- Make stock solutions of sodium phosphate, 4-aminoantipyrine, H2O2, and horseradish peroxidase.
- Perform an assay on the enzymatic rate of horseradish peroxidase.
- Vary the concentration of H2O2, while keeping the concentration of AAP constant, then vary the concentration of APP while keeping the concentration of H2O2 constant.
HRP Assay Solutions
2.3 μM horseradish peroxidase in H2O:
- 1 mg of HRP was dissolved in 1 mL H2O to give a 23 μM HRP solution.
- 1 mL of the 23 μM HRP solution was added to 9 mL of H2O, giving a final concentration of 2.3 μM HRP.
- Note that the molecular weight of HRP is 44 kDA.
0.2 M sodium phosphate buffer in H2O:
- 250 mL of 0.2 M sodium phosphate stock solution requires the following:
MW sodium phosphate: 163.94 g/mol
Mass sodium phosphate:
250 mL × (10-3 L/mL) × 0.2 M × (10-3 mol/mmol) × 163.94 g/mol = 13.4035 g sodium phosphate
Actual mass sodium phosphate: 13.4031 g
13.4031 g × (1 mol/163.94 g) × (1/0.25 L) = 0.327 M in H2O
- 13.4031 g of sodium phosphate was dissolved in about 200 mL of H2O.
- The pH was adjusted to 7.00 by adding 20 mL of 1 N HCl dropwise.
- The solution was then diluted to a final volume of 250 mL with additional H2O.
0.0017 M H2O2 in sodium phosphate buffer:
- 1 mL of 30% H2O2 was added to 100 mL of H2O. Note that the final volume of this solution was 101 mL.
- 1 mL of this solution was added to 49 mL of 0.2 M sodium phosphate buffer at pH 7.00, prepared earlier. The final concentration of H2O2 in this solution was 0.0017 M.
0.0025 M 4-Aminoantipyrine in H2O:
- 0.125 g AAP was dissolved in to 5 mL of H2O.
18 mM 4-Iodophenol in DMSO
- 0.001 g of 4-iodophenol was added to 250 μL of DMSO.
- Please refer to Dhea Patel's entry for notes on how this procedure differs from the standard protocol of dissolving 4-iodophenol in H2O.
Spectrophotometric Analysis of Horseradish Peroxidase Enzyme Activity Rate at 510 nm
- Final concentrations of stock solutions in total volume of 1.5 mL:
||Initial Concentration, M1 [M]
||Volume Added V1 [mL]
||Final Concentration, M2 [M]
- 0.05 mL of horseradish peroxidase was added to each cuvette at the last moment.
- The concentration of H2O2 was varied by serial dilution as follows, in order to produce more stock solutions, while the concentrations of all other solutions were kept constant as shown in the graph.
- The H2O2 solutions were diluted before being added to the cuvette for UV-vis analysis.
- Graph of absorbance over time for enzymatic activity assay of horseradish peroxidase (HRP) at 510 nm. The concentration of HRP in all solutions was 2.3 μM and the signals of all solutions were amplified by adding 10 μL of 18 mM 4-iodophenol in DMSO. The concentration of AAP was fixed at 2.5 mM, while the concentration of H2O2 was varied between 1.7 mM to 106.25 μM. For every 0.75 mL of H2O2 solution, 0.7 mL of AAP solution was added, for a total volume, including that of the 4-iodophenol solution, of about 1.46 mL in the cuvette.
- Graph of absorbance over time for enzymatic activity assay of horseradish peroxidase (HRP) at 510 nm. The concentration of HRP in all solutions was 2.3 μM and the signals of all solutions were amplified by adding 10 μL of 18 mM 4-iodophenol in DMSO. The concentration of H2O2 was fixed at 1.7 mM, while the concentration of AAP was varied between 1.25 mM to 156.25 μM. For every 0.75 mL of H2O2 solution, 0.7 mL of AAP solution was added, for a total volume, including that of the 4-iodophenol solution, of about 1.46 mL in the cuvette.
- The absorbance of only the buffer was included in both graphs to demonstrate that it does not absorb at 510 nm and did not affect the absorbance of the other components of the analyzed solutions.
- The interior of the spectrophotometer was set to a constant temperature of 25°C ± 0.1°C.