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