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RESEARCH PROFILES
If you stick a protein in a test tube with two types of polymers, the protein often doesn’t show a preference for either polymer. But if you stick the protein on a nanoparticle before introducing it into the test tube, the protein will select a polymer to associate with. In the January 15 issue of Analytical Chemistry (pp 379–386), Christine Keating and Scott Long at Pennsylvania State University demonstrate that protein conjugation to gold nanoparticles can change a protein’s behavior toward two polymers from indifference to strong preference. The investigators say that preferential partitioning could be used to purify biomolecule–nanoparticle conjugates developed for diagnostics and materials applications. When two aqueous solutions of polymers are mixed together in a so-called aqueous two-phase polymer system (ATPS), they act like a mixture of oil and water and separate into two distinct phases. Some biomolecules, such as nucleic acids, organelles, and cells, can be purified in an ATPS by partitioning them into a particular polymer phase. However, to get conditions suitable for preferential biomolecule partitioning into a polymer phase, parameters like pH, salt concentration, polymer concentration, and polymer molecular weight have to be adjusted. Keating and Long are limited, however, in how much they can tinker with the ATPS because they would like to pop it inside giant lipid vesicles to act as a model of the cytoplasm (Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 5920–5925). The lipid vesicles can only encapsulate the ATPS under certain experimental conditions, so the composition of the polymers in the ATPS can’t be altered very much. “If you optimize for [protein] partitioning, you’re optimizing away from the conditions where you can put [an ATPS] inside a lipid vesicle,” says Keating. “People put in 4 M salt. We can’t make vesicles in 4 M salt. We can make vesicles in 50 mM salt.”
Keating and Long knew from experience that colloidal gold nanoparticles preferentially go into the poly(ethylene glycol) (PEG) phase of an ATPS made of dextran and PEG polymers. On the
COURTESY OF CHRISTINE KEATING
Protein conjugates pick polymer phase
HRP–gold nanoparticle conjugates partition into the PEG-rich phase of a PEG–dextran ATPS.
basis of this observation, they decided to use gold nanoparticles to instigate protein partitioning. They directly adsorbed the proteins onto the surfaces of the nanoparticles; as nanoparticle conjugates, the proteins now entered either the PEG or dextran phase. “We didn’t have to change the composition of the aqueous two-phase system, which, for us, was really important. This is a method where you can keep [the ATPS] constant and still get good partitioning,” says Keating. The investigators studied the partitioning of three proteins—horseradish peroxidase (HRP), protein A, and bovine serum albumin—in a dextran–PEG ATPS. Free proteins didn’t exhibit a strong preference for either polymer
phase. However, nanoparticle conjugation significantly affected partitioning of the proteins—HRP–gold nanoparticles partitioned into the PEG phase by a factor of 150 more and the protein A–gold nanoparticle conjugates partitioned into the dextran phase by a factor of 2000 more. The bovine serum albumin–gold nanoparticle conjugates also preferentially partitioned into the dextran phase of the ATPS. Keating and Long found that an increase in nanoparticle diameter from 13 to 31 nm improved the partition coefficient by a factor of >40. They also determined that the partitioning of the protein–nanoparticle conjugates is dependent on the polymer concentration, the polymer molecular weight, and, in some cases, the concentration of the nanoparticles. The investigators don’t see why other types of nanoparticles, besides gold, shouldn’t work just as well in promoting the preferential partitioning of proteins in an ATPS. Partitioning of the biomolecule– nanoparticle conjugates could be useful for sorting out active and denatured DNA– and protein–nanoparticle conjugates in a mixture. Keating says, “If you have some [conjugates] that are denatured and some that are not denatured, you might want to think about ways of pulling out the more denatured ones and keeping them away from your sample. This technique is really sensitive to the surface chemistry. It might be a good way to start [asking] ‘Can we do a better purification of these [conjugates] now that they’re becoming more useful in a variety of analyses?’” But for now, Keating and Long are heading back to their simple model of a cell based on the ATPS inside the giant vesicles. “We’re going to use this [system] to partition enzymes so we can look at enzyme–enzyme interactions as a function of local concentration,” explains Keating. The increase in local concentration may change the way the enzymes behave in sequential reactions. a —Rajendrani Mukhopadhyay
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