Introduction: Polyampholytes may be efficiently prepared by copolymerization of cationic and anionic monomers, and exhibit distinct properties such as isoelectric points (pI), anti-polyelectrolyte effects, and often, soluble to insoluble transitions during heating. These protein-like properties give polyampholytes the potential ability to inhibit protein aggregation, and protect mammalian cells during freezing. We are preparing stoichiometric polyampholytes formed from acrylic monomers such as N-(3-aminopropyl) methacrylamide hydrochloride (APM) and acrylic acid (AA), and are exploring their solution properties and potential use as new protein aggregation inhibitors and cryoprotective agents.
Materials and Methods: Polyampholytes were prepared by free radical copolymerization in water at 55°C, with 10% (w/v) total monomer loading and 1:1 ratio of APM and AA or methacrylamidopropyltrimethylammonium chloride (MAPTAc) and AA and 1 mol% initiator (Vazo-56, relative to total monomer), with polymerizations stopped at 60-80% conversion as determined by 1H-NMR. The copolymers were purified by dialysis and isolated by freeze drying. The pI was measured by turbidimetric titration and the LCST by UV-vis spectrophotometry using a heated sample holder. Protein aggregation inhibition was studied by heating 3 mg/ml lysozyme PBS solutions with or without 1 %wt polymer at 90oC for 10 min.
Results and Discussion: The pI of PAPM-co-AA is close to 8.3, with the polymer solution showing cloudiness in a broad pH range around the pI (between pH 7 to 9). The polymer solubility increases with NaCl concentration, and finally soluble with 200 mM NaCl. The reversible soluble to insoluble (LCST) transition during heating was measured by UV-vis at pI with 225 mM NaCl, and the LCST is at 60oC. However, the PMAPTAc-co-AA does have pH or temperature sensitivity. To measure the protein aggregation inhibition, lysozyme was mixed with both PAPM-co-AA and PMAPTAc-co-AA and heated at 90oC for 10 min. Before heating the neat lysozyme solution is clear; the one with PAPM-co-AA is cloudy because the pH is close to pI because the polymer forms a liquid coacervate phase; and the other one with PMAPTAc-co-AA is clear because the PMAPTAc-co-AA is not a pH or temperature sensitive polymer. After heating, the lysozyme solution precipitates, the one with PAPM-co-AA retains similar cloudiness but the other one with PMAPTAc-co-AA becomes cloudy. That indicates that PAPM-co-AA can inhibit lysozyme from aggregating upon heating, and this is attributed to partitioning of the protein into the protective liquid polymer coacervate phase.
Conclusion: The PAPM-co-AA is a protein-like polymer with pH and temperature induced soluble to insoluble transition in water. The solubility in water is pH, [NaCl] and temperature related. Preliminary results shows the polymer has potential ability to protect proteins during heating, and the key factor should be the thermally induced phase separation of PAPM-co-AA.
NSERC (the discovery and CREATE programs); The OCE-RE program of Ontario