pH Optimization

Reaction Mixture

• 20μL Prussian Blue Ferritin (22μg/mL)
• 16μL Substrate (TMB or ABTS, 10mg/mL)
• 32μL Hydrogen Peroxide (30%)
• Buffer (pH 2-11) up to 268μL

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 the vary pHs of 2, 2.73, 3.6, 4, 4.4, 5, 5.6, 8, 9 and 11.

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 pH point (2, 2.73, 3.6, 4, 4.4, 5, 5.6, 8, 9, and 11).
5. Slopes of each experiment was determined and plotted against pH to determine optimum pH.