LABORATORY 11-MEASUREMENT OF ENZYME ACTIVITY-DAY 1
BIOL 105-LAB 11-Exercise 1
GENERATION OF A STANDARD CURVE:
| Tube Number | Concentration of p-Nitrophenol | Optical Density (410 nm) |
| 1 | 5 μM | 0.711 |
| 2 | 10 μM | 1.376 |
| 3 | 20 μM | 2.161 |
| 4 | 40 μM | 2.252 |
Standard curve of p-nitrophenol
scale y axis 1cm rep 0.025
x axis 1cm rep 0.25mg/mL
LABORATORY 11-MEASUREMENT OF ENZYME ACTIVITY-DAY 1
BIOL 105-LAB 11-Exercise 2
EFFECT OF ENZYME CONCENTRATION
| Concentration | Absorption at 410nm |
| Undiluted | 0.508 |
| 1:2 | 0.260 |
| 1:4 | 0.111 |
| 1:8 | 0.051 |
Graph the concentration of p-nitrophenol against the enzyme concentration.
In order to plot the concentration of p-nitrophenol against enzyme concentration, concentration of p-nitrophenol in each tube containing the enzymes must be found. Firstly, the concentration of the enzyme in each tube that is in tubes E1, E2, E3 and E4 is calculated. The concentration of enzyme is calculated by initial concentration divided by the dilution factor (Lochter & Merkle, 2010). The initial concentration is 0.4 mg/mL. The dilution factor is calculated by taking the initial volume divided by the final volume, that is 600μL divided by 300μL which is equal to 2. This means that each tube is 2 times more dilute across all the tubes. Therefore, in tube E1 the concentration is 0.4mg/mL (the concentration of the enzyme remains unchanged) because was is undiluted. The concentration of E2 is 0.4mg/mL divided by 2 which equals to 0.2mg/mL. the concentration of E3 is equal to 0.2mg/mL divided by 2 which equals to 0.1mg/mL. finally, the concentration of E4 is 0.1mg/mL divided by 2 which equals to 0.05mg/mL (Pocker & Stone, 2015).
To determine the concentration of p-nitrophenol in each “E” tube, the concentrations is checked from the standard curve using the absorbances of “E” tubes obtained. Therefore, the concentration of p-nitrophenol in E1 is 4.25 μM, in E2 is 1.75 μM, in E3 is 1 μM and in E4 is o.25 μM.
Conversion of the concentrations from μM to mg/mL
If 10^6 μM=1mg/mL
What about 4.25μM= 4.25 μM*1mg/ml = 4.25*10^-6mg/mL
10^6 μM
1.75 will be 1.75*10^-6 mg/mL
1 μM will be 1*10^-6 mg/mL
0.25 μM will be 2.5*10^-7mg/ml
The table below can then be used to plot the graph of p-nitrophenol against concentration of enzyme.
| Tubes | Concentrations of the enzyme in mg/mL | Concentration of p-nitrophenol in each “E” tube in mg/m |
| E1 | 0.4 | 4.25*10^-6mg/mL |
| E2 | 0.2 | 1.75*10^-6 mg/mL |
| E3 | 0.1 | 1*10^-6 mg/mL |
| E4 | 0.05 | 2.5*10^-7mg/ml |
scale y axis- 1cm rep 5.0*10^-7 mg/mL
X axis 1 cm rep 0.05 mg/mL
Discussion Questions for Exercise 2
How was the concentration of p-nitrophenol related to the concentration of acid phosphatase?
p-nitrophenol is acts as a substrate to Acid phosphatase. The fact that acid phosphatase acts on p-nitrophenol phosphate by cleaving the phosphate group on p-nitrophenol phosphate resulting to p-nitrophenol which is a yellow in color which absorbs maximally at 410 nm allow allows accurate spectrophotometry measurement and estimation of concentration on a standard curve (Yang et al., 2015).
In this part, the concentration of enzyme was manipulated. What would you expect to see happen if the amount of substrate was manipulated?
If the substrate concentration was decreased faint yellow color will be observed and the absorbances will be low. This is because the rate of reaction will be slow leading to formation faint yellow color. Reaction rate increases with the substrate concentration (Shin et al., 2017)
References
Lochter, M., & Merkle, J. (2010). Elliptic curve cryptography (ECC) Brainpool standard curves and curve generation.
Pocker, Y., & Stone, J. T. (2015). The catalytic versatility of erythrocyte carbonic anhydrase. VI. Kinetic studies of noncompetitive inhibition of enzyme-catalyzed hydrolysis of P-nitroPhenyl acetate. Biochemistry, 7(8), 2936-2945.
Shin, S., Lee, J., Lee, S., Kim, H., Seo, J., Kim, D., Hong, J., Lee, S., & Lee, T. (2017). Sensors: A droplet-based high-throughput SERS platform on a droplet-guiding-Track-Engraved Superhydrophobic substrate (Small 7/2017). Small, 13(7), 9-12.
Yang, W., Yu, Z., Pan, B., Lv, L., & Zhang, W. (2015). Simultaneous organic/inorganic removal from water using a new nanocomposite adsorbent: A case study of P-nitroPhenol and phosphate. Chemical Engineering Journal, 268(6), 399-407.
