RESUMEN
Complexation of proteins with polyelectrolytes or block copolymers can lead to phase separation to generate a coacervate phase or self-assembly of coacervate core micelles. However, many proteins do not coacervate at conditions near neutral pH and physiological ionic strength. Here, protein supercharging is used to systematically explore the effect of protein charge on the complex coacervation with polycations. Four model proteins were anionically supercharged to varying degrees as quantified by mass spectrometry. Proteins phase separated with strong polycations when the ratio of negatively charged residues to positively charged residues on the protein (α) was greater than 1.1-1.2. Efficient partitioning of the protein into the coacervate phase required larger α (1.5-2.0). The preferred charge ratio for coacervation was shifted away from charge symmetry for three of the four model proteins and indicated an excess of positive charge in the coacervate phase. The composition of protein and polymer in the coacervate phase was determined using fluorescently labeled components, revealing that several of the coacervates likely have both induced charging and a macromolecular charge imbalance. The model proteins were also encapsulated in complex coacervate core micelles and micelles formed when the protein charge ratio α was greater than 1.3-1.4. Small angle neutron scattering and transmission electron microscopy showed that the micelles were spherical. The stability of the coacervate phase in both the bulk and micelles improved to increased ionic strength as the net charge on the protein increased. The micelles were also stable to dehydration and elevated temperatures.
Asunto(s)
Polielectrolitos/química , Proteínas/química , Animales , Bovinos , Micelas , Modelos Moleculares , Conformación ProteicaRESUMEN
Enzymes have been applied to a variety of industrially and medically relevant chemistries as both catalysts and sensors. Incorporation of proteins and enzymes into complex coacervates has been demonstrated to improve the thermal, chemical, and temporal stability of enzymes in solution. In this work, a neutral-cationic block copolymer and an enzyme, alkaline phosphatase, are incorporated into complex coacervate core micelles (C3Ms) and coated onto a solid substrate to create a biocatalytic film from aqueous solution. The incorporation of photo-cross-linkable groups into the neutral block of the polymer allows the film to be cross-linked under ultraviolet light, rendering it insoluble. The morphology of the film is shown to depend most strongly on the protein loading within the film, while solvent annealing is shown to have a minimal effect. These films are then demonstrated as specific sensors for Zn2+ in solution in the presence of other metals, a model reaction for ion-selective heavy metal biosensing useful in environmental monitoring. They are shown to have low leaching and maintain sufficient activity and response for sensing for 1 month after aging under ambient conditions and at 40 °C and 50% relative humidity. The C3M immobilization method demonstrated can be applied to a wide variety of proteins with minimal chemical or genetic modification and could be used for immobilization of charged macromolecules in general to produce a wide variety of thin-film devices.