# User:Melissa Novy/Notebook/CHEM-571/2012/09/18

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## Objectives

• 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

• Calculations:
• 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
Actual concentration:
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:
 Stock Solution Initial Concentration, M1 [M] Volume Added V1 [mL] Final Concentration, M2 [M] H2O2 0.000034 0.75 0.000017 AAP 0.0025 0.7 0.001166667 Phenol 0.018 0.01 0.00012
• 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, and 1.0625 μM.

• Graph of absorbance over time for enzymatic activity assay of horseradish peroxidase (HRP). 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 concentrations of 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.