Putting enzymes on pause
Figure 3. Scatterplot depicting the concentration of pNPP versus the absorbance of pNP after 5 minutes of a standard curve kinetic assay at 1 μM enzyme concentration. Linear regression analysis was performed, yielding a slope equation and an R² value, which are depicted on the chart. Analysis of the data above influenced the parameters for the standard curve kinetic enzyme assay, with a 1 μM enzyme concentration running for 5 minutes. The assay was run in triplicate, and the data were graphed using the averages for each pNPP concentration. Graphing the data from the assay, as the concentration of pNP in μM versus the recorded absorbance, resulted in a standard curve (Figure 3), which was used to convert absorbance values to concentrations from this point on.
Figure 4. Bar graph comparing the concentration of enzyme present after running an activity assay with and without sodium tungstate as an inhibitor. The error bars represent the standard deviations of the concentration values for the reaction with and without sodium tungstate present. The activity assay measuring the concentration of pNP produced with and without the presence of sodium tungstate showed a significant decrease in activity with the inhibitor present (figure 5). The assay was run in triplicate, with the average concentrations of pNP used to produce the bar graph.
Figure 5: A Michaelis-Menten Curve comparing pNPP concentration and velocity. Velocity was calculated using the pNPP concentration obtained from the sodium phosphate standard curve equation and the time the reaction was run for (5 minutes).
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