Measurement of Ki of a competitive inhibitor (lactose)- For the third part of the experiment, three duplicates of 11 test tubes were set up exactly as in the previous step. The first duplicate was series A while the second duplicate was series B and the last one was series C. Series A did not contained any inhibitors so additional water was added to each tube. Series B contained the 50mM lactose while series C contained the 150mM lactose. The amounts of buffer, ONPG, and water added to each tube in each series are listed in detailed in tables 4, 6, and 8. The Lineweaver- Burk plot of each series was created using the same steps used in the second part of the experiment. Then, the obtained line equation from each plot was used to calculate …show more content…
the Kmapparent and Vmaxapparent of each plot. Using equation 7 along with the Km and the Vmax from series A, the Ki of Series B and C were calculated. In order to use equation 7, the inhibitor concentration used in series B and C had to be determined first using the equation 1. Then the Ki of each series was found by substituting the inhibitor concentration, the Km , and the Vmax into the equation 7. Heat inactivation and pH vs enzyme activity- Investigating the effects of heat inactivation and the pH levels on the enzyme activity was the last section of the experiment.
For the heat inactivation, two sets of 11 tubes were set up. The indicated amounts of buffer, water, and ONPG listed in table 10 were added to each tube. In addition, the control enzyme (0.1ml) was added to each tube of the control set and the same amount of heated enzyme was added to each tube of the heated set. The absorbance readings were taken and recorded in table 10. Finally, two Lineweaver-Burk plots were created. The plot for the heated set is represented by graph 10 and graph 11 represents the control set. The Km and the Vmax for the heated set and the control set were determined. Finally, the last part of the experiment examined the enzyme activity at different pH levels. Four sets of 11 tubes were set up in this part. The procedure for this part is the same as before, but 4 other buffers were substituted for the standard pH 7.3 phosphate buffer. Set A used the 5.5 pH buffer while set B used the 6.5 pH buffer. The buffer of pH 8.5 was used for set B and for set D the pH was 9. The absorbance readings for 4 sets were taken and recorded in table 13. Using the linear equation that the best-fit line gave for each set, the Km and the Vmax of each set were determined. Then, table 15 was made by dividing the Vmax by the Km. of the four pHs. The Vmax and Km of the control set were also used to make
this table. The Vmax /Km values were then plotted as a function of pH levels to demonstrate the catalytic efficient of the enzyme.
In undertaking the experiment, the hypothesis “if the number of Alka Seltzer tablets reacted increases, then the maximum rate of reaction will increase,” was formed. When graphing the relationship between the maximum rate of reaction and the number of Alka Seltzer tablets reacting, Graph 7 produced a line of best fit with a constant increasing slope that passed through the origin (0,0). This is characteristic of linear graphs, which have the general equation, y=mx, where m is the slope, a constant term, and y and x are changing variables that are directly proportional (i.e. y ∝x). Hence, it can be deduced that Graph 7 is a linear graph, and that there is a linear relationship between the maximum rate of reaction and the number of Alka Seltzer tablets, where they are directly proportional. That is, as the number of Alka Seltzer tablets increased, the maximum rate of reaction increased, supporting the hypothesis. As the true value of the maximum rate of reaction per Alka Seltzer tablet was not known, and a value for comparison was unavailable, the accuracy of the results could not be determined. However, due to the scatter in Graphs 2 to 7, it was evident that the results had low precision. In future, repeating the experiment using different and/or new apparatus will aid in detecting systematic errors and improve the accuracy and validity of the results.
Input variables In this experiment there are two main factors that can affect the rate of the reaction. These key factors can change the rate of the reaction by either increasing it or decreasing it. These were considered and controlled so that they did not disrupt the success of the experiment. Temperature-
The control for both curves was the beaker with 0% concentration of substrate, which produced no enzyme activity, as there were no substrate molecules for...
The effect of a change in PH on enzymes is the alteration in the ionic
The purpose of this experiment was to discover the specificity of the enzyme lactase to a spec...
The independent variable for this experiment is the enzyme concentration, and the range chosen is from 1% to 5% with the measurements of 1, 2, 4, and 5%. The dependant variable to be measured is the absorbance of the absorbance of the solution within a colorimeter, Equipments: Iodine solution: used to test for present of starch - Amylase solution - 1% starch solution - 1 pipette - 3 syringes - 8 test tubes – Stop clock - Water bath at 37oc - Distilled water- colorimeter Method: = == ==
Alkaline Phosphatase (APase) is an important enzyme in pre-diagnostic treatments making it an intensely studied enzyme. In order to fully understand the biochemical properties of enzymes, a kinetic explanation is essential. The kinetic assessment allows for a mechanism on how the enzyme functions. The experiment performed outlines the kinetic assessment for the purification of APase, which was purified in latter experiments through the lysis of E.coli’s bacterial cell wall. This kinetic experiment exploits the catalytic process of APase; APase catalyzes a hydrolysis reaction to produce an inorganic phosphate and alcohol via an intermediate complex.1 Using the Michaelis-Menton model for kinetic characteristics, the kinetic values of APase were found by evaluating the enzymatic rate using a paranitrophenyl phosphate (PNPP) substrate. This model uses an equation to describe enzymatic rates, by relating the
Purpose: This lab gives the idea about the enzyme. We will do two different experiments. Enzyme is a protein that made of strings of amino acids and it is helping to produce chemical reactions in the quickest way. In the first experiment, we are testing water, sucrose solution, salt solution, and hydrogen peroxide to see which can increase the bubbles. So we can understand that enzyme producing chemical reactions in the speed. In the second experiment, we are using temperature of room, boiling water, refrigerator, and freezer to see what will effect the enzyme.
To analyze the activity of alkaline phosphatase at different substrate concentrations, a continuous assay was conducted, where the absorbance (at 400 nm) of the enzyme-substrate solution was monitored and recorded over the course of 70 seconds at 10 second increments. This assay was used to determine the rate at which alkaline phosphatase can dephosphorylate p-nitrophenyl phosphate to p-nitrophenol, which then dissociates to phenolate ion, which causes the solution to turn yellow in a solution at a pH of 9.0 (assumed optimum pH) (McCollam-Guilani, p.71). The color change causes the change in absorbance measured by the spectrophotometer.
Enzymes as mentioned before help speed up reactions, they generally work by bonding to a substrate, this bonding occurs at the active site. This link then forms a different molecule which will benefit its respective process. Every enzyme has its own optimum pH level to work under, if too low the enzyme will be very slow. However if too high the enzyme will then denature and be obsolete. This is why it is important to know the optimum pH level for whatev...
The experiment was performed using mutated E. coli, cell lysis, two centrifugations, two dialysis processes, heat denaturation, salting out via ammonium sulfate, anion exchange chromatography, and spot testing. By following these procedures, one should be able to obtain a lot of purified alkaline phosphatase and should see a yellow tint during the spot test.
Each enzymes works with a small range of pH, there is a pH at which its activity is greatest called optimal pH. This is due to the changes in pH can make a break intra and intermolecular bonds, distorting the shape of the enzyme, and its effectiveness. Generally, enzymes have an optimum pH this doesn’t go to say that the optimum is the same for each enzyme. For example the optimum pH for enzyme pepsin found acidic lumen in the stomach is lower than that of the enzyme carbonic anhydrase that works in the cytosol at neural pH.
In this lab, it was determined how the rate of an enzyme-catalyzed reaction is affected by physical factors such as enzyme concentration, temperature, and substrate concentration affect. The question of what factors influence enzyme activity can be answered by the results of peroxidase activity and its relation to temperature and whether or not hydroxylamine causes a reaction change with enzyme activity. An enzyme is a protein produced by a living organism that serves as a biological catalyst. A catalyst is a substance that speeds up the rate of a chemical reaction and does so by lowering the activation energy of a reaction. With that energy reactants are brought together so that products can be formed.
From looking at the results I can conclude that when the pH was 3 and 5. No oxygen was produced, therefore no reactions were taking place. This was because the pH had a high hydrogen ion content, which caused the breaking of the ionic bonds that hold the tertiary structure of the enzyme in place of the syringe. The enzyme lost its functional shape.
The enzyme activity was measured for tubes 4 through 8 depending on their pH level.