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

1. Combine all of the reagents listed in the reaction mixture, minus hydrogen peroxide.
2. Set the spectrophotomer to read the appropriate absorbance (650nm for TMB, 415nm for ABTS) for 10 minutes with 10 second intervals.
3. Add the appropriate amount of hydrogen peroxide, and IMMEDIATELY begin taking readings.
4. Repeat the experiment multiple times for each hydrogen peroxide volume (2, 4, 6, 8, 12, 16, 32 and 64μL)
5. Slopes of each experiment was determined and plotted on a Michaelis-Menten plot.

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

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

1. Combine all of the reagents listed in the reaction mixture, minus hydrogen peroxide.
2. Set the spectrophotomer to read the appropriate absorbance (650nm for TMB, 415nm for ABTS) for 10 minutes with 10 second intervals.
3. Add the appropriate amount of hydrogen peroxide, and IMMEDIATELY begin taking readings.
4. Repeat the experiment multiple times for each substrate volume (0.5, 1, 2, 4, 6, 8 and 10μL)
5. Slopes of each experiment was determined and plotted on a Michaelis-Menten plot.