Correlations between distribution coefficients of various biomolecules in different polymer/polymer aqueous two-phase systems

Distribution coefficients for a variety of proteins and certain other biomolecules (peptides, amino acids, and carbohydrates) (overall 27 different solutes) were measured in aqueous two-phase systems (ATPSs) dextran (Dex)–polyethylene glycol (PEG) and Dex–Ucon 50-HB-5100 (Ucon—a random copolymer of ethylene glycol and propylene glycol) both containing 0.15MNaCl in 0.01Mphosphate buffer, pH 7.4, at 23 ◦C. Distribution coefficients of some selected solutes were also measured in the above two-phase systems at three different polymer concentrations for each system. It was established that the distribution coefficients for all the proteins examined in the ATPSs are correlated according to the so-called Collander linear equation.Fundação para a Ciência e a Tecnologia (FCT)FEDE

“On the collander equation”: protein partitioning in polymer/polymer aqueous two-phase systems

Distribution coefficients of randomly selected proteins were measured in aqueous two-phase systems (ATPSs) formed by different combinations of Dextran-75 (Dex), Ficoll-70, polyethylene glycol-8000 (PEG), hydroxypropyl starch-100 (PES), and Ucon50HB5100 (Ucon, a random copolymer of ethylene glycol and propylene glycol) at particular polymer concentrations, all containing 0.15 M NaCl in 0.01 M phosphate buffer, pH 7.4. Most of the proteins in the PEG-Ucon system precipitated at the interface. In the other ATPSs, namely, PES-PEG, PES-Ucon, Ficoll-PEG, Ficoll-Ucon, and in Dex-PEG and Dex-Ucon described earlier the distribution coefficients for the proteins were correlated according to the solvent regression equation: ln Ki = aio ln Ko + bio, where Ki and Ko are the distribution coefficients for any protein in the ith and oth two-phase systems. Coefficients aio and bio are constants, the values of which depend upon the particular compositions of the two-phase systems under comparison.Fundo Europeu de Desenvolvimento Regional (FEDER)Fundação para a Ciência e a Tecnologia (FCT

Analysis of partitioning of organic compounds and proteins in aqueous polyethylene glycol-sodium sulfate aqueous two-phase systems in terms of solute-solvent interactions

Partition behavior of nine small organic compounds and six proteins was examined in poly(ethylene glycol)-8000-sodium sulfate aqueous two-phase systems containing 0.5 M osmolyte (sorbitol, sucrose, trehalose, TMAO) and poly(ethylene glycol)-10000-sodium sulfate system, all in 0.01 M sodium phosphate buffer, pH 6.8. The differences between the solvent properties of the coexisting phases (solvent dipolarity/polarizability, hydrogen bond donor acidity, and hydrogen bond acceptor basicity) were characterized with solvatochromic dyes using the solvatochromic comparison method. Differences between the electrostatic properties of the phases were determined by analysis of partitioning of sodium salts of dinitrophenylated (DNP-) amino acids with aliphatic alkyl side-chain. It was found out that the partition coefficient of all compounds examined (including proteins) may be described in terms of solutesolvent interactions. The results obtained in the study show that solutesolvent interactions of nonionic organic compounds and proteins in polyethylene glycol-sodium sulfate aqueous two-phase system differ from those in polyethylene glycol-dextran system.Nuno da Silva acknowledges the financial support by Fundacao para a Ciencia e a Tecnologia (FCT) of the Portuguese's Ministry for Science, Technology and Higher Education, in the framework of the Operational Program COMPETE (PTDC/EQU-FTT/120332/2010).Pedro P. Madeira acknowledges the financial support in part provided by (i) FCT/MEC, FEDER under Program PT2020 (Project UID/EQU/50020/2013) and (ii) QREN, ON2 and FEDER, under Program COMPETE (Project NORTE-07-0124-FEDER-0000011).Jose A. Teixeira and Nuno da Silva appreciate the Fundacao para a Ciencia e Tecnologia for the strategic funding of UID/BIO/04469/2013 unit and financial support by the Portuguese Foundation for Science and Technology (FCT) under the scope of the Project RECI/BBB-EBI/0179/2012 (FCOMP-01-0124-FEDER-027462)

Linear relationships between partition coefficients of different organic compounds and proteins in aqueous two-phase systems of various polymer and ionic compositions

Analysis of the partition coefficients of small organic compounds and proteins in different aqueous two-phase systems under widely varied ionic compositions shows that logarithms of partition coefficients for any three compounds or proteins or two organic compounds and one protein are linearly interrelated, although for protein(s) there are ionic compositions when the linear fit does not hold. It is suggested that the established interrelationships are due to cooperativity of different types of solute–solvent interactions in aqueous media. This assumption is confirmed by analysis of distribution coefficients of various drugs in octanol-buffer systems with varied ionic compositions of the buffer. Analysis of the partition coefficients characterizing distribution of variety of drugs between blood and different tissues of rats in vivo reported in the literature showed that the above assumption is correct and enabled us to identify the tissues with the components of which the drug(s) may engage in presumably direct interactions. It shows that the suggested assumption is valid for even complex biological systems.info:eu-repo/semantics/publishedVersio

Hydrogen bond arrangement is shown to differ in coexisting phases of aqueous two-phase systems

Analysis by attenuated total reflection-Fourier transform infrared spectroscopy shows that each coexisting phase in aqueous two-phase systems has a different arrangement of hydrogen bonds. Specific arrangements vary for systems formed by different solutes. The hydrogen bond arrangement is shown to correlate with differences in hydrophobic and electrostatic properties of the different phases of five specific systems, four formed by two polymers and one by a single polymer and salt. The results presented here suggest that the arrangement of hydrogen bonds may be an important factor in phase separation.P.P.M. acknowledges University of Aveiro, CICECO-Aveiro Institute of Materials for funding in the framework of the project UIDB/5011/2020 and UIDP/50011/2020, financed by national funds through the FCT/MEC contract foreseen in the numbers 4, 5, and 6 of the article 23, of the Decree-Law 57/2016, of August 29, changed by Law 57/2017, of July 19.publishe

\u3cem\u3eIn Aqua Veritas\u3c/em\u3e: The Indispensable Yet Mostly Ignored Role of Water in Phase Separation and Membrane-less Organelles

Despite the common practice of presenting structures of biological molecules on an empty background and the assumption that interactions between biological macromolecules take place within the inert solvent, water represents an active component of various biological processes. This Perspective addresses indispensable, yet mostly ignored, roles of water in biological liquid–liquid phase transitions and in the biogenesis of various proteinaceous membrane-less organelles. We point out that changes in the structure of water reflected in the changes in its abilities to donate and/or accept hydrogen bonds and participate in dipole–dipole and dipole–induced dipole interactions in the presence of various solutes (ranging from small molecules to synthetic polymers and biological macromolecules) might represent a driving force for the liquid–liquid phase separation, define partitioning of various solutes in formed phases, and define the exceptional ability of intrinsically disordered proteins to be engaged in the formation of proteinaceous membrane-less organelles

Solvent Interaction Analysis as a Proteomic Approach to Structure-based Biomarker Discovery and Clinical Diagnostics

Proteins have several measurable features in biological fluids that may change under pathological conditions. The current disease biomarker discovery is mostly based on protein concentration in the sample as the measurable feature. Changes in protein structures, such as post-translational modifications and in protein–partner interactions are known to accompany pathological processes. Changes in glycosylation profiles are well-established for many plasma proteins in various types of cancer and other diseases. The solvent interaction analysis method is based on protein partitioning in aqueous two-phase systems and is highly sensitive to changes in protein structure and protein–protein- and protein–partner interactions while independent of the protein concentration in the biological sample. It provides quantitative index: partition coefficient representing changes in protein structure and interactions with partners. The fundamentals of the method are presented with multiple examples of applications of the method to discover and monitor structural protein biomarkers as disease-specific diagnostic indicators

Analytical Applications of Partitioning in Aqueous Two-phase Systems: Exploring Protein Structural Changes and Protein–partner Interactions in Vitro and in Vivo by Solvent Interaction Analysis Method

This review covers the fundamentals of protein partitioning in aqueous two-phase systems (ATPS). Included is a review of advancements in the analytical application of solute partitioning in ATPS over the last two decades, with multiple examples of experimental data providing evidence that phase-forming polymers do not interact with solutes partitioned in ATPS. The partitioning of solutes is governed by the differences in solute interactions with aqueous media in the two phases. Solvent properties of the aqueous media in these two phases may be characterized and manipulated. The solvent interaction analysis (SIA) method, based on the solute partitioning in ATPS, may be used for characterization and analysis of individual proteins and their interactions with different partners. The current state of clinical proteomics regarding the discovery and monitoring of new protein biomarkers is discussed, and it is argued that the protein expression level in a biological fluid may be not the optimal focus of clinical proteomic research. Multiple examples of application of the SIA method for discovery of changes in protein structure and protein–partner interactions in biological fluids are described. The SIA method reveals new opportunities for discovery and monitoring structure-based protein biomarkers

Phase Equilibria, Solvent Properties, and Protein Partitioning in Aqueous Polyethylene Glycol-600-trimethylamine N-oxide and Polyethylene Glycol-600-choline Chloride Two-phase Systems

The phase diagram of a new aqueous two-phase system (ATPS) formed by polyethylene glycol with molecular weight 600 (PEG-600) and trimethylamine N-oxide (TMAO) in 0.01 M sodium phosphate buffer (NaPB), pH 7.4, is determined and hydrophobic, electrostatic and other solvent properties of the phases are characterized. The same properties are determined for the ATPS formed by PEG-600 and choline chloride in 0.01 M sodium phosphate buffer (NaPB), pH 7.4. Solvent properties of water (dipolarity/polarizability, hydrogen bond donor acidity, and hydrogen bond acceptor basicity) in aqueous solutions of polypropylene glycol-400 (PPG-400), polyethylene glycol dimethyl ether -250 (PEGDME-250), and choline chloride are determined at different concentrations. The concentrations of the aforementioned polymers, as well as PEG-600 and PEG-1000 required for phase separation in mixtures with choline chloride reported in the literature are analyzed. It is found that the concentrations of polymers needed for phase separation in mixtures with 35%wt. choline chloride are linearly related with water hydrogen bond donor acidity or hydrogen bond acceptor basicity in the individual polymer solutions at given concentrations. Partition behavior of nine proteins was examined in both systems. The partition coefficients of proteins in PEG-600-choline chloride ATPS exceeded those observed in PEG-600-TMAO ATPS from ca. 2 to ca. 75-fold possibly due to the larger difference between the composition of the coexisting phases in the former ATPS. Analysis of partition coefficients in the two ATPS were compared to those reported in Dextran-PEG ATPS, and proteins likely engaged in direct interactions with choline chloride were identified

What Can Be Learned from the Partitioning Behavior of Proteins in Aqueous Two-Phase Systems?

This review covers the analytical applications of protein partitioning in aqueous two-phase systems (ATPSs). We review the advancements in the analytical application of protein partitioning in ATPSs that have been achieved over the last two decades. Multiple examples of different applications, such as the quality control of recombinant proteins, analysis of protein misfolding, characterization of structural changes as small as a single-point mutation, conformational changes upon binding of different ligands, detection of protein–protein interactions, and analysis of structurally different isoforms of a protein are presented. The new approach to discovering new drugs for a known target (e.g., a receptor) is described when one or more previous drugs are already available with well-characterized biological efficacy profiles