Part:BBa_K1189018:Experience

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Applications of BBa_K1189018

We performed a kinetic analysis of our Prussian blue ferritin. We included a comparison of Prussian blue horse spleen ferritin to regular horse spleen ferritin for both TMB and ABTS (Figures 1, 2). For both of the substrates we can see that normal ferritin has a very low catalytic activity compared to our modified ferritin. Using this data were able to determine the Michaelis-Menten catalytic constants for Prussian blue ferritin with different substrates.

Figure 1. Measurements of the absorbance of the 650nm light by the substrate TMB over a period of 600 seconds. 6 µL of 10 mg/mL substrate was used in a 242 µL reaction volume.Commercial Prussian blue ferritin ( 10 µL of 0.022 mg/mL sample) is represented by the blue data points. Orange data points are a negative control using standard ferritin (10 µL of 0.047 mg/mL sample). Negative controls are TMB and hydrogen peroxide, and TMB only. Standard error of the mean bars are based on a sample size where n=8. Substrate and hydrogen peroxide sample data is not clearly visible as it is in line with the substrate only data.

Figure 2. Measurements of the absorbance of the 415nm light by the substrate ABTS over a period of 600 seconds. 8 µL of 10 mg/mL substrate was used in a 242 µL reaction volume. Commercial Prussian blue ferritin ( 10 µL of 0.022 mg/mL sample) is represented by the blue data points. Orange data points are a negative control using standard ferritin (10 µL of 0.047 mg/mL sample). Negative controls are ABTS and hydrogen peroxide, and ABTS only. Standard error of the mean bars are based on a sample size where n=8.

In order to complete our kinetic analysis we had to determine the catalytic properties of our Prussian blue ferritin according to the Michaelis-Menten kinetic model. For these tests we varied the colourimetric substrate concentrations (ABTS and TMB) (Figures 3,4). We also varied the hydrogen peroxide concentration in association with TMB as this the first chemical compound that will react in the system (Figure 5).

Figure 3. Michaelis-Menten kinetic plot for commercial Prussian blue ferritin based on varying concentrations of ABTS. Absorbance readings were taken at 415 nm. Velocities were generated from the average slope of eight data sets. Standard error of the mean bars are not displayed but are present in the foundational data (eg. Figure 6).

Figure 4. Michaelis-Menten kinetic plot for commercial Prussian blue ferritin based on varying concentrations of TMB. Absorbance readings were taken at 650 nm. Velocities were generated from the average slope of eight data sets. Standard error of the mean bars are not displayed but are present in the foundational data (eg. Figure 5).

Figure 5. Michaelis-Menten kinetic plot for commercial Prussian blue ferritin based on varying concentrations of hydrogen peroxide. Absorbance readings were taken at 650 nm which measure the breakdown of TMB. Velocities were generated from the average slope of eight data sets. Standard error of the mean bars are not displayed but are present in the foundational data.

Table 1. Catalytic constants for our Prussian blue ferritin

Catalyst	Enzyme Concentration (M)	Substrate	Km (mM)	Vmax (Ms-1)	Kcat (s-1)	Kcat/Km (M-1s-1)
Prussian Blue Ferritin	1.31 x 10-9	ABTS	0.448	1.25 x 10-8	9.51	2.12 x 104
Prussian Blue Ferritin	1.31 x 10-9	TMB	0.0432	1.12 x 10-7	85.3	1.97 x 106
Prussian Blue Ferritin	1.31 x 10-9	H2O2 (TMB)	0.0176	1.31 x 10-8	11.1	6.28 x 105

We also performed a pH optimization of our Prussian blue ferritin using the substrates TMB and ABTS (Figure 6, 7).

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Figure 6. pH optimization of commercial Prussian blue ferritin with ABTS. Data is presented as a relative activity based on the highest activity seen during the experiment. Absorbance readings were taken at 415 nm to detect the colourimetric change in a 242 µL solution. Data based on a sample size of n=8. Standard error of the mean bars are not displayed due to their lack of visibility.

Figure 7. pH optimization of commercial Prussian blue ferritin with TMB. Data is presented as a relative activity based on the highest activity seen during the experiment. Absorbance readings were taken at 650 nm to detect the colourimetric change in a 242 µL solution. Data based on a sample size of n=8. Standard error of the mean bars are not displayed due to their lack of visibility.

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