-
Indian Journal of Biotechnology Vol I , January 2002, pp
87-95
Electrosta,tic Interactions, Phase Separation Behaviour and
Partitioning of Proteins in Polyelectrolyte Based Aqueous Two-Phase
Systems
Vandana Gupta, Sunil Nath and Subhash Chand*
Department of Biochemical Engineering and Biotechnology, Indi an
Institute of Technology Delhi , New Delhi 110016, Indi a
Electrostatic interactions playa major role in the purification
of proteins by different methods and they tend to be specific in
nature in the presence of salts and environmental hydrogen ion
concentration (PH). These can be readily exploited as the basis for
protein isolation and purification by aqueous two-phase system
(ATPS) using a polyelectrolyte as one of the polymeric components.
Polyelectrolytes are water-soluble charged polymers and the phase
separation in the polyelectrolyte based ATPS is dependent on the
ionic composition of the system and the charge density of polymers.
This review outlines the mechanism of the phase separation in
non-ionic polymers based A TPS on the basis of water structure in
polymeric solution and validity of the hypothesis is further
discussed for polyelectrolyte-based systems. Partitioning of
proteins in polyeletrolyte based A TPS is dominantly controlled by
the electrostatic interactions. Several factors that influence the
partitioning of proteins include the properties of the polymers,
pH, salt type and concentration and the charge on protein
molecules. Low polymer concentration requirement for the phase
separation and high specificity of protein purification in
polyelectrolyte based A TPS holds a great promise for their large
scale applications in isolation and purification of proteins.
Keywords: polyelectrolyte, electrostatic, bioseparation,
proteins, aqueous two-phase
Introduction Advances in genetic engineering, recombinant-
DNA technology, cell fusion techniques in biotechnology have
made it possible to produce protein based therapeutic products and
vaccines on a large scale. The difficulties , however, lie in the
separation of these bioproducts in the desired conformation in an
ultra pure and stable form (Sadana & Beelaram, 1994; Lienqueo
& Asenjo, 2000) . Since the purification steps are so
cost-intensive, emphasis has been on improving purification
strategies, or analyzing and developing new and more promising
strategies for the separation of valuable proteins based on their
physico-chemical properties and bulk environment.
Major interactions that govern various protein isolation and
purification schemes are electrostatic , hydrophobic, van der
Waals' and hydrogen bonding (Scopes, 1994). A clear understanding
of the physico-chemical interactions involved can help pave the way
for not only the development of newer processes but also for the
improvement of existing purification methods. The efficiency of the
separation methods can be improved by regulating these interactions
in
* Author for correspondence: Tel: + 91 -11-6591004; Fax: +
91-11-6868521 E-mail: [email protected]
different ways. The current review is focused on the role of
electrostatic interactions in partitioning of proteins in aqueous
two phase system (A TPS ) including the phase separation behaviour
and the partitioning of proteins in the polyelectrolytes based
ATPS.
Electrostatic Interactions Electrostatic interactions are
important in biological
systems as most biomolecules are charged under physiological
conditions. Proteins are charged molecules that catTy ionizable
amino ac ids imparting surface charge on them. The charge on the
protein molecules can be varied by changing simple solution
parameters li ke p H and ionic strength (Creighton , 1993).
Moreover, quantification of the charge on the protein can be
performed by techniques like isoelectric focusing , titrati on
(Wilson & Goulding, 1986) or by partition ing in ATPS
(Johansson, 1994).
The importance of electrostatic interactions lies in the fact
that they tend to be highly specific in nature. Only those proteins
that are oppositely charged to th at of the pmify ing agent
interact with them and by changing the pH or ionic strength of the
solution further, even proteins carrying similar kind of the charge
on thei r surface can be separated from each other. In comparison
to electrostatic interactions , other non-covalent interactions
lack the high
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88 fNDIAN J BIOTECHNOL, JANUARY 2002
specificity level s. Various bioseparation processes based on
electrostatic interactions are:
(i) Ion exchange chromatography (Karlsson et aI, 1989; Belter et
aZ, 1988);
(ii) Ultrafiltration using charged membranes (Huisman et aZ,
2000; Chaufer & Rabiller-Baudry, 2001; Pouliot et aI,
1999);
(iii) Electrophoresis with various modifications (Nath et ai,
1990a; 1990b; 1993; 1996; Corthals et ai, 1997; Denton & Tate,
1997; Chidambra Raj, 1994; Shimura, 1990; Andrews et ai, 2000; Lim
et ai, 1993; Yang et aI, 1998; Kontturi et aI, 1994; Regnier et aZ,
1999);
(iv) Precipitation by charged polymers (Nath, 1995 ; Nath et ai,
1995; Gupta et ai, 2000; Clark & Glatz, 1992; Sternberg &
Hershberger, 1974);
(v) Partitioning in ATPS (Alberts son, 1986).
Role of Water Structure in Phase Separation in ATPS
The ATPS's are obtained by mixIng two incompatible
polymers/polymer-salt solutions above a critical concentration.
Proteins tend to partition into these A TPS' s on the basis of
their differential affinity for the phases, which is dependent on
the properties of the phase-forming polymers. ATPS's offer several
advantages for purification of proteins such as mild conditions;
short processing times, ease of scale up, simple process
equipment/instrumentation requirement, and its application at the
initial stage of the protein purification (Albertsson, 1986; Baskir
et ai, 1989). The bioseparation system performance is usually
described in terms of phase volume ratio and partition coefficient
(defined as the ratio of concentration of the desired protein in
the two-phases).
The structure and/or state of water in an aqueous polymer system
are of fundamental importance for phase separation (Zaslavsky et
ai, 1983; 1989). The phase separation in an aqueous mixture of two
polymers results from effect of polymers on the water structure and
the phase separation is due to the incompatibility of these
polymer-modified water structures. This hypothesis is validated by
studies on a number of water structure perturbing factors on the
phase separation process e.g., inorganic salts, temperature and
urea (Zaslavsky, 1995).
The dfect of different inorganic salts on the phase behavior
could be attributed to their role in affecting
the structure of water in the system. The salts have been
classified as "structure breaking" and "structure making" to
describe the effects of different ions on the structure of water.
By these terms it is impli ed that the effect of a
structure-breaking ion on water is qualitatively similar to that of
an increase in temperature, while a structure-making ion produces
an opposite effect like that of a decrease in temperature. The
structure-affecting properties of the ions are displayed in such
properties of their aqueous solutions as the viscosity
(structure-breaking ions reduce it), the rate of exchange of water
molecules between the hydration shell and bulk water
(structure-breaking ions decrease its energy of activation), the
longitudinal relaxation rate of the water molecule measured by NMR
(structure-breaking ions increase it), etc . According to the
various measures of the effects of ions on the structure of water,
the water structure making category of ions include cations,
h L ·+ N + NH + C ?+ M ?+ d' suc as I , a, 4, a- , g-, etc., an
amons, such as F, SO/, cot, P043-, CH3COO-, etc. , and the
structure breaking ions are K+, Rb+, Cs+, cr, Br", 1", SCN-, N03-,
CI04-, etc (Zaslavsky, 1995).
At the molecular level , a reorganization of the water molecules
or H-bonds can be perceived either as structure making or breaking
(Conway, 1981; Edsall & Mckenzie, 1978; 1983; Kjellander &
Florin; 1981 ; Florin et ai, 1984). This was confirmed by measuring
the rate of exchange of water molecules between the hydration shell
of the ion and bulk water and the longitudinal relaxation rate of
the water molecules as measured by NMR (Krestov, 1984; Marcus,
1985). Therefore, in the presence of water structure making salts
like sulphate the concentration of the polymers to form two phases
decreased whereas water structure breaking anions like bromide or
chloride required higher concentration of the polymers to form two
phases in dextran/ficoll , or polyethyleneglycol (PEG) based ATPS's
(Zaslavsky, 1995). However, a clear understanding of the
differential behaviour of these two categories of ions is still
lacking.
The effect of temperature and urea (water structure breaking
compound) also match with the hypothesis that the phase separation
is due to the incompatibility of the polymer-modified water
structures. To further confirm the validity of this hypothesis ,
phase diagrams were also analyzed using the Flory-Huggins theory
and the aqueous solution thermodynamics approach (Zaslavsky et aI,
1989). The Flory-Huggins interaction parameter (X) was measured for
polymer-
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GUPTA et al: POLYELECTROLYTE BASED AQUEOUS TWO-PHASE SYSTEM
89
polymer and polymer-water systems in the presence of various
water structure perturbing factors to understand the importance of
the state or structure of the water in phase separation. The
results showed that addition of a salt or urea, or an increase in
the temperature producing significant alterations of the phase
diagram, does not affect the parameter XDex- PVP value showing that
there are no direct interactions between the phase polymers and the
ions or urea present in the system. On the other hand, the
interaction parameters for the polymer-solvent
interactions X Dex-H20 and XPVP-H
20 values varied
depending on the type of the additive present and on the
temperature of the system (Zaslavsky et at, 1989). Such evidences
while cover the non-ionic polymer based ATPS, the information in
respect for polyelectrolyte based A TPS is lacking. The work
conducted in our laboratory confirmed the existence of similar
mechanism in polyelectrolyte based A TPS' s irrelevant of the kind
of charge on the polyelectrolytes (Gupta, 2001) . In order to probe
the role of water structure in the phase separation in
polyelectrolytes [{ polyacrylic acid (PAA) or polyethylenimine(PEI)
}-polyethylene glycol(PEG)] based A TPS' s, the effect of different
inorganic salt additives that influence the water structures
differently on the phase separation was studied. As mentioned
earlier, nitrate is classified as a water structure breaking ion
and sulphate and phosphate as water structure making ions. It can
be seen from Fig.1(a & b) that the addition of the nitrate
resulted in the elevation of the binodial line or the higher
polymer requirement for the phase separation. On the other hand, in
the presence of sulphate or phosphate a depression in the binodial
line was observed implying the lower polymer concentration
requirement for the phase separation.
This trend was observed in the case of both positively as well
as negatively charged polyelectrolytes (PEl and PAA, respectively)
based ATPS contradicting the hypothesis that salts which act as the
efficient counter ions to the polymers promote the phase separation
(Dissing & Mattiasson, 1994). However, the similar behaviour
shown by both PAA as well as PEl in the presence of ions indicate
the role of water structure on the phase separation in the
polyelectrolyte based ATPS's and the data amply conclude that the
phase separation behaviour is strongly influenced by the structure
and/or state of water in a polymeric mixture and their
incompatibility.
Parameters Influencing Phase Separation in Polyelectrolyte Based
A TPS
Phase separation in polyelectrolyte based ATPS ' s depends on a
number of factors , major being the following ionic composition and
charge density on the polyelectrolyte of the system:
(a) Ionic Composition Phase separation in mixtures
containing
polyelectrolyte as one of the polymer depends highly on both the
ionic strength and the kind of ions present. This is clearly shown
by the systems containing Na-dextran sulphate-PEG and PAA-PEG. A
mixture of Na-dextran sulphate-PEG that gave a homogenous solution
below polymer concentrations of 20 % resulted in the phase
separation by the addition of 0.15 M NaCl. Moreover, with increase
in the salt
4.0,-------------------, 3' '3 3.5 ~ 3.0 -C o 1.S
:;::;
~ 1.0 C Q) () 1.5 C o o 1.0 (9 W 0.5 0..
•
0.0 +----,-----,----_,__--_,__---.-------1 0.0 0.5 1.0 1.5 1.0
1.5 3.0
PAA Concentration (% w/w)
Fig. I (a). Effect of anion s on the phase behavior in PAA- PEG
based aqueous two-phase system (salt concentration - 0.5 M). (e)
Sodium nitrate; C.) sodium sulphate; ( .... ) sodium phosphate.
8
:i ?;
;:,R 0 6 '-" C 0
:;::; co L... 4 ...... c Q) () C 0 0 1 (9 W 0..
0
0 1 134
PEl Concentration (%w/w)
Fig. I (b). Effect of anion on the binodial in PEI- PEG based
ATPS. ( .... ) Nitrate; ce) Sulphate; (.) Phosphate. The
concentration of the salt additive was I M in all systems.
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90 INDIAN J BIOTECHNOL, JANUARY 2002
concentration, a lower concentration of the polymers was
required for the phase separation e.g., in a mixture of 5%
sodium-dextran sulphate in 0.15 M NaCI, at least 8% PEG was
required for the phase separation, while in the presence of 1M NaCI
only 1.5 % PEG 6000 was necessary for the phase separation
(Albertsson, 1986). Similarly, in PEG-PAA based aqueous two-phase
system, the addition of 0.15 M NaCI resulted in the lowering of the
PAA concentration from 8% to 5%. Further addition of 1 M NaCl
resulted in a decrease in the PAA concentration to as low as 1.5%
(Perrau et ai, 1989). Similar results were also obtained with the
polyvinylpyrrolidone (PYP)-PAA as well as gum acacia-PEG based A
TPS in the presence of NaCl (Perrau et ai, 1989; Rastogi &
Chand, 1989). In the case of PEI-hydroxyethylcellulose (REC) based
aqueous two-phase system, with the addition of salt (0.3 mmol/g
sodium sulphate) the PEl concentration required to form two phases
decreased to 0.75% from 3% (Dissing & Mattiasson, 1993). The
above experimental evidences clearly illustrate the role of salts
in lowering the concentration of polymers required to form two
phases. However, these studies on the polyelectrolyte based ATPS
lack the mechanistic details of the phase behaviour.
The concentration of PEG, which is necessary for the phase
separation with a certain amount of dextran sulphate, depends also
on the kind of salt added (Dissing & Mattiasson, 1993). The
tendency to favour phase separation in a dextran sulphate - PEG
mixture increases with the following series of cations (as
chlorides): Li+ < NH4+ < Na+ < Cs+ < K+. The phase
separation also depends on the kind of anions ; the tendency to
favour phase separation decreases with the following series of
anions(as sodium salts): N03- > cr > Y2 HPOt > Ih sot
(Albertsson, 1986). In the PEI-HEC based ATPS, anions (in the
decreasing order, PO/' > S042- > Cl) are efficient in
promoting phase separation, the effect of which is attributed to
the polyvalent anions being better counter ions to the
positively-charged PEl chai ns to crosslink them and excl uding the
PEG into the other phase (Dissing & Mattiasson, 1993). However,
quantitative data to prove this hypothesis are lacking.
(b) Charge Density of the Polyelectrolytes The charge density on
the polyelectrolyte also plays
a major role in the phase separation in polyelectrolyte-based
PEG and can be controlled by varying the pH of the system. In
PEI-HEC based
ATPS, as the pH was increased from 4 to 9, the charge on the PEl
was decreased . This decreased charge resulted in single-phase
formation as the pH was increased to 9.0 (Dissing & Mattiasson
, 1993). In the case of PAA-PEG based ATPS , with increase in the
degree of neutralization of the PAA, the compatibility of the
polymers decreases i.e., with increase in the ionic form of the
polyelectrolyte, i~compatibility of the polymers to form two phases
increases (Perrau et ai, 1989; Minh et aI, 1982).
Although, these experimental evidences clearly indicate the
importance of the ionic composition and the charge density on the
polyelectrolyte as major parameters controlling phase separation in
the polyelectrolyte based ATPS, limited studies on the phase
separation behaviour of the polyelectrolyte based A TPS has
hampered the deve lopment of these systems for studying the
partitioning of proteins.
Partitioning of Proteins in Polyelectrolyte Based ATPS
The partitioning of the proteins 111 the polyelectrolyte based
ATPS as a result of electrostatic interactions is controlled by
several factors like the properties of the polymers, pH, salt and
the charge on the protein molecules. Their gross effect can be
quantified in terms of the electrostatic potential difference
between two phases.
Nature of the Phase Forming Polymers The charge on the polymer
is the major factor that
influences the partitioning of the proteins. Bovine Serum
Albumin(BSA), when negatively charged partitioned into the
positively charged bottom PEl rich phase in the
PEI-hydroxyethylcellulose (PEI-HEC) based ATPS (Dissing &
Mattiasson, 1994). Similarly, in PEG-gum acacia (a
positively-charged plant based polysaccharide) based system, BSA
(when negatively charged) partitioned in favour of positi
vely-charged bottom gum acacia-rich phase (Rastogi & Chand,
1989). On the other hand, in the case of PEG-cashew-nut tree gum
(an acidic heteropolysaccharide) , BSA (when negatively charged) is
repelled by the bottom negatively-charged cashew-nut tree gum rich
phase to the top PEG-rich phase (Sarubbo et ai, 2000). In the case
of the A TPS composed of two net anionic acrylic co-polymers (pI 4
.8 and pI 4.1) trypsin from the crude bovi ne pancreatic extracts
partitioned almost exclusively into the lower pI rich phase (K =
0.05) at pH 6.6 presumably because of an electrostatic
interaction
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GUPTA el (Ii: POL YELECTROL YTE BASED AQUEOUS TWO-PHASE SYSTEM
91
between the positively charged trypsin and the more negatively
charged of the two polymers. In the case of a polyampholyte (pi 4
.1) and polyvinyl alcohol biphasic system, at pH 8.2, a 3.4 fold
enrichment of the trypsin was recorded in the lower phase. In an
oppositely charged polyampholyte-polyvinylalcohol (PV A) system
comprising of a polymer of pI 6.5 and PY A at pH 6.2, an almost
two-fold enrichment was obtained in the upper phase (Hughes &
Lowe, 1989). Therefore, as expected a protein partitioned into the
polyelectrolyte-rich phase only when the protein molecules were
oppositely charged to that of the polyelectrolyte, the higher the
charge on the polymer the higher was the degree of electrostatic
interaction between the protein and the polymer.
The role of the hydrophobic component/polymer in polyelectrolyte
based ATPS is not significant in comparison to the non-ionic
polymers based ATPS. In the case of PEl-PEG based ATPS, a variation
in the concentration of PEG did not influence the partitioning of
the BSA as shown in Fig. 2 (Gupta, 2001). Earlier workers have
shown that PEG controlled the partitioning of proteins in
PEG-dextran based ATPS by changing the hydrophobicity of the
system. However, in comparison to those systems, the concentration
of PEG employed in our PEl-PEG based A TPS are much lower and
therefore it is unlikely that hydrophobic interactions/components
become significant.
pH of the System The charge on the polyelectrolyte and on
the
protein's surface varies with the pH of the solution .
6T---------------------------------~
4
2
300 400 50
-2
-4
~~------~~~------~~~~------~ Salt Concentration (mM)
Fig. 2. Effect of PEG concentration on the partitioning of the
BSA in PEl (3%)-PEG based ATPS at pH 7.0. PEG concentration: (.)
4%; ( .... ) 5%; (e) 6%.
Therefore, the pH of the system is an important parameter for
the partitioning of proteins in the polyelectrolyte-based ATPS. In
PEI-HEC based ATPS , BSA (pI 4.9) favoured the lower positively
charged PEl-rich phase above pH 4.9 and moved into the top PEG-rich
phase below pH 4.9 when it is repelled by the similarly charged
PEl-rich bottom phase (Dissing & Mattiasson, 1994). In PEl-PEG
based ATPS, a simulated mixture of proteins of varying isoelectric
points i.e. BSA (pI 4.8), myoglobin (pI 7) and a-chymotrypsin (pi
8.75) could be purified selectively by changing the pH as shown in
Fig. 3. These results showed that BSA partitioned into the lower
PEl-rich phase above pH 4.8 when it i s negatively charged and
partitioned into the top PEG-rich phase below pH 4.8 when
positively charged. Similar partitioning trends were also observed
for myoglobin and a-chymotrypsin i.e., below their isoelectric
point they partitioned into the top PEG-rich phase and above the
isoelectric point into the bottom PEl rich phase. As can be seen
between pH 5-6, myoglobin and a- chymotrypsin being positively
charged are repelled by the positively charged PEI-rich phase into
the top PEG-rich phase. BSA, however, remains in the bottom
PEl-rich phase under these conditions (Gupta, 2001) .
In biphasic systems comprising a net negativel y charged
co-polymer (pI 4.4) and PYA, albumin partitioning was regulated by
the pH. At pH 5-6, both serum albumin and total protein were
directed almost quantitatively towards the lower ampholytic
polymer-rich phase. At more alkaline pH values, both albumin and
total protein partitioned toward the upper PYA
6 ~----------------------------.
4 .... -------1.----- .....
2
~ ~ 0 .s
6 7 -2
-4
-6----------------------------~
pH
Fig. 3. Partitioning of a mixture of proteins of different
isoelectric points in PEl (4%)-PEG (4%) based ATPS. (.) Bovine
serum albumin; ( .... ) Myoglobin ; (e) a-chymotrypsin.
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92 INDIAN J BIOTECHNOL, JANUARY 2002
phase. At pH 7.0, however, an approximate 7-fold increase in the
partition coefficient for albumin was accompanied by a 3-fold
increase iQ total protein. This resulted in a 1.35-fold
purification of human serum albumin with almost quantitative
recovery in the upper phase (Hughes & Lowe, 1989). In the
PEG-cashew gum biphasic system, partitioning of the protein
increased as the pH was increased from 6 to 8. This resulted in the
electrostatic repulsion of the negatively charged BSA by the
negatively charged bottom cashew gum rich-phase into the top
PEG-rich phase (Sarubbo, 2000).
Role of Salt A number of studies have shown that the
partition
of proteins in polyelectrolyte-based ATPS's depends on type and
concentration of the salt. For the partitioning of BSA in the
PEI-HEC based ATPS at pH 5.5 (BSA is negatively charged at pH 5.5),
supplementation of NaCI did not significantly influence the
partitioning of the BSA. Phosphate salts on the other hand strongly
increased the partition in favour of the HEC-rich top phase.
Phosphate ions were more efficient in screening the charge on the
PEl molecules and, therefore, decreased the electrostatic
attraction between the protein and the PEl resulting in an increase
in the partitioning in favour of the top PEG-rich phase (Oissing
& Mattiasson, 1994).
A suitable selection of the type and concentration of the salt,
can lead to an increase in the specificity of the purification of a
protein from a mixture of proteins. In PEI-HEC based A TPS , in a
mixture of BSA, lactate dehydrogenase and myoglobin, BSA and
lactate dehydrogenase prefer the top HEC-rich phase at 0.4 M of
sodium phosphate whereas myoglobin remains in the bottom PEl-rich
phase at pH 6.5 (Oissi ng & Mattiasson, 1994). In a system
composed of two net anionic copolymers, in the presence of 1M NaCI
the specific activity almost doubled in the lower phase since the
partition coefficient of the total protein was increased to 2.4,
while for trypsin it remai ned less then 1. In an anionic
copolymer-PYA system at pH 5.8, in the presence of 1M NaCI, the
trypsi n partition coefficient was increased almost 10-fo ld while
the partition coefficient for total protein decreased 4-fold. This
resulted in the 3.4 fold purification of the enzyme in the upper
phase. Similarly, in the case of a cationic copolymer PYA based
ATPS at pH 6.0, inclusion of the salt in the system resulted in a
3-fold increase in the specific activity (Hughes & Lowe, 1989).
Therefore, the
inclusion of the salt in the sys tem can be used for the
increased and specific partitioning of proteins.
Electrostatic Potential Difference Proteins, being charged
molecules, experience
electrostatic ion-ion and ion-dipole solute-solvent interactions
in the ATPS, in addition to all intermolecular interactions
experienced by non-ionic species. Electrochemical partitioning
occurs when a charged solute encounters an interfacial potential
difference generated by the uneven distribution of the salts or
polyelectrolytes in the phase system (Luther & Glatz, 1994).
This unequal partitioning of ions along with the requirement of
electroneutrality in the phases gives ri se to an electrostatic
potential difference (an interfacial potentia l) between the phases
(Brooks et ai, 1984). The partitioning effect increases with the
strength of the distribution potential, which depends on the choice
of the added salt or other ionic species like polyelectrolytes
(Haynes et ai, 1991). Albertsson (1986) developed a general
relationship between the partition coefficient of a charged
biomolecule and the electrostatic potential difference as:
where Kp is the partition coefficient of the biomolecule, zP is
the charge on the protein and ~ is the parti tion coefficient of
the biomolecule when 6. = o or zP = O.
The role of electrostatic potential difference on the
partitioning of proteins has been studied by a number of workers in
non-ionic polymer based ATPS but such studies are completely
lacking in the polyelectrolyte based ATPS. Technical procedures
used in experimental measurements of an interfacial electrostatic
potential difference in ATPS 's are generally based on two basic
approaches : (i) analysis of the partitioning of a solute of known
and variable charge (Johansson, 1985); and (ii ) direct
measurements with reversi ble, non-polarizable electrodes
(Bamberger et aI, 1984). In our work on polyelectrolytes based
ATPS, the electrostatic potential difference measurements were
carried out based on the method used by Bamberger et al (1984) to
elucidate its role in the partitioning of proteins. The
electrostatic potenti al difference and partitioning coefficients
for the partitioning of BSA in PEr-PEG based ATPS were measured in
the presence of salts (Fig. 4).
-
GUPTA el al: POL YELECTROL YTE BASED AQUEOUS TWO-PHASE SYSTEM
93
Salt Concentration (mM)
_SL-----------------------------~-4
Fig. 4. Partitioning of BSA and electrochemical potential
difference as a fun ction of salt concentration in PEl-PEG based
ATPS at pH 7.0. Dashed lines represent the partition coefficient
and solid lines represent the electrostatic potential difference
between two phases. Salt: (. ) Sodium chloride; ( ... ) Sodium
sulphate; (e) Sodium phosphate.
BSA, which is negatively charged at pH 7.0, partitioned
completely into the bottom positively charged PEl-rich phase in the
presence of NaCI even at concentrations as high as 500 mM. However,
in the presence of divalent and polyvalent anions, BSA partitir;ned
into the bottom phase at lower salt concentration such as 100 mM.
With further increase in the salt concentration of
divalent/polyvalent ions, BSA also started moving into the top
PEG-rich phase. PEl, being the positively-charged polymer tends to
interact with negatively-charged ionic species. Therefore, anions
interact with the positively-charged PEl and bind/screen the charge
on the polymer molecules . The ability of the anions to
10n-bind/screen the charge on the PEl molecules decreased in the
order, phosphate > sulphate > chloride. Therefore, PEl stayed
maximally positively charged in the presence of NaCI and hence
attracted negatively charged BSA more efficiently, resulting in
100% partitioning of the BSA into the bottom phase. This was confi
rmed by measuring the electrostatic potential difference between
the two phases in the presence of these salt ions. In the presence
of NaCI , lesser screening of the PEl molecules resu Iled in a net
positive electrostatic difference between the two phases even till
500 mM salt concentration , which was higher th an that observed
with sulphate or phosphate salts. Su lphate and phosphate, on the
other hand, reduced/screened the positive charge on the PEl more
effic iently as can be seen from the lower values of the
electrostatic potential difference and reduced the electrostatic
attraction with the negatively-charged
BSA molecules. This resulted in a decrease in the partitioning
of the BSA into the bottom PEl ri ch phase. This was confirmed by
the fact as the concentration of the sulphate or phosphate
increased ; the concentration of the BSA was also increased in the
top PEG rich phase (Fig. 4). These results clearly imply that
partitioning of proteins in polyelectrolyte based ATPS shows an
inverse relationship with the electrostatic potential difference
(Gupta, 2001).
Conclusion Polyelectrolyte based ATPS's serve as an attracti
ve
method of protein purification . The formation of two-phases at
low polymer concentrations (as low as 0.5 %) in comparison to the
traditionally used PEG-dextran/salt based systems (10-12%) are
especially useful for the large-scale applications where cost of
polymers is a major concern. The strong dependence of
polyelectrolyte based ATPS on the electrostatic interactions
results in the separation of proteins on the basis of their
differential surface charge and results in their high partitioning
coefficients .
Acknowledgement One of the authors thank the authorities of Indi
an
Institute of Technology Delhi for providing the facilities to
peruse the studies in the area of the review and financial support
for carrying out this research work. The authors also thank Dr B Y
Zaslavsky (Analiza Inc., USA) for helpful discussion (via e-mail)
on the role of water structure in phase separation.
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Macromolecules (3 rd
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2000. Affinity gel electrophoresis as a predicti ve techn ique
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