Hydrogen Peroxide Variation

Reaction Mixture

• 10μL Prussian Blue Ferritin (CONCENTARION)
• 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%.

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 (CONCENTARION)
• 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.

Temperature Optimization

Reaction Mixture

• 10μL Prussian Blue Ferritin (CONCENTARION)
• 6μL 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 at temperatures ranging from 25-45˚C, with 5˚C intervals. Reaction continued for 10 minutes and absorbance values were taken in 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.

pH Optimization

Reaction Mixture

• 20μL Prussian Blue Ferritin (CONCENTARION)
• 16μL Substrate (TMB or ABTS, 10mg/mL)
• 32μL Hydrogen Peroxide (30%)
• Buffer (pH 3.6) up to 268μL
WHAT PH DID WE DO

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 with buffers of varying pH, ranging from ___________WHAT WERE THEY-________.