Prussian Blue Ferritin Michaelis-Menten Kinetic Analysis

Hydrogen Peroxide Variation

Reaction Mixture

• 10μL Prussian Blue Ferritin (22μg/mL)
• 10μL Substrate* (TMB or ABTS, 10mg/mL)
• 2-64μL Hydrogen Peroxide (1% or 0.05%)*
• Sodium Acetate-Acetic Acid Buffer (pH 3.6) up to 220μL

*Experiments conducted with TMB used stock hydrogen peroxide concentrations of 1%, while ABTS experiments used stock concentrations of 0.05%.

• Combine all of the reagents listed in the reaction mixture, minus hydrogen peroxide.
• Set the spectrophotomer to read the appropriate absorbance (650nm for TMB, 415nm for ABTS) for 10 minutes with 10 second intervals.
• Add the appropriate amount of hydrogen peroxide, and IMMEDIATELY begin taking readings.
• Repeat the experiment multiple times for each hydrogen peroxide volume (2, 4, 6, 8, 12, 16, 32 and 64)

The reaction was conducted in room temperature for 10 minutes and the absorbance value was recorded at 10 second intervals. Absorbance for TMB was taken at 650nm, and absorbance for ABTS was taken at 415nm. Eight replicates were conducted. Substrates were the last reagent to be added to the mix. The experiment was conducted multiple times for the addition of 2, 4, 6, 8, 12, 16, 32, and 64μL of hydrogen peroxide. Resulting slopes of these experiments were then used to generate a Michaelis-Menten plot.

Substrate Variation

Reaction Mixture

• 10μL Prussian Blue Ferritin (22μg/mL)
• 0.5-10μL Substrate (TMB or ABTS, 10mg/mL)
• 32μL Hydrogen Peroxide (30%)
• Sodium Acetate-Acetic Acid Buffer (pH 3.6) up to 242μL



The reaction was conducted in room temperature for 10 minutes and the absorbance value was recorded at 10 second intervals. Absorbance for TMB was taken at 650nm, and absorbance for ABTS was taken at 415nm. Eight replicates were conducted. Substrates were the last reagent to be added to the mix. The experiment was conducted multiple times for the addition of 0.5, 1, 2, 4, 6, 8, and 10μL of substrate. Resulting slopes of these experiments were then used to generate a Michaelis-Menten plot.