User:Keyun Wang/Notebook/Experimental Biological Chemistry I/2012/09/19

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  • To determine enzymatic activity of horseradish peroxidase via the usage of a luminol chemiluminescence assay.


  • Stock solutions were pre-made 24 hours ago before samples were mixed and run under fluorometer. See 2012/09/18 for details in making stock solutions on concentration of horseradish peroxidase and 4-Idophenol that's used in solutions.
  • Luminol stock solution was diluted to 2.5mM and mixed with carbonate buffer. See Melissa's Notebook for details of the dilution and reasoning behind making the luminol stock.
  • First sample was mixed with 4-Idophenol, H2O2, luminol, and horseradish peroxidase with the concentration below, respectively, before measurements were taken. For details in concentration of stocks added, see below table:
Solution Initial Concentration, Mi [mM] Initial Volume, Vi [uL] Final Concentration, Mf [mM] Final Volume in cuvette, Vf [uL]
H2O2 0.034 13.2 0.0002244 2000
Luminol 1.25 920 0.575 2000
Phenol 18 990 8.91 2000
Horseradish Peroxidase 0.0023 66 0.0000759 2000
  • Samples with varying concentrations of luminol or H2O2 were diluted from those stock solutions. To make 625uM luminol, 50uL of 1.25uM was mixed with 50uL of distilled water. To make 850uM of H2O2, 50uL of 1.7mM H2O2 was mixed with 50uL of distilled water. For the final concentration of the substrate in each sample, please refer to Melissa's Notebook for the calculated final concentration of substrate in each sample after mixture.
  • Fluorometer was set to wavelength 430nm. All data were collected for a period of ~300 seconds to make sure samples has reached equilibrium and had completed in fluorescing.


  • Fluorescence at 510nm was plotted against time in seconds. Only three samples were run: 1.25mM luminol and 1.7mM H2O2, 625uM luminol and 1.7mM H2O2, and 625uM luminol and 850uM H2O2. Within the samples also added 18mM 4-iodophenol and 2.3uM horseradish peroxidase.

Fluorescence at 510nm versus time-s-.png

  • In the above graph, the time in seconds axis was chopped to about 125 seconds to focus on activity before fluoresences decrease. The 1.25mM luminol and 1.7mM H2O2 was the first sample tested. The curve remained constant in fluorescence until around 70 seconds before decrease to no fluorescence. This indicated low fluorescence rate at such concentration of reactants. This also indicated that reaction acted too fast to be recorded by fluorometer by the time sample was loaded. Concentrations of reactants were changed in response to low fluoresence and fast reaction. Then concentration of luminol was diluted down by half (625uM) while remaining the concentration of H2O2 constant in the hope of slowing down reaction. This change was made solely on the conclusion that a decrease in AAP with H2O2 decreases enzymatic activity from 09/18/12. The detector slit in fluorometer was also raised from 10 to 20 in attempt to increase level of fluorescence assuming an increase in detector slit increases detector sensitiviy to fluorescence from samples. As one see in the sample 625uM luminol and 1.7mM H2O2, the curve showed no significant difference from the first sample. This indicates that intensity of fluorescence remained the same and enzymatic activity remained the same. The next prediction was to alternate the concentration of H2O2, predicting from previously HRP data with AAP and H2O2 that a decrease in H2O2 also decreases enzymatic activity. Shown in above graph, the plot for 625uM Luminol and 850uM H2O2 indicated a sharp increase in enzymatic activity and a sharp increase in fluorescence intensity.
  • Further work could be conducted on HRP assay with luminol and H2O2 with fluorometer. A range of luminol or H2O2, namely 1.25mM luminol and 850uM H2O2 sample could be run to see the effect of reactants at different concentrations. Other work suggestion could be running other reactants with H2O2 to test horseradish peroxidase activity.