- 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.
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.
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, while the concentrations of all other solutions were kept constant:
- 8.5 μM, 4.25 μM, 2.125 μM, 1.0625 μM.