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

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  • To determine enzymatic activity of horseradish peroxidase via a luminol chemiluminescence assay measured with a fluorimeter.


  • The stock solutions of HRP, H2O2, and phenol that were made yesterday were mixed and run under the fluorometer with an emission at 430nm.
  • 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.


Fluorescence at 510nm versus time-s-.png

  • This graph depicts 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).
    • The graph indicates that no significant difference in the reaction occurs when the concentration of Luminol is halved. The third curve with 625μM Luminol and 850μM hydrogen peroxide indicates that a significant loss in enzymatic activity occurs when the concentration of hydrogen peroxide is halved. However, this data needs to be supplemented with intermediate concentrations of Luminol and hydrogen peroxide to ensure that the observations hold true for varying concentration ratios.