P-Nitrophenyl Phosphate Lab Report

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The effect of p-nitrophenyl phosphate on the rate of an enzymatic reaction Kathy D. Chin, Sarah E. Arbaugh, Alexandra C. Mendelsohn, Tira T. Oskoui Biology 13 Lab, Section I, Tufts University, Boston, MA, USA Results The effect of substrate concentration of p-nitrophenyl phosphate (pNPP) on enzyme activity was studied. The phosphatase enzyme was incubated with different concentrations of pNPP, ranging from 0.2 to 10.67 mM. After 15 minutes of incubation at 35°C, the amount of p-nitrophenol (pNP) was measured using a spectrophotometer. The results were expressed in a Substrate-Velocity curve (Figure 1). This curve indicated that reaction rate (velocity) increases with increasing substrate concentration of pNPP. More specifically, the curve…show more content…
Recent research into enzyme activity has highlighted the importance of various concentrations of substrate molecules. For example, a recent study investigated how various factors affect the calf intestinal alkaline phosphatase (CIAP) enzyme (Chaudhuri et al. 2013). This study reported that increasing the substrate concentration of pNPP from 1 mM to 1.5 mM saturates the CIAP enzyme (Chaudhuri et al. 2013). These observations are consistent with the results of our experiment. Moreover, these observations allow us to explore ways in which to improve our experiment. For example, increasing the enzyme concentration will increase the rate of reaction given that there will be more active sites available to bind with pNPP. Similarly, an increase in temperature will increase the rate of reaction given that molecules will be able to move faster, which results in an increase of high energy collisions. In addition, the manipulation of enzyme concentration and temperature could lead to maximum conformational flexibility to accommodate substrate molecules, which in turn, leads to maximum catalysis. Now, this idea of achieving maximum catalysis prompt us to think about what happens if we increase pressure. Would an increase in pressure lead to maximum conformational flexibility that will allow substrate molecules to accommodate or bind to the active site? This question is a future direction in enzymology. By understanding how pressure affects enzyme activity we can have a better understanding of how enzymes function and at what point the enzyme reaches a maximum conformational flexibility, which leads to maximum catalysis. Finally, achieving maximum catalysis is important given that the enzyme will be able to carry out its job without difficulties. Again, we need enzymes to perform at its maximum in order for the cell to perform all its metabolic processes,

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