Larissa M. Mikheeva

Solvent Properties Governing Solute Partitioning in Polymer/Polymer Aqueous Two-Phase Systems: Nonionic Compounds

The solvatochromic solvent parameters characterizing the solvent polarity (pi*), solvent hydrogen-bond donor acidity (alpha), and solvent hydrogen-bond acceptor basicity (beta) of aqueous media were measured in the coexisting phases of nine different aqueous polymer/polymer two-phase systems (ATPS), containing 0.15 M NaCl in 0.01 M phosphate buffer, pH 7.4. Partitioning coefficients of six neutral compounds were measured in the nine ATPS at particular polymer concentrations. The solvatochromic equation was used to describe the partitioning of each compound. Three descriptors of the solvent properties of the phases could describe adequately the partitioning of the solutes in all the ATPS employed.

Solvent properties governing protein partitioning in polymer/polymer aqueous two-phase systems

Distribution coefficients of various proteins were measured in aqueous Dextran-Ficoll, Dextran-PES, and Ficoll-PES two-phase systems, containing 0.15M NaCl in 0.01 M phosphate buffer, pH 7.4. The acquired data were combined with data for the same proteins in different systems reported previously and known solvatochromic solvent properties of the systems to characterize the protein-solvent interactions. The relative susceptibilities of proteins to solvent dipolarity/polarizability, solvent hydrogen bond acidity, solvent hydrogen bond basicity, and solvent ability to participate in ion-ion and ion-dipole interactions were characterized. These parameters, which are representative of solute-solvent interactions, adequately described the partitioning of the proteins in each system. It was found that the relative susceptibilities of proteins to solvent dipolarity/polarizability are interrelated with their relative susceptibilities to solvent hydrogen bond acidity and solvent hydrogen bond basicity similarly to those established previously for small nonionic organic compounds.

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 0.15M 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: lnKi=aiolnKo+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.

Effect of salt additives on partition of nonionic solutes in aqueous PEG–sodium sulfate two-phase system

Partition of 12 nonionic organic compounds in aqueous PEG-8000-Na(2)SO(4) two-phase system was examined. Effects of four salt additives (NaCl, NaSCN, NaClO(4), and NaH(2)PO(4)) in the concentration range from 0.027 up to ca. 1.9 M on binodal curve of PEG-sulfate two-phase system and solute partitioning were explored. It was found that different salt additives at the relatively high concentrations display different effects on both phase separation and partition of various nonionic solutes. Analysis of the results indicates that the PEG-Na(2)SO(4) ATPS with the up to 0.215 M NaCl concentration may be viewed as similar to the ATPS without NaCl in terms of the Collander equations predictive ability of the partitioning behavior of nonionic compounds. All ATPS with each of the salt additive used at the concentration of 0.027 M may be viewed as similar to each other as the Collander equation holds for partition coefficients of nonionic solutes in these ATPS. Collander equation is valid also for the compounds examined in the ATPS with additives of NaSCN and NaClO(4) at the concentrations up to 0.215 M. The observed similarity between these ATPS might be explained by the similar effects of these two salts on the water structure. At concentrations of the salt additives exceeding the aforementioned values, different effects of salt additives on partitioning of various nonionic solutes are displayed. In order to explain these effects of salt additives it is necessary to examine the intensities of different solute-solvent interactions in these ATPS within the framework of the so-called Linear Solvation Energy Relationship (LSER) model.

Effect of salt additives on protein partition in polyethylene glycol–sodium sulfate aqueous two-phase systems

Partitioning of 15 proteins in polyethylene glycol (PEG)-sodium sulfate aqueous two-phase systems (ATPS) formed by PEG of two different molecular weights, PEG-600 and PEG-8000 in the presence of different buffers at pH7.4 was studied. The effect of two salt additives (NaCl and NaSCN) on the protein partition behavior was examined. The salt effects on protein partitioning were analyzed by using the Collander solvent regression relationship between the proteins partition coefficients in ATPS with and without salt additives. The results obtained show that the concentration of buffer as well as the presence and concentration of salt additives affects the protein partition behavior. Analysis of ATPS in terms of the differences between the relative hydrophobicity and electrostatic properties of the phases does not explain the protein partition behavior. The differences between protein partitioning in PEG-600-salt and PEG-8000-salt ATPS cannot be explained by the protein size or polymer excluded volume effect. It is suggested that the protein-ion and protein-solvent interactions in the phases of ATPS are primarily important for protein partitioning.

Effect of NaCl additive on properties of aqueous PEG-sodium sulfate two-phase system

The concentrations of all components in the phases of aqueous two-phase polyethylene glycol-sodium sulfate system of a fixed composition with different concentrations of NaCl additive were determined. Solvatochromic solvent features of aqueous media in the phases of all the systems were characterized in terms of solvent dipolarity/polarizability, solvent hydrogen bond donor acidity and hydrogen bond acceptor basicity. Partitioning of a homologous series of dinitrophenylated amino acids with aliphatic alkyl side chain was examined in all the systems, and the differences between the relative hydrophobicity and electrostatic properties of the phases were quantified. These differences were described in terms of solvatochromic solvent features of the phases. The previously reported partition coefficients of twelve different nonionic compounds in all the systems were expressed in terms of solute descriptors. It is demonstrated that two solvatochromic solvent descriptors (solvent dipolarity/polarizability, and solvent hydrogen bond donor acidity) could adequately describe the partitioning of the solutes in all the systems employed.

Origin of salt additive effect on solute partitioning in aqueous polyethylene glycol-8000-sodium sulfate two-phase system

Partitioning of a homologous series of dinitrophenylted (DNP-) amino acids with aliphatic side chains was examined in aqueous polyethylene glycol (PEG)-8000-sodium sulfate two-phase systems (ATPS) with the additives NaSCN, NaClO4, and NaH2PO4 at concentrations varied from 0.025M up to 0.54M. The differences between the relative hydrophobicities and electrostatic properties of the two phases in all ATPS were estimated. Partitioning of adenine, adenosine mono-, di- and tri-phosphates was also examined in all ATPSs, including those with NaCl additive. Partition coefficients for these compounds and for nonionic organic compounds previously reported [L.A. Ferreira, P. Parpot, J.A. Teixeira, L.M. Mikheeva, B.Y. Zaslavsky, J. Chromatogr. A 1220 (2012) 14.] were analyzed in terms of linear solvent regression relationship. The results obtained suggest that the effects of the salts additives are related to their influence on the water structure.

Prostate-specific Antigen/Solvent Interaction Analysis: A Preliminary Evaluation of a New Assay Concept for Detecting Prostate Cancer Using Urinary Samples

OBJECTIVE To provide preliminary clinical performance evaluation of a novel prostate cancer (CaP) assay, prostate-specific antigen/solvent interaction analysis (PSA/SIA) that focused on changes to the structure of PSA. METHODS Two-hundred twenty-two men undergoing prostate biopsy for accepted clinical criteria at 3 sites (University Hospitals Case Medical Center in Cleveland, Cleveland Clinic, and Veterans Administration Boston Healthcare System) were enrolled in institutional review board-approved study. Before transrectal ultrasound-guided biopsy, patients received digital rectal examination with systematic prostate massage followed by collection of urine. The PSA/SIA assay determined the relative partitioning of heterogeneous PSA isoform populations in urine between 2 aqueous phases. A structural index, K, whose numerical value is defined as the ratio of the concentration of all PSA isoforms, was determined by total PSA enzyme-linked immunosorbent assay and used to set a diagnostic threshold for CaP. Performance was assessed using receiver operating characteristic (ROC) analysis with biopsy as the gold standard. RESULTS Biopsies were pathologically classified as case (malignant, n=100) or control (benign, n=122). ROC performance demonstrated area under the curve=0.90 for PSA/SIA and 0.58 for serum total PSA. At a cutoff value of k=1.73, PSA/SIA displayed sensitivity=100%, specificity=80.3%, positive predictive value=80.6%, and negative predictive value=100%. No attempt was made in this preliminary study to further control patient population or selection criteria for biopsy, nor did we analytically investigate the type of structural differences in PSA that led to changes in k value. CONCLUSION PSA/SIA provides ratiometric information independently of PSA concentration. In this preliminary study, analysis of the overall structurally heterogeneous PSA isoform population using the SIA assay showed promising results to be further evaluated in future studies.

ΔG(CH2) as solvent descriptor in polymer/polymer aqueous two-phase systems

Phase diagrams were determined for aqueous two-phase systems (ATPSs) formed by different paired combinations of Dextran (Dex-75), Ficoll-70, polyethylene glycol (PEG-8000), hydroxypropyl starch (PES-100), and Ucon50HB5100 (a random copolymer of ethylene glycol and propylene glycol) all containing 0.15M NaCl in 0.01M phosphate buffer, pH 7.4, at 23 degrees C. Partition coefficients of a series of dinitrophenylated (DNP) amino acids with aliphatic side-chains were studied in all the ATPSs at particular polymer concentrations. Free energies of transfer of a methylene group between the coexisting phases, DeltaG(CH(2)), were determined as measures of the difference between the hydrophobic character of the phases. Furthermore, partition coefficients of tryptophan (Trp) and its di- and tri-peptides and a set of p-nitrophenyl (NP)-monosaccharides were measured in all the two-phase systems, and the data obtained compared with the DeltaG(CH(2)) values obtained in the systems. It was established that for eight out of 10 of two-phase systems of different polymer compositions the partition coefficients for Trp peptides correlate well with the DeltaG(CH(2)) values. Similar correlations for NP-monosaccharides were valid for seven out of 10 two-phase systems. These observations indicate that the difference between the hydrophobic characters of the coexisting phases represented by the DeltaG(CH(2)) value cannot be used as a single universal measure for comparison of the ATPSs of different polymer compositions.

Structural features important for differences in protein partitioning in aqueous dextran-polyethylene glycol two-phase systems of different ionic compositions

Partitioning of 15 proteins in dextran-70-polyethylene glycol (PEG)-8000 aqueous two-phase systems (ATPSs) in the presence of 0.01M sodium phosphate buffer, pH7.4 was studied. The effect of salt additives (NaCl, CsCl, Na2SO4, NaClO4 and NaSCN) at different concentrations on the protein partition behavior was examined. The salt effects on protein partitioning were analyzed by using the Collander solvent regression relationship between the protein partition coefficients in ATPSs with and without salt additives. The results obtained show that the presence and concentration of salt additives affect the protein partition behavior. Analysis of ATPSs in terms of the differences between the relative hydrophobicity and electrostatic properties of the phases does not explain the protein partition behavior. The differences between protein partitioning could not be explained by the protein size. The structural signatures for the proteins were constructed from partition coefficient values in four ATPSs with different salt additives, and the structural distances were calculated using cytochrome c as the reference structure. The structural distances for all the examined proteins (except lysozyme) were found to be interrelated. Analysis of about 50 different descriptors of the protein structures revealed that the partition behavior of proteins is determined by the peculiarities of their surfaces (e.g., the number of water-filled cavities and the averaged hydrophobicity of the surface residues) and by the intrinsic flexibility of the protein structure measured in terms of the B-factor (or temperature factor).