An Enzyme is a macromolecule that helps decrease the time for a reaction to take place [1]. They are very important to cells as they reduce the EA barrier, or the activation energy required to get a reaction en route [1]. The enzyme does not get consumed by a reaction it facilitates, and thus can be reused [1].
The enzyme used in this lab is Catechol Oxidase. This lab is separated into two experiments: one measuring the effects of temperature on the enzyme, and one testing the change of pH on the enzyme. To observe the effects of temperature and pH, spectrophotometry is used to test the absorbance of light in intervals of thirty seconds over a time period of three minutes. For the first experiment I hypothesize that when the temperature is
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After 5 minutes the substrate catechol is added and the solution placed in the spectrophotometer. Table 1 consists of my group’s data and another group’s data. My group’s ice bath trial failed so I will use the other group’s data for that temperature category. In table 1, my group’s room temperature absorbance reading initially started at 2.288, then proceeded to increase to the 2.4 range where the level stayed for the remaining time. For the boiling temperature, the initial reading was 0.220 with a very minimal increase in absorbance. At the end of three minutes the absorbance level was only 0.234. The hot temperature seems to have slowed the enzyme’s ability. The other group’s ice bath reading started at a low 1.326 it then grew rapidly over thirty seconds to the 2.4 range that the room temperature was also at. The cold temperature seemed to slow the enzymatic activity. The difference between the cold and room temperatures can be observed in graph 1 …show more content…
My group’s data is in column 2, the pH we tested the enzyme with was 4. In table 2 column 1 the pH used was a very acidic 2. At a pH of 2 the absorbance had a very minimal change gravitating around ~0.74. The pH 2 solution slowed the enzyme down significantly. In the next column the solution’s pH was 4. Initially the absorbance was 1.410, and steadily increased by ~0.1 every thirty seconds. At 180 seconds the absorbance hit 2.070 which is the largest change of absorbance level out of the pH experiment. With a pH of 7 the absorbance levels started at 1.186. From there it increased to 1.325, then decreased to 1.023. The pH 7 solution did not have much effect on enzymatic activity as the absorbance maxed out early on. Water stayed a consistent ~0.87 throughout the 180 seconds. In column 5 with a pH of 8, the absorbance started at 0.425 and then dropped to 0.402 at the 180 second mark. The solution with a pH of 10 initially started at 0.524, it too decreased in value ending at 0.474. The more basic solutions seem to stop enzymatic activity at the very
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 values of color absorbance are effective because color absorbance has a linear relationship with concentration values, which in turn, allows us to easily find concentration values for many solutions. Beer’s law describes this phenomenon since the absorbance is directly proportional to concentration. We observed that as the color absorbance increased, the concentration of the FeSCN2+ complex ion increased. This is because as the FeSCN2+ concentration increases, the blood-red color becomes darker due to more presence of the blood-red FeSCN2+ ion. Therefore, the color absorbance increases because there is more blue color absorbed by the darker red color. We then graphed the absorbance and concentration values and created a line of best fit. Using the line of best fit, we were able to predict the equilibrium concentrations of the FeSCN2+ solutions and find the change required to reach equilibrium. Since we already knew the initial concentration of FeSCN2+ and since we already found the equilibrium concentration of FeSCN2+, we can calculate the change in equilibrium. Using this data, we were able to calculate the equilibrium concentration of all of the species in this lab, since we already knew the change from the initial concentration to the equilibrium change. Q is less than K because there was no initial concentration of FeSCN2+, but after the system reached
This experiment was used to measure the buildup of the colored product, benzoquinone, to observe the change in the absorbance of the mixture in a spectrophotometer at a wavelength of 486 nm. It was hypothesized that over a longer period catechol oxidase activity and reaction in the mixture will continue, creating more benzoquinone resulting in an increase in the absorbance. The hypothesis was supported by the data as seen in figure 2 which shows the positive relationship between time and absorbance. As can be seen, with more time the catechol oxidase can catalyze the reaction and turn catechol (substrate) into benzoquinone (product). This in turn increased absorbance of the blue light at 486 nm by the solution containing benzoquinone which has a dark brown
Figure 1. Results recorded from experiment. After every 15 seconds for four minutes, the absorbance was recorded.
Step 1: Label the test tubes 1, 2, 3 and 4, where test tube 1 represents the sample placed on the counter at room temperature, test tube 2 represents the tube placed in the refrigerator, test tube 3 represents the tube placed in the freezer, and test tube 4 represents the sample exposed to boiling water. You will expose catalase to each of these four conditions.
The two experiments investigated measure the activity of the enzyme, catechol oxidase. Specifically, they investigate the effect of decreasing amounts of enzyme on rate of reaction and effect of decreasing amounts of substrate on rate of reaction. Enzymes are proteins with specific structures determined by the sequence of amino acid used to accelerate and regulate biochemical processes; enzyme activity can be measured using the rate at which the reaction catalyzes and can be expressed in concentration of substrate or product. Catechol oxidase, the enzyme used in the experiments, can be found in potatoes and catalyzes the oxidation of catechol to ortho-quinone. This substance turns fruits brown when cut open. The ortho-quinone produced in the experiments was used to determine the reaction rate. The experiments involved enzyme dilutions mixed with water and catechol in experiment II and .026M catechol with water and potassium phosphate in experiment III. Both experiments were measured using a spectrophotometer. My hypothesis for experiment II stated that as the enzyme concentration increased the rate of reaction would also increase at a constant rate, and my hypothesis for experiment III stated that the substrate concentration would increase as the rate of reaction increased at a constant rate. The results concluded that in experiment II the enzyme concentration increased at a constant rate, while the reaction rate increased. The experiment III concluded that the
Within the experiment, pure catechol was mixed with different concentrations of catechol oxidase and the rate at which each solution produced benzoquinone was measured. The amount of benzoquinone made throughout the trials was measured by using a colorimeter to measure the level of “brownness” of the liquid. The colorimeter worked by shining a light through the liquid and then measuring that light on the other side to see how much of it was absorbed. In this experiment, absorbance of blue light was measured because blue light is absorbed by the color brown. The amount of blue light absorbance was measured every 15 seconds for five minutes. Because enzymes speed up reactions, more enzymes would cause the reaction to be even faster.1
Enzymes are a key aspect in our everyday life and are a key to sustaining life. They are biological catalysts that help speed up the rate of reactions. They do this by lowering the activation energy of chemical reactions (Biology Department, 2011).
Enzymes are central to every biochemical process. Due to their high specificity they are capable of catalyzing hundreds of reactions that signifies their vast practical importance.
reaction rate increases. If the temperature of an enzyme gets to high the reaction rate will slow
An increase in enzyme concentration will increase enzyme substrate speed up the rate of reaction until the saturation point is reached. (Enzymes and...) Meaning that all the enzyme active sites are occupied by substrate and can no longer enhance in activity. For the next experiment we tested on was temperature. I hypothesis, that the enzyme energy will increase as the temperature rises and have little energy as it lowers. When the temperature rises the activity of the enzyme and substrate collisions increases as well. But, it will hit its maximum point where its bonds will begin to break down. We first measured the absorbance rate at 0 Celsius giving a us .001405 (1/s) and giving the product a light shade of brown. The reaction rate became more active as the temperature rose and the color would darken. But, when reaching over 40 Celsius that’s when the enzyme began to denature. Also, when reaching 100 Celsius the enzyme reaction rate was less than .0005 (1/s) and the solution stayed transparent. Next, we tested the pH, it’s a scale that measures 0 to 14, 0 being the most acidic and 14 being the most
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.
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
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.
To find the effect of temperature on the activity of an enzyme, the experiment deals with the steps as follows. First, 3 mL if pH 7 phosphate buffer was used to fill three different test tubes that were labeled 10, 24, and 50. These three test tubes were set in three different temperature settings. The first test tube was placed in an ice-water bath for ten minutes until it reached a temperature of 2° C or less. The second tube’s temperature setting was at room temperature until a temperature of 21°C was reached. The third tube was placed in a beaker of warm-water until the contents of the beaker reached a temperature setting of 60° C. There were four more test tubes that were included in the procedure. Two of the test tubes contained potato juice were one was put in ice and the other was placed in warm-water. The other two test tubes contained catechol. One test tube was put in ice and the other in warm water. After