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

Objective

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

• Abigail E. Miller 20:27, 7 October 2012 (EDT):via a luminol chemiluminescence assay measured with a fluorimeter(not via a fluorimeter under 510nm, what does that mean?)

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).
  • 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.