Part:BBa_K1189018:Experience
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Applications of BBa_K1189018
Kinetic Analysis of Prussian Blue Ferritin
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.
| 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.
Another aspect of our analysis was determining the optimal temperature for catalytic activity of Prussian blue ferritin (Figure 8, 9).
Figure 8. Temperature 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 9. Temperature optimization of commerical 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.
The next aspect of our analysis was to see how Prussian blue ferritin would act in a catalytic sense on nitrocellulose (Figures 10,11). From these results we can that TMB is a better substrate on for use on nitrocellulose(Figure 11). With this substrate we saw a result from only 5 ng of Prussian blue ferritin present on the nitrocellulose.
Figure 10. Blots of Prussian blue ferritin on nitrocellulose (20 µL samples) that are reacted with ABTS (10 mg/mL). Concentrations of Prussian blue ferritin used are indicated in the figure. Results indicate colour change after 6 minutes. Controls include the substrate by itself, unmodified ferritin and bovine serum albumin. Four replicates are present per sample trial.
Figure 11. Blots of Prussian blue ferritin on nitrocellulose (20 µL samples) that are reacted with TMB (10 mg/mL). Concentrations of Prussian blue ferritin used are indicated in the figure. Results indicate colour change after 6 minutes. Controls include the substrate by itself, unmodified ferritin and bovine serum albumin. Four replicates are present per sample trial.
We applied a scaled down Prussian blue synthesis experiment to our own ferritinOur own Prussian blue ferritin was then exposed to the TMB substrate (Figure 12). From the results we can see that the ferritin with the E-coil attached had excellent catalytic activity.
Figure 12. Measurements of the coloured substrate TMB (10 mg/mL) at 650 nm over a 600 second time period for our own Prussian blue ferritin and unmodified ferritin. Sample volume was 242 µL. Controls for this experiment include bovine serum albumin (1 mg/mL)and the substrate solution by itself. Due to limitations on the protein available only one replicate was performed. Zero time points do not have low absorbance as colour change was rapid and began before measurements started.
In the case of the coils we were interested to see if the K-coil fused to TALE proteins (BBa_K1189029, BBa_K1189030) could bind to the E-coil found on one of our Prussian blue ferritin constructs (BBa_K1189018). To complete this task we placed the TALE on the membrane, washed and blocked the membrane. The ferritin protein with the complimentary coil was then added to the membrane. If this coil successfully binds to the other coil then the ferritin will not be washed off during the next wash step. We can then see if Prussian blue ferritin is bound by adding a TMB substrate solution that will cause a colour change. To this extent we saw a blue ring in this trial indicating a positive result. This suggests that our coils are actually binding in an in vitro system.
Another interesting element of this assay is why we used two variants of the TALE K-coil negative control. A blue ring on our TALE negative control confirmed our fear that during the second protein application and wash step that some of the ferritin with coil proteins would drift over and bind to the TALE K-coils on the nitrocellulose. This did not occur for our separate negative control (Figure 13).
Figure 13. This basic qualitative assay was used to inform us whether certain elements of our system are able to bind to each other. Our TALE proteins were mounted to the membrane along with positive controls of three Prussian blue variants; two recombinant ferritins and one commercial protein. The membranes were then washed and blocked. Prussian blue ferritin with a coil was added to our TALE protein containing a coil. Prussian blue ferritin with a TALE that could bind to the DNA held by another TALE on the membrane was also added. A TMB substrate solution was added to cause a colourimetric change over 5 minutes. Positive results are indicated by dark rings of colour. Negative controls include a TALE with a coil on the same membrane and the same TALE and bovine serum albumin on separate membranes that were treated separately. Image contrast was altered to make the results more clear on a digital monitor; the same changes were applied to each element of the figure.
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