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Team:Calgary/Sandbox/Project/Reporter/PrussianBlueFerritin
From 2013.igem.org
Prussian Blue Ferritin
What is Ferritin?
Ferritin is a ubiquitous iron storage protein found in both prokaryotes and eukaryotes allowing cells to keep iron in a soluble and non-toxic form. Ferritin across different species has very similar architecture and function. This is in despite of variations at the primary structure level (Harrison and Arosio, 1996). The 450 kDa protein shell consists of 24 subunits that can be composed of both heavy and light chains (Lawson et al., 1991) (Figure 1). Inside of this shell is room for an iron core composed of up to 4500 Fe (III) atoms stored as ferrihydrite phosphate (Ford et al., 1984). The goal of our project is to make use of this natural nanoparticle as both a scaffold and a reporter system.
Figure 1. Ribbon visualization of a fully assembled ferritin protein.
How can Ferritin be a Reporter?
Iron has naturally attracted attention for its ability to participate in Fenton chemistry. In this reaction iron acts as a catalyst in order to cause the disproportionation of hydrogen peroxide into oxygen-radical species (Figure 2). The resulting hydroxyl radical can cause the oxidation of common horseradish peroxidase substrates such as 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) (ABTS) and 3,3’,5,5’-tetramethylbenzidine (TMB) to produce a colourimetric output. The standard iron core of ferritin however is not very suitable for catalyzing this process. The catalytic activity of ferritin can be increased by synthesizing ferritin with a magnetite core. The synthesis of this ”magnetoferritin” however is time consuming and is prone to disruption due to oxidation.
Figure 2. Chemical equation for the disproportionation of hydrogen peroxide into oxygen radical species using iron as a catalyst; Fenton chemistry.
An alternative to increasing the catalytic activity of ferritin would be to provide the necessary ferrous ions for the Fenton chemistry via the iron complex Prussian blue. This compound could act as a surface modification agent to give the ferritin core a Prussian blue surface. Prussian blue has previously displayed high catalytic activity and is believed to act as a source of negative charge at appropriate pH levels in order to yield an affinity with positively charged peroxidase substrates such as TMB (Zhang et al., 2010).
Synthesizing Prussian Blue Ferritin
The creation of Prussian blue ferritin is a relatively simple process that involves the surface modification of the iron core of normal ferritin. In order to accomplish this purified ferritin is combined with the compound potassium ferrocyanide under acidic conditions in order to produce the iron complex Prussian blue (Zhang et al., 2013). This chemical reaction can be seen below:
Figure 3. Chemical equation for the synthesis of Prussian blue ferritin using potassium ferrocyanide.
The chemical process can easily be observed as there is a visible colour change from the colour of ferritin to the blue colour of Prussian blue ferritin (Figure 4). This reaction takes place overnight and then the Prussian blue ferritin is collected via dialysis.
Figure 4. Comparison image of commercial ferritin to Prussian blue ferritin after the synthesis reaction. The synthesis reaction took place over a 12 hour time period.
Kinetic Testing of Prussian Blue Ferritin
BLAH BLAH BLAH, FINISH GRAPHS, TABLE OF KINETIC STUFF, ADD EXPLOSIONS
Optimization of Prussian Blue Ferritin Reaction Conditions
EXPLOSION NOISE! HERE! KABOOM! KAPOW AND BAM BAM BAM!!! I <3 SPICE GIRLS
