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| − | <p>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 4, 5). 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.</p>
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| − | <figure>
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| − | <img src="https://static.igem.org/mediawiki/2013/3/36/UCalgary2013TRTmb6ulgraph.png" alt="Prussian Blue Ferritin and TMB" width="800" height="439">
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| − | <figcaption>
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| − | <p><b>Figure 4.</b> 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. </p>
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| − | </figcaption>
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| − | </figure>
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| − |
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| − | <figure>
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| − | <img src="https://static.igem.org/mediawiki/2013/1/15/UCalgary2013TRABTS8ulgraph.png" alt="Prussian Blue Ferritin and ABTS" width="800" height="433">
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| − | <figcaption>
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| − | <p><b>Figure 5.</b> 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.</p>
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| − | </figcaption>
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| − | </figure>
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| − |
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| − | <p>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 6,7). We also varied the hydrogen peroxide concentration in association with TMB as this the first chemical compound that will react in the system (Figure 8).</p>
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| − |
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| − | <figure>
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| − | <img src="https://static.igem.org/mediawiki/2013/2/20/UCalgary2013TRPBFABTSmichaelismentengraph.png" alt="Michaelis-Menten Plot for Prussian Blue Ferritin with ABTS" width="800" height="436">
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| − | <figcaption>
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| − | <p><b>Figure 6.</b> 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).</p>
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| − | </figcaption>
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| − | </figure>
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| − |
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| − | <figure>
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| − | <img src="https://static.igem.org/mediawiki/2013/c/c7/UCalgary2013TRPBFTMBmichaelismentengraph.png" alt="Michaelis-Menten Plot for Prussian Blue Ferritin with TMB" width="800" height="435">
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| − | <figcaption>
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| − | <p><b>Figure 7.</b> 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).</p>
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| − | </figcaption>
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| − | </figure>
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| − |
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| − | <figure>
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| − | <img src="https://static.igem.org/mediawiki/2013/e/e5/UCalgary2013TRPBFTMBGHydrogenperoxidemichaelismentengraph.png" alt="Michaelis-Menten Plot for Prussian Blue Ferritin Based on Hydrogen Peroxide (with TMB)" width="800" height="434">
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| − | <figcaption>
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| − | <p><b>Figure 8.</b> 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.</p>
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| − | </figcaption>
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| − | </figure>
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| − |
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| − | <center><b>Table 1.</b> Catalytic constants for our Prussian blue ferritin</center>
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| − |
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| − | <center><table width="800" border="1">
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| − | <tr>
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| − | <td>Catalyst</td>
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| − | <td>Enzyme Concentration (M)</td>
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| − | <td>Substrate</td>
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| − | <td>K<sub>m</sub> (mM)</td>
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| − | <td>V<sub>max</sub> (Ms<sup>-1</sup>)</td>
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| − | <td>K<sub>cat</sub> (s<sup>-1</sup>)</td>
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| − | <td>K<sub>cat</sub>/K<sub>m</sub> (M<sup>-1</sup>s<sup>-1</sup>)</td>
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| − | </tr>
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| − | <tr>
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| − | <td>Prussian Blue Ferritin</td>
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| − | <td>1.31 x 10<sup>-9</sup></td>
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| − | <td>ABTS</td>
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| − | <td>0.448</td>
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| − | <td>1.25 x 10<sup>-8</sup></td>
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| − | <td>9.51</td>
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| − | <td>2.12 x 10<sup>4</sup></td>
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| − | </tr>
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| − | <tr>
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| − | <td>Prussian Blue Ferritin</td>
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| − | <td>1.31 x 10<sup>-9</sup></td>
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| − | <td>TMB</td>
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| − | <td>0.0432</td>
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| − | <td>1.12 x 10<sup>-7</sup></td>
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| − | <td>85.3</td>
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| − | <td>1.97 x 10<sup>6</sup></td>
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| − | </tr>
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| − | <tr>
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| − | <td>Prussian Blue Ferritin</td>
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| − | <td>1.31 x 10<sup>-9</sup></td>
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| − | <td>H<sub>2</sub>O<sub>2 </sub> (TMB)</td>
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| − | <td>0.0176</td>
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| − | <td>1.31 x 10<sup>-8</sup></td>
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| − | <td>11.1</td>
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| − | <td>6.28 x 10<sup>5</sup></td>
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| − | </tr>
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| − | </table></center>
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| − |
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| − |
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| − | <br>
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| − | </html>
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This construct can be used as a reporter through a modification of the iron core to form Prussian Blue (Figure 2). The resulting molecule can then catalyze the formation of radicals from hydrogen peroxide, which can then cause a colour change in substrates such as TMB or ABTS (Figure 3).
