Bioanalytical Applications of Partitioning in Aqueous Polymer Two-Phase Systems Boris Y. Zaslavsky Department of Physiology and Biophysics Cornell University Medical College 1300 York Ave. New York, NY 10021
The rapid development of biotechnology requires new analytical methodologies for activities such as characterizing cell-expressed recombinant proteins and glycoproteins, monitoring cell population homogeneity, and assessing lot-to-lot consistency. Current methods of protein characterization include lectin affinity chromatography; MS; NMR spectroscopy; HPLC; and other chromatographic, chemical, and enzymatic methods. Most methods are time- and laborconsuming and rather expensive in terms of equipment and personnel costs. A simple, highly efficient characterization method is partitioning in aqueous polymer two-phase systems {1-3). This method may complement and sometimes even replace much more expensive techniques used in quality control and analytical laboratories where biotechnology research is performed. When two particular polymers— such as dextran (Dex) and Ficoll (Fie) or Dex and poly(ethylene glycol) (PEG)—or one polymer and a certain salt (e.g., PEG and sodium phosphate) are mixed at certain concentrations in an aqueous solution, the solution separates into two immiscible phases. One phase is rich in one polymer, and the second phase is rich in the other polymer (or salt). A clear interfacial boundary exists between the phases (1-3). Biological materials added to such systems distribute between the two 0003-2700/92/0364-765A/$03.00/0 © 1992 American Chemical Society
phases without any loss of biological activity. The partition behavior of a solute in a given two-phase system is governed by t h e solute's specific properties and by the types, molecular weights, and concentrations of the phase polymers; types and amounts of salts present; pH; and temperature. Pairs of polymers capable of phase separation in water are listed in Reference 1, and new polymer combinations are constantly being introduced (3). Partitioning of biological materials (from enzymes and nucleic acids to cells and viruses) has proved to be a universal and highly sensitive method for their separation and fractionation. Particularly important is the fact that the labile biomaterials
REPORT remain intact during partition experiments (1-3). Different modes of performing partitioning in aqueous two-phase systems are used. The simplest mode is the batch one-step procedure, which c o n s i s t s of m i x i n g a p p r o p r i a t e amounts of aqueous stock polymer solutions and salt-buffer solutions by weight. The solute solution is added, and the entire system is vigorously mixed. The system is usually centrifuged at ~ 4500 g for 10-15 min to speed phase settling. Aliquots of the settled phases are withdrawn from the system and processed to isolate the required product. Commonly used multistage chromatographic procedures include countercurrent distribution (CCD), centrifugal partition chromatography (CPC) (4, 5), and liquid column
chromatography (6, 7). To enhance partitioning of proteins or cells, an affinity ligand can be attached to one of the phase polymers. This procedure, known as affinity partitioning (1, 2), is similar to affinity column chromatography. Numerous examples of using the partition technique for separation and purification of biomaterials appear in a recent review (8). Because of the high sensitivity, reproducibility, versatility, and low cost of the technique, it is increasingly being used for biotechnology applications (8-10). The goal of preparative chromatography is to isolate or purify compounds; in analytical work, the goal is to obtain information about the sample (11). The requirements for the method depend on its application. Particularly important for analytical work are the n a t u r e of the information obtained and the sensitivity, time, and cost of a single analysis. To discuss these p a r a m e t e r s with regard to the partition method, the general features of aqueous twophase systems and of solute partitioning in these systems must be outlined.
Physicochemical features of aqueous two-phase systems The composition of the two phases is shown graphically in a phase diagram (Figure 1). The polymer compositions of the coexisting phases are represented by t h e corresponding points on the binodal curve that are joined by the so-called tie-line. The polymer concentration may be varied along a given tie-line to produce changes in the volumes of the phases with the same compositions. Any change in polymer concentra-
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REPORT tion away from a given tie-line pro duces two phases of different compo s i t i o n s . U s i n g t h e a n a l o g y of a water-organic solvent system, the polymer systems corresponding to the same tie-line are similar to a mixture of water with a given or ganic solvent, and the systems corre sponding to different tie-lines are similar to those formed by water with different solvents. Phase diagrams are different for different polymer pairs; they depend on molecular weight distributions (MWDs) of the polymers, tempera ture, low molecular weight additives, and other factors (1-3). Addition of an inorganic salt to a given polymer system usually changes the polymer composition of the coexisting phases. Polymer and salt compositions of the two phases are interrelated (12) ln([salt] 1 /[salt] 2 ) = β8 χ ( [ P l i - [ P ] a ) (1) where [salt] and [P] represent the concentrations (in weight percent) of a salt and phase polymer, P, respec tively; subscripts 1 and 2 denote the phases; and ββ is a constant that de pends on the type and total concen tration of the salt and on the types of both phase polymers. Any change in the polymer compo sition of the two phases will lead to a change in their salt composition. The salt additive seems to be similar to the organic modifier in solvent twophase systems. The modifier is usu ally added to change the phase prop erties that govern solute partitioning (4) and may be distributed between both phases or mostly in one phase.
The amount and type of modifier reg ulate partitioning properties of a ter nary solvent system, as does a salt additive in aqueous polymer systems. Physical properties of the phases (e.g., density, refractive index, and interfacial tension) vary with their polymer concentrations. Differences among the properties of the two phases are very small. Low interfacial t e n s i o n s of 0 . 1 - 1 0 0 μΝ/m make it possible to partition materi als such as labile enzymes and frag ile cells. A small density difference, however, complicates the CCD and CPC procedures (4, 5). An increase in the viscosity of the phases resulting from increasing polymer concentra tions limits the range of polymer compositions that can be used in the multistage chromatographic proce dures. These limitations, however, are much less important when the one-step procedure is used. The physicochemical properties of the phases also vary with their poly mer and salt compositions. To decide which properties are important for solute partitioning in aqueous poly mer systems, the mechanisms of par titioning must be understood. Differ ent theoretical approaches to the mechanisms of phase separation and partitioning of solutes in the systems have been reviewed (8, 13). These mechanisms are not completely un derstood. The operative forces clearly are those of solute interactions with system components (two polymers and water). The crucial question is, "How many components does the sol ute interact with—one, two, or all?" The answer seems to emerge from the following reasoning. Partitioning of a solute between the two phases is characterized by the partition coefficient, K, which is defined as the ratio of the solute con centration in the first polymer-rich phase to that in the second polymerrich phase: Κ = [solute] i/tsolutela
Figure 1. Phase diagram for an aqueous Dex-PEG system. The curved (binodal) line represents the borderline concentrations for formation of two-phase systems. Polymer mixtures below the line form single phases, and those above the line form two coexisting immiscible phases whose compositions are represented by the corresponding points on the binodal line.
(2)
where [solute] is the solute concen tration in a given phase and sub scripts 1 and 2 denote the corre sponding phases. The partition coefficient of a solute is k n o w n to v a r y w i t h different batches of phase polymers. Presum ably, this variation reflects lot-to-lot variations in the MWD of the poly mers. Therefore the system must be calibrated each time a new lot of polymers is used. Calibration also must be done if different pairs of polymers are used. Calibration consists of partitioning a randomly chosen set of 10-15 dif
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ferent chemical compounds, such as peptides, carbohydrates, proteins, and nucleic acids, in each system. Partition coefficients for a given sol ute in the two systems are related ac cording to In Κϋ = a, x In Koj + 6,
(3)
where Kj is the partition coefficient of the /th compound, subscripts i and ο denote the two aqueous polymer two-phase systems being compared, and at and 6, are constants (14). Coefficient a{ represents the ratio between the free energies of transfer of a methylene group between the two phases in the given systems a,· = AG(CH2),-/AG(CH2)0
(4)
where AG(CH2) is the free energy of transfer of a methylene group be tween the two phases (14,15). Coeffi cient a{ specifies the difference in the ability of the two phases of the sys tems being compared to hydrate nonpolar groups, and coefficient b{ speci fies the difference in their ability to interact with ionic and polar groups of the solutes being partitioned in the systems (15). Equation 3 is commonly known as an empirical extrathermodynamic Collander equation, which is used for comparing solute partition coeffi cients in different water-organic sol vent systems (16). In contrast to aqueous polymer two-phase systems, coefficient 6, for the solvent twophase systems depends not only on the systems being compared but also on the types of solutes being parti tioned (16). This dependence is at tributable to the difference in solutesolvent interactions in chemically different organic solvents. In aqueous polymer t w o - p h a s e systems, coefficients o ; and bt are in dependent of the chemical n a t u r e and structure of the compounds be ing partitioned; they depend only on the systems being compared. Chemi cally different solutes (e.g., proteins, nucleic acids, or carbohydrates) are not likely to interact similarly with chemically different phase polymers (e.g., PEG and Fie). Hence it was suggested (14) that there are no di rect solute-polymer interactions—at least in certain aqueous two-phase systems, such as D e x - P E G and D e x Fic systems. This assumption clearly does not cover the affinity partition technique or certain cases in which a given biopolymer may interact with one or another phase polymer. Once the values of a, and bt are de termined by partitioning a set of the same compounds in both systems, the partition coefficient value for any
REPORT solute in the ith system can be calcu lated if its value in the oth system is known. This simple technique en ables one to switch from one polymer batch to another or even from one type of two-polymer system to an other with little difficulty. It follows from the above that at least in some polymer systems, parti tioning of a solute between the two phases is governed by solute interac tions with only one component—wa ter—in both phases. Hence the physicochemical features of the water component in the two phases seem to be most important for solute parti tioning between the phases. The re sults obtained from complex permit tivity measurements, partition techniques, pH measurements, and the solvatochromic technique indi cate that the physicochemical prop erties of water in the two phases of a given polymer system are different from each other and from those of pure liquid water. The state and/or structure of water and its ability to hydrate solutes in the two phases are different. Partitioning of a solute is governed by all types of intermolecu-
lar forces (e.g., hydrogen bonds, elec trostatic ion-dipole and dipoledipole i n t e r a c t i o n s , h y d r o p h o b i c forces, and repulsive forces) involved in the solute-aqueous medium inter actions in the coexisting phases. Two different measures have been sug gested (17) to quantitate the differ ence in solvation ability of the aque ous media in the two phases. First, the free energy of transfer of a methylene group between the two phases, AG(CH 2 ), can be used to quantitate the difference in relative hydrophobicity of the two phases. The value of AG(CH2) is determined experimentally from the difference in partition coefficients in a homolo gous series of compounds between solutes differing in one alkyl chain length (-CH 2 -) (see Figure 2). The second method involves the solvent polarity parameter, ΕΎ, which is de termined from the transition energy for the solvatochromic absorption band of a dye employed as a probe for a given solvent (17, 18). The differ
ence in ET values for the two phases of a given system characterizes the difference in total solvation (hydra tion) ability of the aqueous media in the phases (17). Both scales (Figures 3a and 3b) can be used to characterize some aqueous polymer and water-organic solvent two-phase systems. The positions of aqueous polymer two-phase systems on solvent hydrophobicity (Figure 3a) and polarity (Figure 3b) scales are close to those of pure water. An enlarged view of each scale for the D e x - P E G system on the right in dicates the positions of the phases when different polymer concentra tions are used. The small difference between the properties of the phases seems to explain the ability of the systems to separate closely related biological molecules and is the reason these systems are used for separat ing biological materials (1-3, 6-10). The same high sensitivity of the par tition method that allows separation of biological materials in aqueous
Table 1. Partition coefficients of closely related compounds in various aqueous polymer two-phase systems
Figure 2. Logarithm of the partition coefficient, K, as a function of the length of the aliphatic chain, Nc, of a homologous series of sodium salts of (1) dinitrophenylated (DNP) amino acids with aliphatic alkyl side-chains; (2) sodium alkyl sulfates; and (3) alkyltrimethylammonium bromides in the aqueous Dex-PEG two-phase system of a fixed polymer and salt composition. Partitioning of each series of compounds is described as In Κ = C + ENC, where C and Ε are constants. Parameter £ represents the slope of a linear curve in the plot (i.e., the contribution of a CH2 group to the In K); it is independent of the chemical nature of the compounds partitioned in the system (the curves are parallel to each other). Parameter Ε is related to the free energy of transfer of a CH2 group from one phase to the other of a given two-phase system, AG(CH2), as AG(CH2) = -RTE, where R is the universal gas constant and Γ is the temperature.
Phase system*
Compound
Κ
Peptides Gly-Leu Leu-Gly lle-Gly Val-Gly Gly-Asp Asp-Gly
0.592 ±0.018 0.687 ± 0.021 0.714 ±0.021 0.511 ±0.015 0.247 ±0.010 0.387 ±0.012
System 1
Tyr-o-Ala-Gly-Phe-D-Leu-L-Arg Tyr-D-Ala-Gly-Phe-D-Leu-O-Arg
1.121 ±0.021 1.053 ±0.018
System 2
Tyr-D-Ala-Gly-Phe-Leu-Arg-Lys-Arg Tyr-Gly-Gly-Phe-Leu-Arg-Lys-Arg
0.900 ±0.019 0.814 ±0.020
System 2a
Nucleotides AMP dAMP CAMP 5'-ApA 2'-Apa 5'-UpU 2'-UpU
0.937 ±0.015 0.981 ±0.015 1.252 ±0.018 1.418 ±0.027 0.940 ± 0.020 1.362 ±0.025 1.008 ±0.015
System 2a
Glycosides 4-Nitrophenyl-p-D-galactopyranoside 4-Nitrophenyl-a-D-galactopyranoside 4-Nitrophenyl-a-D-glucopyranoside 4- Nitrophenyl -α-D- mannopyranoside
1.076 ±0.020 0.801 ±0.015 0.958 ±0.017 0.807 ±0.015
System 2
Proteins Insulin (intact) Insulin (iodinated, 1:1) Glucagon (intact) Glucagon (iodinated, 1:1) Glucagon (iodinated, 1:2.1)
1.00 0.38 1.15 0.45 0.24
System 3"
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polymer two-phase systems can be exploited for the analysis of biologi cal materials.
Applications of the partition technique for solute analysis and characterization The partition coefficient, K, for a given solute in a two-phase system with a fixed polymer and salt compo sition is a constant feature of the sol ute (as in water-organic solvent sys tems [19]) similar to other variables such as, for example, the specific ab sorption coefficient and the dielectric constant. The Κ values given in Table I are quite different for closely re lated compounds—even chiral pairs. Differences between Κ values for re versed dipeptides (20), hexapeptides differing only in enantiomers of the Arg residue (21), and conformationally different dinucleosidephosphates are large enough t h a t these com pounds can be separated by CCD or CPC techniques (4, 5, 18). A few recent examples include iso
Compound Proteins (continued) Albumin bovine (with lipid traces) Albumin bovine (lipid-free) Albumin human-Ie Albumin human-IIe Gamma-globulin human Alpha-globulin human Albumin human (HSA) (100% pure) HSA-warfarin (1:1) complex HSA-cloxacillin (1:1 ) complex HSA-cloxacillin (1:2) complex HSA-sulfamoxole (1:1) complex HSA-sulfamoxole (1:2) complex HSA-sulfisoxazole (1:1) complex Concavalin A Concavalin A complexes (1:1) with a-D-glucopyranose α-D- mannopyranose a- Methyl -D-glucopyranoside a-Methyl -D- mannopyranoside
lation of xanthine oxidase from milk by CCD in an aqueous D e x - P E G two-phase system (22), isolation of plasmid and high molecular weight DNA from eukaryotic or prokaryotic sources by extraction in a P E G - a m monium sulfate system (23), and pu rification of formate dehydrogenase from a crude extract of Candida boidinii by using affinity column chroma tography with a LiParGel 750 sup port equilibrated with a D e x - P E G system and using the PEG-bound affinity ligand Procion Red HE3b dye (24). Differences in p a r t i t i o n coeffi cients of peptides, proteins, and gly coproteins following r a t h e r slight chemical modifications (25) or con formational changes induced by for mation of 1:1 or 1:2 complexes with low molecular weight compounds (see Table I) support the generally ac cepted opinion (1-3) that the parti tion coefficient value is a highly sen sitive c h a r a c t e r i s t i c of a solute. Typically, albumin samples from dif-
κ
Phase system*
0.486 ± 0.022 0.558 ± 0.020 0.482 ± 0.026 0.540 ± 0.021 1.549 ±0.020 1.974 ±0.027
System 2
0.388 ± 0.009 0.589 ±0.018 0.486 ±0.017 0.715 ±0.020 0.549 ±0.018 1.019 ±0.015 0.641 ±0.012 0.450 ±0.018
System 4
0.230 ± 0.020 0.298 ± 0.024 0.267 ±0.018 0.366 ±0.010
" Polymer and salt compositions of the aqueous two-phase systems are as follows. System 1 : - 8.67% (w/w) PEG-3400; 13.36% (w/w) potassium phosphate (a salt mixture with the ratio of 306.9 g K2HPO4 to 168.6 g KH2P04)—no exact total composition of the system em ployed is given in Reference 20; System 2:10.8% (w/w) Dex-70; 12.5% (w/w) Fic-400; 0.15 mol kg"-1 NaCl; 0.01 mol kg~1 sodium phosphate buffer, pH 7.40 (21); System 2a: polymer composition as in System 2 but different salt composition—0.11 mol kg"1 sodium phosphate buffer, pH 7.40; System 3: 12.0% (w/w) Dex-500; 6.0% (w/w) PEG-6000; 0.02 M citrate-phosphate buffer, pH 5.0 (25); System 4:11.6% (w/w) Dex-70; 13.5% (w/w) Fic-400; 0.15 mol kg-1 NaCl; 0.01 mol kgr1 sodium phosphate buffer, pH 7.4. 6 Partition coefficients are taken from Reference 25 (presented as a plot with no experimental er ror indications). 'Human albumin-l and human albumin-ll are from ICN Pharmaceuticals and Central Institute of Haematology and Blood Transfusion, Moscow, respectively; samples of these two albumins con tained different lipids traces (
