Partitioning Of Biomolecules Research Articles

Affinity-Controlled Partitioning of Biomolecules at Aqueous Interfaces and Their Bioanalytic Applications.

All-aqueous phase separation systems play essential roles in bioanalytical and biochemical applications. Compared to conventional oil and organic solvent-based systems, these systems are characterized by their rich bulk and interfacial properties, offering superior biocompatibility. In particular, phase separation in all-aqueous systems facilitates the creation of compartments with specific physicochemical properties, and therefore largely enhances the accessibility of the systems. In addition, the all-aqueous compartments have diverse affinities, with an important property known as partitioning, which can concentrate (bio)molecules toward distinct immiscible phases. This partitioning affinity imparts all-aqueous interfaces with selective permeability, enabling the controlled enrichment of target (bio)molecules. This review introduces the basic principles and applications of partitioning-induced interfacial phenomena in a typical all-aqueous system, namely aqueous two-phase systems (ATPSs); these applications include interfacial chemical reactions, bioprinting, and assembly, as well as bio-sensing and detection. The primary challenges associated with designing all-aqueous phase separation systems and several future directions are also discussed, such as the stabilization of aqueous interfaces, the handling of low-volume samples, and exploration of suitable ATPSs compositions with the efficient protocol.

Ionic Effect on the Microenvironment of Biomolecular Condensates.

Biomolecules such as proteins and RNA could organize to form condensates with distinct microenvironments through liquid-liquid phase separation (LLPS). Recent works have demonstrated that the microenvironment of biomolecular condensates plays a crucial role in mediating biological activities, such as the partition of biomolecules, and the subphase organization of the multiphasic condensates. Ions could influence the phase transition point of LLPS, following the Hofmeister series. However, the ion-specific effect on the microenvironment of biomolecular condensates remains unknown. In this study, we utilized fluorescence lifetime imaging microscopy (FLIM), fluorescence recovery after photobleaching (FRAP), and microrheology techniques to investigate the ion effect on the microenvironment of condensates. We found that ions significantly affect the microenvironment of biomolecular condensates: salting-in ions increase micropolarity and reduce the microviscosity of the condensate, while salting-out ions induce opposing effects. Furthermore, we manipulate the miscibility and multilayering behavior of condensates through ion-specific effects. In summary, our work provides the first quantitative survey of the microenvironment of protein condensates in the presence of ions from the Hofmeister series, demonstrating how ions impact micropolarity, microviscosity, and viscoelasticity of condensates. Our results bear implications on how membrane-less organelles would exhibit varying microenvironments in the presence of continuously changing cellular conditions.

Investigation of non-equilibrium separation time on the partitioning of cephalexin in an aqueous two-phase system composed of glucose and acetonitrile

Abstract In order to commercialize aqueous two-phase systems (ATPSs), not only the equilibrium data is essential, but also the knowledge of separation mechanisms, kinetics, settling time, and operational conditions are needed. Mixing duration and settling time are the most critical factors affecting separation and biomolecule partitioning in terms of economic aspects. This research aimed to find the desired conditions for separating cephalexin in an ATPS consisting of acetonitrile, glucose, and water. Firstly, the evolution of the interphase region was observed. Hereafter, to examine the effect of time on the experimental tie-lines and partition coefficient in non-equilibrium states, the settling time was varied from 2 min to 24 h. In addition, centrifugation was applied to help the separation at different time intervals and rotational speeds. The results of tie-lines slope and partitioning coefficients showed that the system approaches equilibrium after 5 h. However, using the centrifuge separation at 4000 rpm improved the separation time to 45 min, reaching 80 % of the actual partition coefficient. It can be concluded that with an acceptable tolerance in the partition coefficient, a remarkably diminished settling time is available for economic productivity in industrial units.

Multimodal peptide ligand extracts parvovirus from interface in affinity aqueous two-phase system.

Aqueous two-phase systems (ATPS) have found various applications in bioseparations and microencapsulation. The primary goal of this technique is to partition target biomolecules in a preferred phase, rich in one of the phase-forming components. However, there is a lack of understanding of biomolecule behavior at the interface between the two phases. Biomolecule partitioning behavior is studied using tie-lines (TL), where each TL is a group of systems at thermodynamic equilibrium. Across a TL, a system can either have a bulk PEG-rich phase with citrate-rich droplets, or the opposite can occur. We found that porcine parvovirus (PPV) was recovered at a higher amount when PEG was the bulk phase and citrate was in droplets and that the salt and PEG concentrations are high. To improve the recovery, A PEG 10kDa-peptide conjugate was formed using the multimodal WRW ligand. When WRW was present, less PPV was caught at the interface of the two-phase system, and more was recovered in the PEG-rich phase. While WRW did not significantly increase the PPV recovery in the high TL system, which was found earlier to be optimal for PPV recovery, the peptide did greatly enhance recovery at a lower TL. This lower TL has a lower viscosity and overall system PEG and citrate concentration. The results provide both a method to increase virus recovery in a lower viscosity system, as well as provide interesting thoughts into the interfacial phenomenon and how to recover virus in a phase and not at the interface.

Ionic Liquid-Assisted Selective Extraction and Partitioning of Biomolecules from Macroalgae.

Macroalgae are a promising feedstock for several industries due to their large content of proteins and carbohydrates and the high biomass productivities. A novel extraction and fractionation concept based on ionic liquids (ILs) using Ulva lactuca as model organism is presented. Biomolecules are first extracted by means of IL-assisted mechanical shear, followed by two-phase partitioning or ultrafiltration in order to fractionate proteins and carbohydrates and to recover the IL. Ethyl methyl imidazolium dibutyl phosphate ([Emim][DBP]) is strongly selective to proteins, leading to extraction yields up to 80.4% for proteins and 30.7% for carbohydrates. The complete process, including extraction and ultrafiltration, allowed protein recovery of up to 64.6 and 15.4% of the carbohydrates in the retentate phase, while a maximum of 85.7% of the IL was recovered in the permeate phase. The native structure of the extracted proteins was preserved during extraction and fractionation as shown by gel electrophoresis. Selective extraction of proteins from macroalgae under non-denaturing conditions using ILs followed by the recovery of IL using ultrafiltration is for the first time reported. The proposed extraction-fractionation approach is simple and can be potentially applied for the biorefinery of macroalgae at the commercial scale.

Artificial neural network modeling on the polymer-electrolyte aqueous two-phase systems involving biomolecules

Polymer-electrolyte aqueous two-phase systems (ATPS) have demonstrated their superior performance in the separation and purification of high-value biomolecules. However, these powerful platforms are still a major academic curiosity, without their acceptance and implementation by industry. One of the major obstacles is the absence of models to predict the partition of biomolecules in ATPS in an easy and predictive way. To address this limitation, modelling studies on the binodal curve behavior of polymer-electrolyte ATPS and the partitioning of biomolecules in these aqueous electrolyte solutions are carried out in this work. First, a comprehensive database targeting the studied systems is established. In total, 11,998 experimental binodal data points covering 276 polymer-electrolyte ATPS at different temperatures (273.15 K-399.15 K) and 626 experimental partition data points involving 22 biomolecules in 42 polymer-electrolyte ATPS at different temperatures (283.15 K-333.15 K) are included. Then, a novel modeling strategy that combines a well-known machine learning algorithm, i.e., artificial neural network (ANN) and group contribution (GC) method is proposed. Based on this modeling strategy, an ANN-GC model (ANN-GC model1) is built to describe the binodal curve behavior of polymer-electrolyte ATPS, while another ANN-GC model (ANN-GC model2) is developed to predict the partition of biomolecules in these biphasic systems. ANN-GC model1 gives a mean absolute error (MAE) of 0.0132 and squared correlation coefficient (R2) of 0.9878 for the 9,598 training data points, and for the 1,200 validation data points they are 0.0141 and 0.9858, respectively. Meanwhile, it also gives a MAE of 0.0143 and R2 of 0.9846 for the 1,200 test data points. On the other hand, ANN-GC model2 gives root-mean-square deviation (RMSD) of 0.0577 for 501 training data points, and for the 62 validation data points and 63 test data points their RMSD are 0.0849 and 0.0885, respectively. Furthermore, the obtained results also indicate that the tie-line length of polymer-electrolyte ATPS calculated from ANN-GC model1 can be directly used in ANN-GC model2 for predicting the partition performance coefficient of biomolecules in these ATPS. The developed models offer the possibility to predict the partition of biomolecules in ATPS without any requirement of experimental data. Based on the developed ANN-GC models, some high-performance ATPS are identified to partition four well-known biomolecules.

An Image Analysis Pipeline for Quantifying the Features of Fluorescently-Labeled Biomolecular Condensates in Cells.

Biomolecular condensates are cellular organelles formed through liquid-liquid phase separation (LLPS) that play critical roles in cellular functions including signaling, transcription, translation, and stress response. Importantly, condensate misregulation is associated with human diseases, including neurodegeneration and cancer among others. When condensate-forming biomolecules are fluorescently-labeled and examined with fluorescence microscopy they appear as illuminated foci, or puncta, in cells. Puncta features such as number, volume, shape, location, and concentration of biomolecular species within them are influenced by the thermodynamics of biomolecular interactions that underlie LLPS. Quantification of puncta features enables evaluation of the thermodynamic driving force for LLPS and facilitates quantitative comparisons of puncta formed under different cellular conditions or by different biomolecules. Our work on nucleoporin 98 (NUP98) fusion oncoproteins (FOs) associated with pediatric leukemia inspired us to develop an objective and reliable computational approach for such analyses. The NUP98-HOXA9 FO forms hundreds of punctate transcriptional condensates in cells, leading to hematopoietic cell transformation and leukemogenesis. To quantify the features of these puncta and derive the associated thermodynamic parameters, we developed a live-cell fluorescence microscopy image processing pipeline based on existing methodologies and open-source tools. The pipeline quantifies the numbers and volumes of puncta and fluorescence intensities of the fluorescently-labeled biomolecule(s) within them and generates reports of their features for hundreds of cells. Using a standard curve of fluorescence intensity versus protein concentration, the pipeline determines the apparent molar concentration of fluorescently-labeled biomolecules within and outside of puncta and calculates the partition coefficient (Kp) and Gibbs free energy of transfer (ΔGTr), which quantify the favorability of a labeled biomolecule partitioning into puncta. In addition, we provide a library of R functions for statistical analysis of the extracted measurements for certain experimental designs. The source code, analysis notebooks, and test data for the Punctatools pipeline are available on GitHub: https://github.com/stjude/punctatools. Here, we provide a protocol for applying our Punctatools pipeline to extract puncta features from fluorescence microscopy images of cells.

Insights into the Partitioning of DNA in Aqueous Biphasic System Containing Ammonium-based Ionic Liquid and Phosphate Buffer

Aqueous biphasic system (ABS), as an efficient separation process, can be applied in the extraction and purification of DNA which is an integral part of life science studies. Ammonium-based ionic liquids (ILs) were synthesized, and ABS formation was evaluated with phosphate buffer [K2HPO4-KH2PO4 (1.82:1.00)] as the second phase forming component. The structural influences of the cation of ILs, including alkyl chain length and the effect of functional groups on phase formation, were methodically assessed. The experimental binodal data were correlated with Merchuks’ equation and showed adequate fitting. The constituents of ABS were carefully selected to obtain a biocompatible system with controlled pH to use for the partitioning of biomolecules. The proposed system was applied in model DNA partitioning and resulted in a single step, highly efficient extraction at 298.15 K. The structural variations of the selected ILs influenced the partitioning, and thus, ABS containing benzyltributylammoniumchloride ([BTBA]Cl) IL was found to be efficient among the studied systems. Furthermore, the partition conditions were optimized in terms of the concentrations of IL and buffer using [BTBA]Cl/K2HPO4-KH2PO4 (1.82:1.00)/water system. Finally, the extraction mechanism involved in the partitioning and stability of DNA was investigated through FT-IR and circular dichroism spectra.

Performance of Bacillus subtilis D3d xylanase separated through optimized aqueous two-phase system in bio-bleaching of sugar beet pulp

Aqueous two-phase systems (ATPS) are potent, biocompatible, and cost-effective techniques for partitioning, concentration, or purification of biomolecules. In this work, the direct recovery of Bacillus subtilis D3d xylanase from the fermentation broth by the polymer-salt ATPS was optimized using response surface methodology (RSM). The 96% of xylanase recovery in the polyethylene glycol phase with the maximum purification factor (2.17 ± 0.02) and partition coefficient (69.87 ± 2.10) were the optimum responses that obtained at 9.5% (w/w) of polyethylene glycol, 20% (w/w) of sodium citrate, and pH 10. A good agreement between experimental and predicted results verified the adequacy of the RSM models. Furthermore, the efficiency of the crude and ATPS-recovered enzyme were evaluated in the biobleaching of sugar beet pulp. The ATPS-recovered enzymatic treatment of sugar beet pulp resulted in a higher release of reducing sugars (10.29 and 21.83 mg g−1) and caused 4.5% and 10% reduction in kappa number over crude-enzyme and control, respectively. This study indicated that ATPS-recovered xylanase compared to the crude enzyme has a promising application in the pulp and paper industry.

Aqueous Two-Phase System (ATPS)-Based Polymersomes for Particle Isolation and Separation.

Aqueous two-phase systems (ATPSs) have been widely used in the separation, purification, and enrichment of biomolecules for their excellent biocompatibility. While ultracentrifugation and microfluidic devices have been combined with ATPS to facilitate the separation of biomolecules and achieve high recovery yields, they often lack the ability to effectively isolate and separate biomolecules in low concentrations. In this work, we present a strategy that leverages the preferential partitioning of biomolecules in ATPS droplets to efficiently separate model extracellular vesicle (EV) particles. We demonstrate that the additional oil phase between the inner ATPS droplets and the aqueous continuous phase in triple emulsion droplets resolves the size controllability and instability issues of ATPS droplets, enabling the production of highly monodisperse ATPS-based polymersomes with enhanced stability for effective isolation of ATPS droplets from the surrounding environment. Furthermore, we achieve separation of model EV particles in a single dextran (DEX)-rich droplet by the massive production of ATPS-based polymersomes and osmotic-pressure-induced rupture of the selected polymersome in a hypertonic solution composed of poly(ethylene glycol) (PEG).

Dysregulated ribonucleoprotein granules promote cardiomyopathy in RBM20 gene-edited pigs.

Ribonucleoprotein (RNP) granules are biomolecular condensates-liquid-liquid phase-separated droplets that organize and manage messenger RNA metabolism, cell signaling, biopolymer assembly, biochemical reactions and stress granule responses to cellular adversity. Dysregulated RNP granules drive neuromuscular degenerative disease but have not previously been linked to heart failure. By exploring the molecular basis of congenital dilated cardiomyopathy (DCM) in genome-edited pigs homozygous for an RBM20 allele encoding the pathogenic R636S variant of human RNA-binding motif protein-20 (RBM20), we discovered that RNP granules accumulated abnormally in the sarcoplasm, and we confirmed this finding in myocardium and reprogrammed cardiomyocytes from patients with DCM carrying the R636S allele. Dysregulated sarcoplasmic RBM20 RNP granules displayed liquid-like material properties, docked at precisely spaced intervals along cytoskeletal elements, promoted phase partitioning of cardiac biomolecules and fused with stress granules. Our results link dysregulated RNP granules to myocardial cellular pathobiology and heart failure in gene-edited pigs and patients with DCM caused by RBM20 mutation.

Pre-purification of genipin from genipap using aqueous-two-phase systems composed of protic ionic liquids + polymers + water at 298 K and atmospheric pressure

The downstream processing still represents the cost bottleneck of obtaining biomolecules. Aqueous two-phase systems (ATPS) composed of alternative constituents have been presented as a cost-effective platform for the concentration, partition and/or purification of biomolecules. In this sense, this work provides a series of new ATPS composed of polymers (PPG-400, PEG-300, -400, -550 and -750 g gmol−1) + protic ionic liquids (PILs) [based on ethylene glycols (mono-, di- - and tri-ethylene glycol) as cation and inorganic anions (H2PO4−, HSO4−, Cl−, NO3−)] + water at 298 K and atmospheric pressure. An initial screening evaluated the effect of the type of polymer, molecular weight of the polymer, type of cation and anion of the ionic liquid on phases’ separation, while the second series of experiments studied their application as effective platforms for the pre-purification of genipin from genipap. The results showed that the hydrophilic/hydrophobic balance between phase forming components is the driving force for the formation of ATPS, as well as for the pre-purification of genipin from genipap. The system constituted of PEG-750 (40 wt%) + [MEA][H2PO4] (30 wt%) + H2O (30 wt%) exhibited the highest genipin pre-purification performance, i.e., purification factor of 6.03, as confirmed chromatographically.

Cholinium chloride as a weak salting-out agent to tune the biomolecules partition behavior in polymer-salt aqueous two-phase systems

Aqueous two-phase systems (ATPS) tend to be a suitable method for the extraction and purification of biomolecules. In this work, the cholinium chloride was applied to replace part of the salt concentration, achieving ions at equivalent concentrations, in ATPS composed of salt (NaH2PO, K2HPO4, K3PO4, K2CO3 or K3C5H5O7), poly(propylene)glycol (molecular weight of 400 g mol−1) and water in order to tune the phase properties and behavior and consequently the biomolecules partition behavior. For this, the liquid-liquid experimental data were obtained to evaluate the cholinium chloride effect on the phases diagram in comparison with the ternary system without cholinium chloride. In the same way alkaloids (caffeine and nicotine), phenolic compounds (gallic acid and vanillic acid) and amino acids (l-tryptophan, l-tyrosine, and l-phenylalanine) were used as molecular probes. The obtained result suggests a low kosmotropic characteristic from the cholinium chloride, which resulted in a decrease of the biphasic region when this salt was present in the systems. Beyond that, the partial substitution of salt by cholinium chloride was able to tune the partition behavior of the evaluated biomolecules, whether changing the preferential phase or increasing the extraction efficiency.

Effect of flow patterns on bovine serum albumin and ampicillin partitioning using aqueous two-phase systems in microdevice

The separation and purification of biomolecules via aqueous two-phase extraction (ATPE) has encountered a series of obstacles to its industrial implementation. Recently, the miniaturization of ATPE has attracted interest due to the increase of performance, the possibility of operating in a continuous mode, and easy to scale-up by numbering-up approach. However, there is a gap in the literature with regard to the effects of flow patterns on the partitioning of biomolecules using microdevices. Thus, the present study investigated the ATPE of bovine serum albumin (BSA) and ampicillin, a beta-lactam antibiotic, under different flow patterns (slug, droplet and parallel) by means of microdevices. The microdevices were manufactured with circular tubes in a senoidal shape and the ATPE were prepared from the mixture between aqueous solutions of polyethylene glycol (PEG) and dibasic potassium phosphate. Using 1.80 mm tubes, slug, droplet and parallel flow patterns were observed only in the PEG 1500/dibasic potassium phosphate system, while the parallel flow pattern was not stabilized in systems involving PEG 4000 and PEG 6000. Experiments using a droplet flow pattern showed higher values of recovery (56% after 10 min) and partition coefficient of BSA compared to the slug and parallel flow patterns. In experiments involving ampicillin, the extraction performance was practically independent of the flow patterns. The BSA recovery behaved as an inverse function of the diameter tubes, which achieved slightly superior performance than the batch experiment (67% after 10 min) using droplet flow pattern, 0.51 mm tubes and residence time of 10 min (69%).

The influence of zwitterions on the partition of biomolecules in aqueous biphasic systems

The use of hydrophilic zwitterionic compounds (ZI) to act as phase forming components of aqueous biphasic systems (ABS) has been attracting increased interest. Although previous works studied the phase behavior of ZI, there is still a lack of knowledge on the partition behavior of compounds in ZI-based ABS. This work reports a study on the influence of ZI structure for the extraction of 7 different biomolecules selected as molecular probes – 2 alkaloids (nicotine and caffeine), 2 phenolic compounds (gallic acid and vanillic acid) and 3 amino acids (L-tryptophan, L-tyrosine and L-phenylalanine). The partition and extraction efficiencies of these biomolecules were evaluated in ternary systems composed of hydrophilic ZI constituted by ammonium, imidazolium, pyridinium and piperidinium cationic and SO3− anionic groups, K2CO3 and water. The obtained results show that all the biomolecules preferentially partition to the ZI-rich phase, and the extent of their partition was closely related with the octanol–water partition coefficients of both biomolecules and ZI. It was demonstrated that the partition of biomolecules in ZI-based ABS is firstly ruled by hydrophobic/hydrophilic effects and only in some particular cases specific interactions between the biomolecule and the ZI affect their partition. Furthermore, despite the similarity between ZI and ionic liquids behavior as phase forming compounds of ABS for the extraction of biomolecules, ZI-based ABS presented a better performance in the extraction of phenolic compounds in alkaline environment.

Epigenetic feedback and stochastic partitioning during cell division can drive resistance to EMT.

Epithelial-mesenchymal transition (EMT) and its reverse process mesenchymal-epithelial transition (MET) are central to metastatic aggressiveness and therapy resistance in solid tumors. While molecular determinants of both processes have been extensively characterized, the heterogeneity in the response of tumor cells to EMT and MET inducers has come into focus recently, and has been implicated in the failure of anti-cancer therapies. Recent experimental studies have shown that some cells can undergo an irreversible EMT depending on the duration of exposure to EMT-inducing signals. While the irreversibility of MET, or equivalently, resistance to EMT, has not been studied in as much detail, evidence supporting such behavior is slowly emerging. Here, we identify two possible mechanisms that can underlie resistance of cells to undergo EMT: epigenetic feedback in ZEB1/GRHL2 feedback loop and stochastic partitioning of biomolecules during cell division. Identifying the ZEB1/GRHL2 axis as a key determinant of epithelial-mesenchymal plasticity across many cancer types, we use mechanistic mathematical models to show how GRHL2 can be involved in both the abovementioned processes, thus driving an irreversible MET. Our study highlights how an isogenic population may contain subpopulation with varying degrees of susceptibility or resistance to EMT, and proposes a next set of questions for detailed experimental studies characterizing the irreversibility of MET/resistance to EMT.

A mechanism for epithelial-mesenchymal heterogeneity in a population of cancer cells.

Epithelial-mesenchymal heterogeneity implies that cells within the same tumor can exhibit different phenotypes—epithelial, mesenchymal, or one or more hybrid epithelial-mesenchymal phenotypes. This behavior has been reported across cancer types, both in vitro and in vivo, and implicated in multiple processes associated with metastatic aggressiveness including immune evasion, collective dissemination of tumor cells, and emergence of cancer cell subpopulations with stem cell-like properties. However, the ability of a population of cancer cells to generate, maintain, and propagate this heterogeneity has remained a mystifying feature. Here, we used a computational modeling approach to show that epithelial-mesenchymal heterogeneity can emerge from the noise in the partitioning of biomolecules (such as RNAs and proteins) among daughter cells during the division of a cancer cell. Our model captures the experimentally observed temporal changes in the fractions of different phenotypes in a population of murine prostate cancer cells, and describes the hysteresis in the population-level dynamics of epithelial-mesenchymal plasticity. The model is further able to predict how factors known to promote a hybrid epithelial-mesenchymal phenotype can alter the phenotypic composition of a population. Finally, we used the model to probe the implications of phenotypic heterogeneity and plasticity for different therapeutic regimens and found that co-targeting of epithelial and mesenchymal cells is likely to be the most effective strategy for restricting tumor growth. By connecting the dynamics of an intracellular circuit to the phenotypic composition of a population, our study serves as a first step towards understanding the generation and maintenance of non-genetic heterogeneity in a population of cancer cells, and towards the therapeutic targeting of phenotypic heterogeneity and plasticity in cancer cell populations.

Formation of Multiphase Complex Coacervates and Partitioning of Biomolecules within them.

Biological systems employ liquid-liquid phase separation to localize macromolecules and processes. The properties of intracellular condensates that allow for multiple, distinct liquid compartments and the impact of their coexistence on phase composition and solute partitioning are not well understood. Here, we generate two and three coexisting macromolecule-rich liquid compartments by complex coacervation based on ion pairing in mixtures that contain two or three polyanions together with one, two, or three polycations. While in some systems polyelectrolyte order-of-addition was important to achieve coexisting liquid phases, for others it was not, suggesting that the observed multiphase droplet morphologies are energetically favorable. Polyelectrolytes were distributed across all coacervate phases, depending on the relative interactions between them, which in turn impacted partitioning of oligonucleotide and oligopeptide solutes. These results show the ease of generating multiphase coacervates and the ability to tune their partitioning properties via the polyelectrolyte sharing inherent to multiphase complex coacervate systems.

Cholinium chloride effect on ethanol-based aqueous biphasic systems: Liquid-liquid equilibrium and biomolecules partition behavior

Alcohols-based aqueous biphasic systems (ABS), such as ethanol-based ABS, are of great interest to the industry due to its low viscosity and suitability in separation and purification of biomolecules. Ethanol-based ABS using several salts were well described in the literature. In this work, the cholinium chloride ([N111(2OH)]Cl) was combined with the salts (NaH2PO4, K2HPO4, K3PO4, K2CO3, and K3C6H5O7) to obtain ions at equivalent concentrations. The cholinium chloride influence was evaluated on the ABS formation and biomolecules partition. For all systems, the [N111(2OH)]Cl affects the liquid-liquid equilibrium, and a decrease of the biphasic region was observed due to the reduction of the salt ionic strength when part of this was replaced by the cholinium chloride. Moreover, the top and bottom phases properties were properly described using analytical techniques to quantify all the phases’ components. Concerning the [N111(2OH)]Cl effect on biomolecules separation and purification process, several biomolecules namely alkaloids (caffeine and nicotine), amino acids (l-tryptophan, l-phenylalanine and l-tyrosine) and phenolic compounds (gallic acid and vanillic acid) were used as molecular probes. The partition coefficient (K) showed a strong influence of the [N111(2OH)]Cl presence on the partition behavior. Besides, in some cases, specific interaction between the [N111(2OH)]Cl and the biomolecules overcome the electrostatic interactions as the main driving force to the partition behavior. The obtained results suggest that [N111(2OH)]Cl can act in ABS to modulate the phases properties and partition behavior.

Charge-Based Separation of Proteins Using Polyelectrolyte Complexes as Models for Membraneless Organelles.

Membraneless organelles are liquid compartments within cells with different solvent properties than the surrounding environment. This difference in solvent properties is thought to result in function-related selective partitioning of proteins. Proteins have also been shown to accumulate in polyelectrolyte complexes, but whether the uptake in these complexes is selective has not been ascertained yet. Here, we show the selective partitioning of two structurally similar but oppositely charged proteins into polyelectrolyte complexes. We demonstrate that these proteins can be separated from a mixture by altering the polyelectrolyte complex composition and released from the complex by lowering the pH. Combined, we demonstrate that polyelectrolyte complexes can separate proteins from a mixture based on protein charge. Besides providing deeper insight into the selective partitioning in membraneless organelles, potential applications for selective biomolecule partitioning in polyelectrolyte complexes include drug delivery or extraction processes.