December 12, 2003
A Monolayer of Ferritin Proteins at a Nanofilm Aqueous-Aqueous Interface
M. Li1, D.J. Chaiko2, and M.L. Schlossman3
1Department of Physics, University of Illinois, Chicago, IL;
2Argonne National Laboratory, Argonne, IL;
3Departments of Physics and Chemistry, University of Illinois, Chicago, IL
The formation of thin aqueous films on top of an aqueous subphase is demonstrated. The films form via a complex spreading process that results in the coexistence of macroscopic lenses and films that are several nanometers thick. Biomolecules, such as proteins, can be trapped at the aqueous-aqueous interface or in the thin film. This idea is demonstrated by an x-ray reflectivity study of ferritin proteins that form a two-dimensional assembly at the interface. It is shown that the electron density interfacial profile of the ferritin trapped in this thin film is consistent with the known crystal structure of ferritin.
Although interfaces between bulk liquids are commonly formed
between immiscible liquids, such as oil and water, it is also
possible to form an interface between two aqueous solutions. For
example, aqueous mixtures of polymers, salts, and water are known to
separate into two or more equilibrium phases. Aqueous biphase
systems (aqueous mixtures that form two phases) are used for the
purification and separation of biological materials. This is
possible because, although biological materials are soluble in both
aqueous phases, they prefer to be in one or the other, and are
therefore separated into one of the two. We have chosen to use
mixtures of polyethylene glycol (PEG), potassium phosphates, and
water to investigate the formation of protein assemblies in layers
of aqueous solutions that are approximately four nanometers thick.
Both the PEG and the potassium phosphates are compatible with many
proteins, making the mixture an appropriate choice.
We have shown that thin layers of aqueous solutions supported on a bulk aqueous subphase can be formed from biphase mixtures by the following procedure: Upon mixing the PEG, salt, and water, the resulting bulk liquid separates into a lower salt-rich phase and an upper PEG-rich phase. After extracting the salt-rich phase into a separate container, a single drop of the PEG-rich phase is placed onto the surface of the salt-rich phase. The drop spreads across the entire surface, thins rapidly, and then breaks up into smaller drops that coarsen to form flat lenses with diameters on the order of one centimeter (Figure 1). Then, using a pipette, any lenses in the path of the x-ray beam are either aspirated off the surface or moved off to the side (at least one lens remains on the surface during the x-ray measurements). These macroscopic lenses function as reservoirs to supply the PEG-rich thin film.
We used x-ray reflectivity to demonstrate that a four nanometer-thick film exists in the region between the macroscopic lenses. This region is a thin layer of the bulk PEG-rich phase, rather than a monolayer of PEG. Since proteins retain their natural conformation in PEG solutions and protein dimensions are typically similar to the film thickness, it is natural to ask if this thin film can be used to collect proteins into two-dimensional assemblies.
In an experiment to test the proof of this principle, we demonstrated that ferritin proteins can be assembled into the PEG-rich thin film. Ferritin is used for the storage of iron, and consists of a nearly spherical organic shell surrounding a nearly spherical core in which the iron is stored. The ferritin is prepared in a solution of water and PEG, whose composition mimics that of the PEG-rich solution in the biphase system. A few drops of the ferritin solution are added to the lower salt-rich bulk phase. Then, they rise to spread into the PEG-rich thin film. Figure 2 illustrates the electron density profile of this film, as measured using x-ray reflectivity, and compares it to the predicted profile of a layer of ferritin calculated from the known molecular structure of the protein. The near-match of measured and calculated profiles indicates that the ferritin forms a two-dimensional film that is ordered normal to the interface.
The overall method described here allows proteins or other biomolecules to be assembled two-dimensionally and studied with x-rays. This is significant because many proteins do not crystallize in three dimensions, or may be difficult to crystallize and, therefore, cannot be studied that way. Allowing proteins to assemble at an aqueous surface often results in protein denaturation, causing the loss of the protein’s biological activity. Our method retains the natural shape of the protein at a liquid surface. Moreover, the formation of these two-dimensional arrays may be useful technologically because the arrays allow the biomolecules to interact with other molecules. This could be utilized in chemical sensing or catalytic applications.
BEAMLINE
X19C
FUNDING
National Science Foundation Division of Materials Research
Petroleum Research Foundation administered by the American Chemical Society
Chemical Engineering Division at Argonne National Laboratory
PUBLICATION
Ming Li, David J. Chaiko, and Mark L. Schlossman, “An X-ray
Reflectivity Study of a Monolayer of Ferritin Proteins at a Nano-film
Aqueous-Aqueous Interface”, Journal of Physical Chemistry B
107, 9079-9085 (2003).
FOR MORE INFORMATION
Mark Schlossman
Department of Physics
University of Illinois at Chicago
Chicago, IL
Email: schloss@uic.edu