LABORATORY 12-MEASUREMENT OF ENZYME ACTIVITY-DAY 2
BIOL 105-LAB 12-Exercise 2
EFFECT OF TEMPERATURE
| Temperature | Absorption at 410nm | Concentration of pNItrophenol |
| 4°C | 0.106 | 0.875 μM |
| 25°C | 0.191 | 1.375 μM |
| 37°C | 0.463 | 3.375 μM |
| 60°C | 0.679 | 5 μM |
To calculate the concentration of p-nitrophenol in each “E” tube, the absorbances obtained to check the concentrations from the standard curve of p-nitrophenol. The concentration found are as follows 0.875μM, 1.375μM, 3.375μM and 5μM of E1, E2, E3 and E4 respectively. These values will then be used to plot a graph of p-nitrophenol against temperature.
Graph showing concentration of p-nitrophenol against temperature
Discussion Questions for Exercise 2
- How did the temperature effect the level of enzyme activity? How was the enzyme physically effected?
Enzyme activity increased with the increase in temperature. as temperature increases the rate at which acid phosphates reacts with p-nitrophenol phosphate increases liberating p-nitrophenol and free phosphate. From the graph above, at 4˚C the enzyme is less active; it reacts slowly with the p-nitrophenol thereby removing only few phosphate groups in the p-nitrophenol phosphate leading to formation of few p-nitrophenol. On the other hand, at 70˚C the acid phosphatase activity increases thus it increase the removal of phosphate groups leading to formation of more p-nitrophenol. This explains why the color intensity in the tubes increases with the increase in temperature (Daniel & Danson, 2010).
The shape of enzyme was affected by increase in temperature. as temperature increases the shape acid phosphatase changes to increase the active sites for p-nitrophenol to react with it.
- At very high temperatures p-nitrophenol phosphate spontaneously (without the need for an enzyme) breaks down into p-nitrophenol. How would this effect the measurement of enzyme activity at high temperatures? How would you control for this?
When p-nitrophenol phosphate spontaneously breaks down into p-nitrophenol without the need of enzyme at very high temperature will give a false result of the enzyme activity hence optimum enzyme activity will not be obtained. At very high temperature the enzyme activity is expected to be low. This is because very high temperatures denature the enzyme thus reducing the reaction of enzyme the enzyme with the substrate (Fernandez-Lopez et al., 2017).
This issue can be controlled by addition of citrate buffer. Citrate buffer prevent spontaneous hydrolysis of citrate by resisting change in pH. Therefore, citrate buffer aids in maintaining pH of the solution in the tubes (Dickinson et al., 2011).
References
Daniel, R. M., & Danson, M. J. (2010). undefined. Trends in Biochemical Sciences, 35(10), 584-591.
Dickinson, G. H., Ivanina, A. V., Matoo, O. B., Portner, H. O., Lannig, G., Bock, C., Beniash, E., & Sokolova, I. M. (2011). Interactive effects of salinity and elevated CO2 levels on juvenile eastern oysters, Crassostrea virginica. Journal of Experimental Biology, 215(1), 29-43.
Fernandez-Lopez, L., Pedrero, S. G., Lopez-Carrobles, N., Gorines, B. C., Virgen-Ortíz, J. J., & Fernandez-Lafuente, R. (2017). Effect of protein load on stability of immobilized enzymes. Enzyme and Microbial Technology, 98(2), 18-25.
