User:Dhea Patel/Notebook/Experimental Biological Chemistry Notebook/2012/09/19

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Objective

  • To determine enzymatic activity of horseradish peroxidase via the usage of fluorometer under 510nm.

Description

  • The stock solutions of HRP, H2O2, and phenol that were made yesterday were mixed and run under the fluorometer at 510nm.
  • 10mL of 2.5mM Luminol stock was prepared using the following calculations:
    • MW luminol: 177.16 g/mol
    • Mass luminol: 10 mL × (10-3 L/mL) × (2.5 × 10-3 M) × (10-3 mol/mmol) × 177.16 g/mol = 0.00443 g luminol
    • Actual mass luminol: 0.0044 g
    • Actual concentration: 0.0044 g × (1 mol/177.16 g) × (1/0.010 L) = 2.5 × 10-3 M in H2O
      • Because the luminol did not completely dissolve in H2O, 0.08g Na2CO3 was added to aid in its dissolution.
        • Ratio: 4g sodium carbonate/500mL solution
      • After the luminol dissolved with the sodium carbonate, 0.48g Na2HCO3 was added to the solution to neutralize the pH of the solution to about pH 7.
  • The first sample was mixed with 4-Iodophenol, H2O2, luminol, and HRP. Please refer to Kay's entry for details in initial concentration, final concentration, and volumes added.

Data

  • Please refer to Kay's entry for the graph produced from the data obtained from 3 samples during fluorescence at 510nm over about 100 seconds. The three samples that ran contained 18mM 4-iodophenol and 2.3uM horseradish peroxidase with varying concentrations of luminol and hydrogen peroxide (1.25mM luminol and 1.7mM H2O2, 625uM luminol and 1.7mM H2O2, and 625uM luminol and 850uM H2O2).