backbone of dark melanoid pigments (Helms et al., 1998). However, not all fruits and plants darken at the same rate. This leads to question the enzymatic strength of catecholase and how nearby surroundings affect its activity. The catecholase enzyme has an optimal temperature of approximately 40°C (Helms et al., 1998). Anything above that level would denature the tertiary or primary structure of the protein and cause it to be inoperable. At low temperatures, enzymes have a slower catalyzing rate. Enzymes also function under optimal pH level or else they will also denature, so an average quantity of ions, not too high or low, present within a solution could determine the efficiency of an enzyme (Helms et al., 1998). Also, if more enzymes were added to the concentration, the solution would have a more active sites available for substrates and allow the reaction rate to increase if excess substrate is present (Helms et al., 1998). However, if more
Figure 3: The absorbance of peroxidase reactions over two minutes using pH 3, pH 5, pH 7, and pH 9.
Jim Clark. (2007). The effect of changing conditions in enzyme catalysis. Retrieved on March 6, 2001, from http://www.chemguide.co.uk/organicprops/aminoacids/enzymes2.html
Investigating Factors that Affect the Rate of Catalase Action Investigation into the factors which affect the rate of catalase action. Planning Aim: To investigate the affect of concentration of the enzyme catalase on the decomposition reaction of hydrogen peroxide. The enzyme: Catalase is an enzyme found within the cells of many different plants and animals. In this case, it is found in celery.
How the Concentration of the Substrate Affects the Reaction in the Catalase Inside Potato Cells Introduction Enzymes are made of proteins and they speed up reactions, this means that they act as catalysts. Hydrogen peroxide is a byproduct of our cell's activities and is very toxic. The enzymes in our bodies break down the hydrogen peroxide at certain temperatures they work best at body temperature, which is approximately 37 degrees. At high temperatures, the cells begin to denature. This means that the hydrogen peroxide is prevented from being broken down because they will not 'fit' into the enzyme.[IMAGE] Objective I am going to find out how the concentration of the substrate, hydrogen peroxide affects the reaction in the catalase inside the potato cells.
Enzyme peroxidase is essential in any cell metabolic reaction as it breaks down the harmful hydrogen peroxide to harmful products in the body. The report analyzed its effect on changes in temperatures by determining the optimum temperatures and the effects of its reversibility. Through the method of extracting the enzyme by blending it with potato tissue in phosphate buffer, the effects were analyzed on the effect of the dye guaiacol and the activity measured under different temperatures. The optimum temperature was obtained at 22.20C and above this temperature, the enzyme was denatured. Conclusively, increase in temperature increases
Therefore there is a slight decrease in oxygen produced. I believe that the inhibitor in the nitrate solution was a competitive one. This because if it was a non-competitive the reaction rate would have risen as concentration rises only for a short while, and then
Comparing the Reaction Rates Between Potato and Hydrogen Peroxide Against Liver and Hydrogen Peroxide Through Loss in Mass
We were not given any instructions either to shake or not to shake the test tubes with the coloured solutions before inserting them in the spectrophotometer to read the absorbance. By shaking each test tube a certain number of times before putting it in the spectrophotometer could have improved the accuracy of the of absorbance of the solutions.
In this experiment increasing amounts of Catechol and L.Dopa where used to determine the rate of enzyme catalysis of PPO. Also an inhibitor, PTU was analyzed with the substrate, Catechol and the enyme PPO. The results were then plotted using a Michaelis-Menten plot and PPO’s affinity (Km) for a particular substrate was determined. The PTU results were also plotted on a Michaelis-Menten plot to see whether or not PTU was a competitive or non-competitive inhibitor. A constant amount of PPO extract from potatoes and Phosphate buffer was used in each of the experiments with varying amounts of DI water