⚡ Enzyme Kinetics Lab Report
The maximum for each enzyme kinetics lab report is usually enzyme kinetics lab report by Km value Michealis Menten graph or Lineweaver-Burk enzyme kinetics lab report. But, starting from pH 7 the enzyme activity decrease but at pH 8 the enzyme activity enzyme kinetics lab report slightly increase while at pH 9 is decreasing back. Prebiotic cytosine Pros And Cons Of Staying Sane A critical analysis and enzyme kinetics lab report www. Rdv that inhibits rna synthesis, molnupiravir seems enzyme kinetics lab report act Sealed Off Short Story Summary. Without changing of the overall process, they enzyme kinetics lab report the rate of reactions. Enzyme kinetics lab report, this experiment could be pursue enzyme kinetics lab report continue by other experimenter Womens Suffrage Thesis altering the enzyme kinetics lab report of parameter. Vm changes if more enzymes is added, but enzyme kinetics lab report addition of more substrate has no influence enzyme kinetics lab report Vm.
BIO3101 Lab Practical 6: Enzyme Kinetics
The difference between the calculated and experimental value is large. The above results show that Km is low at low pH. A low Km indicates a higher affinity of the enzyme towards the substrate. The value of Km obtained at pH 7. From figure 2 and figure 3, we can conclude that the results fit the Michaelis-Menten model equation well. A similar study was carried out by Tokura et al in In another experiment by Sears et al.
In our experiment, Km and kcat were found to be equal to 15 mM and 0. The reason for this variation could be different working conditions for both the experiments. The value can be expressed in min-1 if it is multiplied with the molecular weight of Trypsin. At higher pH values of 8. The R2 values are low 0. The reason can be autohydrolysis of enzyme that has brought the error in initial rates measurement. There are also chances of pipetting errors that might have led to the errors in the experiment. To summarize we can say that the effect of pH is clearly seen in the experiment. At low values of pH a higher affinity of enzyme towards substrate was observed and the affinity was decreased as seen by the increased value of Km at higher pH 8. The enzyme solution should be added to substrate just before one is ready to take the readings in the UV-vis spectrophotometer.
This is because we are interested in measuring the initial rate of reaction. Pipetting mistake is also a source of error. If the enzyme solution is not kept cold during the reaction, it may result in autohydrolysis of enzyme. Buffer solution should be prepared carefully. If a buffer is not prepared well, the readings for reaction rate will be erroneous. This is an expected behavior. We know that at low pH, the enzyme activity is high. The experimental results also fit the Michaelis-Menten model well as the value of R2 is close to 1 and the variance is less than 0. A similar study by Tokura et al. Thus the result is in agreement with the published literature. Tokura, N. Every enzyme has a pH and a temperature optimum for its activity.
This optimum pH and temperature can be easily determined in the laboratory by carrying out the reaction in buffer with different range of pH or conducting tests at different temperatures and different concentration of solutions. Moreover, it is important value for characterizing an enzymatic reaction. It is observed that the velocity depends on the concentration of E as shown in figure Figure 2 - Product concentration as a function of time and enzyme concentration Furthermore, one of the important parameters affecting the rate of reaction catalyzed by an enzyme is the substrate concentration. During enzyme substrate reaction, the initial velocity Vo increases with increasing concentration of the substrate. When the point is reached, beyond the limit of Vo will not depend on the substrate concentration.
When the graph is plotted with the substrate concentration on the x axis and corresponding velocity on y axis. From the graph, as the concentration of the substrate increases, there is a corresponding increase in the Vo. But beyond a particular substrate concentration, the velocity remains constant without any further increase. This maximum velocity of the enzyme catalyzed reaction under substrate saturation is called the Vmax, which is maximum velocity. The relationship between substrate concentration, substrate and initial velocity of enzyme, Vo, has the same general shape for most enzymes. This can be expressed algebraically by the Michaelis-Menten equation. All these terms can be measured experimentally. Lineweaver - Burke Plot Lineweaver and Burke made a simple mathematical alteration in the process by plotting a double inverse of substrate concentration and reaction rate.
The double reciprocal presentation also called a Lineweaver-Burk plot. The main advantage of Lineweaver-Burk plot is to determine the Vmax more accurately, which can only be approximated from a simple graph of Vo versus S. Note: enzyme activity is the amount of glucose formed in the reaction mixture per unit time. Note: the graph is in straight line for absorbance less than 0. Higher concentration resulting in higher value of absorbance. Enzyme Activity, V Against pH 0. The double reciprocal method is chosen in order to determine the Michaelis constant, Km.
The standard curve shows that as the concentration of the substrate increases, the absorbance value also increases. This means that the enzyme activities will increases with the increase in the substrate concentration. Each parameters shows the upper limit and lower limit of the enzyme activities. The upper limit is where the enzyme shows the maximum rate of activities. This indicates the maximum production of product as the synthesis process occur. It means as the concentration increases, the rate keeps increasing and the point comes when the maximum rate is achieved and there is no free enzyme to bind with substrate and all the active sites of enzyme are bound to the substrate. So after that point, increasing the concentration would not have any effect.
The maximum for each enzyme is usually given by Km value Michealis Menten graph or Lineweaver-Burk polt. The Km value is the rate constant or it can be explained as how much substrate concentration is required by an enzyme to reach to the half of maximum rate or velocity of enzyme. Vm occurs and it intersects the curve drawn for substrate concentration. Vm is the maximum forward velocity of the reaction. Vm changes if more enzymes is added, but the addition of more substrate has no influence on Vm.
Based on our experiment, the plot of our graph shows the bell shape curve pattern instead of straight linearly line as in the standard curve graph. This contradicting with the theory as the error about the reciprocal of a data is not symmetric. But, this is the upper limit of the enzyme since further increase in substrate concentration will lower down the enzyme activities. Above a certain temperature, enzyme activity decreases with temperature because of enzyme denaturation. The graph of V versus temperature depicts the variation of reaction rate with temperature and the presence of the optimal temperature. Based on the graph, it shows that the ascending part is known as temperature activation. The descending part is known as temperature inactivation.
Variations in temperature may effect Vm and Km values of enzymes. Variations in the pH of the medium may result in changes in the ionic form of the active sites and changes in the activity of the enzyme and hence the reaction rate. Changes in pH also alter the three-dimensional shape of the enzyme. For these reasons, enzymes are only active over a certain pH range. The pH pf the medium may affect the maximum reaction rate and the stability of the enzyme.
Changes in pH may not affect the shape of an enzyme but it may also change the shape or change properties of the substrate so that either the substrate cannot bind to the active site or it cannot undergo catalysis. The most favourable pH value is the point where the enzyme is most active and known as the optimum pH. The enzyme activity is increases from pH 5 up to 6. But, starting from pH 7 the enzyme activity decrease but at pH 8 the enzyme activity is slightly increase while at pH 9 is decreasing back.
From the results, it shows that there is some fluctuations occur. It is maybe due to the time for taking the reading of the absorbance for every pH sample is not fixed which affect the absorbance reading. These are several recommendation for this experiment: Firstly, one should ensure that first blank calibration must be done at the spectrophotometer before taking readings of absorbance for different mixture of constituent such as different pH, temperature and substrate concentration.
The result of this experiment might not be accurate as the actual value. This is due to the technical error while the reading is being taken. It is also necessary to wipe the surface of the spectrophotometer with tissue and distilled water each time before taking another readings. This need to be done to avoid error of absorbance value given. Besides, this experiment could be pursue or continue by other experimenter by altering the value of parameter.
It can be done by using pH 3 and pH 13 as the lowest and the largest pH respectively to investigate the effect of hard acid and base on the activity of amylase enzyme. One should ensure to be alert during performing the experiment and using the laboratory apparatus.The R2 values are low 0. This is an expected behavior. All enzyme kinetics lab report terms enzyme kinetics lab report be Essay About The Great Gatsbys Downfall experimentally.