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…
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
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...
Enzymes are proteins that increase the rate of chemical reaction by lowering their activation energy. The enzyme glucose oxidase is one of the most widely used enzyme as an analytical reagent due to its ability to identify the presence of glucose, its low cost and good stability. This report discusses the role of enzymes concentration in biological reactions and the catalytic activity of glucose oxidase on D-Glucose. The activity was studied by spectrophotometry and the results were first tabulated and then plotted. The results of this experiment indicate that the enzyme concentration has no major affect on the rate of
The purpose of this experiment was to discover the specificity of the enzyme lactase to a spec...
In this experiment as a whole, there were three individual experiments conducted, each with an individualized hypothesis. For the effect of temperature on enzyme activity, catalase activity will be decreased when catalase is exposed to temperatures greater than or less approximately 23 degrees Celsius. For the effect of enzyme concentration on enzyme activity, a concentration of greater or less than approximately 50% enzymes, the less active catalase will be. Lastly, the more the pH buffer deviates from a basic pH of 7, the less active catalase will be.
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
The Vmax values, as determined from the Lineweaver-Burk plot, for the uninhibited, half uninhibited, and inhibited enzymes were, 0.3647, 0.1262, and 0.3087 μmol/min respectively. The non-linear regression V¬max¬ values for the same enzyme were 0.3343 (9.09% error as compared to Lineweaver-Burk plot), 0.1264 (0.16% error), and 0.2694 μmol/min (14.6% error) respectively. The differences in the values are due to the presence of error introduced by a Lineweaver-Burk plot, where data points at higher and lower substrate concentrations are weighed differently (Tymoczko, p.115). This error is the reason why a Michaelis-Menten plot is preferred.
The enzyme assay was repeated in water baths at four temperatures: an ice bath (approximately 4 degrees celsius), room temperature (approximately 23 degree celsius), 32 degree celsius, and 48 degree celsius. Test tube 9 was obtained and labeled 19. The appropriate solutions were added to each test tube. All tubes were preincubated at the appropriate temperature prior to the mixing of tubes. The tubes were then set aside to acclimate for 15 minutes. After the equilibrium was reached and the spectrophotometer was adjusted with the control (tube 1) the pairs 2 & 3, 4, & 5, 6 & 7, and 8, & 9 were mixed one at a time. The absorbance changes at 15 second intervals for 60 seconds for each temperature were
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.
: The enzyme activity was measured for tubes 3 through 6 over the 20 minutes that they were exposed to 22 degrees Celsius room temperature, 4 degrees Celsius in ice and 50 degrees Celsius in water bath.
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 effect of a change in PH on enzymes is the alteration in the ionic
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.