Journal of Bioscience and Bioengineering VOL. xx No. xx, 1e6, 2014 www.elsevier.com/locate/jbiosc
Characterization of bovine serum albumin partitioning behaviors in polymer-salt aqueous two-phase systems Yin Hui Chow,1 Yee Jiun Yap,2 Chin Ping Tan,3 Mohd Shamsul Anuar,1 Bimo Ario Tejo,4 Pau Loke Show,5 Arbakariya Bin Ariff,6 Eng-Poh Ng,7 and Tau Chuan Ling8, * Department of Process and Food Engineering, Faculty of Engineering, Universiti Putra Malaysia, UPM Serdang 43400, Selangor, Malaysia,1 Department of Applied Mathematics, Faculty of Engineering, University of Nottingham Malaysia Campus, Jalan Broga, Semenyih 43500, Selangor Darul Ehsan, Malaysia,2 Department of Food Technology, Faculty of Food Science and Technology, Universiti Putra Malaysia, UPM Serdang 43400, Selangor, Malaysia,3 Center for Infectious Diseases Research, Surya University, JI. Scientia Boulevard Block U/ 7, Gading Serpong, Tangerang 15810, Banten, Indonesia,4 Manufacturing and Industrial Processes Division, Faculty of Engineering, Centre for Food and Bioproduct Processing, University of Nottingham Malaysia Campus, Jalan Broga, Semenyih 43500, Selangor Darul Ehsan, Malaysia,5 Department of Bioprocess Technology, Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia, UPM Serdang 43400, Selangor, Malaysia,6 School of Chemical Sciences, Universiti Sains Malaysia, Minden 11800, Malaysia,7 and Institute of Biological Sciences, Faculty of Science, University of Malaya, Kuala Lumpur 50603, Malaysia8 Received 26 September 2014; accepted 26 November 2014 Available online xxx
In this paper, a linear relationship is proposed relating the natural logarithm of partition coefficient, ln K for protein partitioning in poly (ethylene glycol) (PEG)-phosphate aqueous two-phase system (ATPS) to the square of tie-line length (TLL2). This relationship provides good fits (r2 > 0.98) to the partition of bovine serum albumin (BSA) in PEG (1450 g/mol, 2000 g/mol, 3350 g/mol, and 4000 g/mol)-phosphate ATPS with TLL of 25.0e50.0% (w/w) at pH 7.0. Results also showed that the plot of ln K against pH for BSA partitioning in the ATPS containing 33.0% (w/w) PEG1450 and 8.0% (w/w) phosphate with varied working pH between 6.0 and 9.0 exhibited a linear relationship which is in good agreement (r2 [ 0.94) with the proposed relationship, ln K [ a0 pH D b0 . These results suggested that both the relationships proposed could be applied to correlate and elucidate the partition behavior of biomolecules in the polymer-salt ATPS. The influence of other system parameters on the partition behavior of BSA was also investigated. An optimum BSA yield of 90.80% in the top phase and K of 2.40 was achieved in an ATPS constituted with 33.0% (w/w) PEG 1450 and 8.0% (w/w) phosphate in the presence of 8.5% (w/w) sodium chloride (NaCl) at pH 9.0 for 0.3% (w/w) BSA load. Ó 2014, The Society for Biotechnology, Japan. All rights reserved. [Key words: Aqueous two-phase system; Statistical mechanics; Bioseparation; Protein recovery; Purification; Bovine serum albumin]
Aqueous two-phase systems (ATPS), a liquideliquid extraction technique, have drawn the interest of the biotechnological industry for its potential in separation and purification of biomolecules (1). This system of two mutually immiscible phases is built by combining the aqueous solution of polymerepolymer or polymersalt above a specific concentration. With each of these components concentrated in one of the phases, a selective environment favoring the isolation and concentration of target biomolecules into one of the phases is generated. This technique has been extensively applied for the purification and recovery of biomolecules such as cells, proteins, nucleic acids and antibodies. The high water content (80e85%) and low interfacial tension of an ATPS create a relatively mild environment that ensures the biological activity of biomolecules is preserved during the purification process (2). As compared to conventional multi-step biomolecules purification techniques, the ATPS is easier to be scaled-up, economically viable, and provides a platform for faster biomolecules recovery since simultaneous recovery of target biomolecule and removal of contaminants can be integrated into one step. Hence, the ATPS offers a better alternative to serve as a primary purification step.
* Corresponding author. Tel.: þ60 3 79674104; fax: þ60 3 79674178. E-mail address: [email protected] (T.C. Ling).
The partition of biomolecules in the ATPS is a complex phenomenon. The selective partition of biomolecule is influenced by the physico-chemical properties of the biomolecules (e.g., surface hydrophobicity, isoelectric point, molecular size and mass) and the system parameters of the ATPS (e.g., polymer molecular weight, tieline length (TLL), volume ratio (VR), pH and the presence of neutral salt). To elucidate the thermodynamics of biomolecules partitioning in the ATPS, several models have been derived. Empirical results show that the partition coefficient, K, can be expressed as a function of hydrophobicity, electrochemical, biospecificity, size and conformation of the biomolecules (1,3). The complexity of this type of model, difficulty in measurement of the parameters involved and the amount of information required have restricted its application to predict biomolecules partitioning in engineering scale-up especially. Among all the parameters which will affect the partitioning of protein in the ATPS, the effect of phase-forming component composition, in particular, is most obvious and should be firstly taken into consideration. Models that relate the biomolecules partitioning to the concentration difference of one of the phase-forming components between the two phases have been established by Brönsted (4), Zaslavsky et al. (5) and Diamond and Hsu (6). In fact, the selective partition of biomolecules in the ATPS involves its interaction with both phase-forming components. Thus, these models are incomplete since the contribution of second
1389-1723/$ e see front matter Ó 2014, The Society for Biotechnology, Japan. All rights reserved. http://dx.doi.org/10.1016/j.jbiosc.2014.11.021
Please cite this article in press as: Chow, Y. H., et al., Characterization of bovine serum albumin partitioning behaviors in polymer-salt aqueous two-phase systems, J. Biosci. Bioeng., (2014), http://dx.doi.org/10.1016/j.jbiosc.2014.11.021
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phase-forming component is not considered simultaneously. Hartounian et al. (7) showed that the K can be described as a function of TLL, but this model is only limited to the polymerepolymer ATPS with low salt and protein concentrations. Furthermore, Forciniti et al. (8) suggested that the effect of polymer molecular weight and size of the biomolecules should not be isolated from polymer concentration. Thus, Lin et al. (9) developed a model which takes into account the simultaneous effect of these parameters, but this model is yet to be supported experimentally. The working pH of the ATPS is usually manipulated to further enhance the selective partition of charged biomolecules. In this case, an additional term which accounts for the contribution from electrostatic effect is necessary in the model. Based on experimental studies, Johansson (10) deduced that K can be expressed as a function of the net charge of the biomolecules. Models similar to this kind of approach can be difficult to realize in practice since the determination of electrostatic dependence of biomolecules surface on the cell surface free energy difference has been proven to be problematic (11). Eiteman (12) developed a mathematical model to predict the partition of charged biomolecules as a function of the pH. However, the assumption of the phases’ properties remain unaffected when the pH is varied is flawed since it has been shown that phase composition changes with the variation in pH (13). The present work is aimed at the development of relationships to correlate the effect of the TLL to K and the effect of pH to K. Building on the work in Chow et al. (14), we further extended the model to elucidate the K of protein partitioning in the ATPS by evaluating the effect of TLL and pH. We have attempted to apply the derived relationships to elucidate and correlate the protein partitioning behavior of a model protein, bovine serum albumin (BSA), in the polymer-salt ATPS. The dependence of the partition of BSA in the PEG-salt ATPS on other system parameters such as PEG molecular weight, VR, protein load, and the presence of neutral salt to achieve favorable conditions for the ATPS protein extraction was also investigated. MATERIALS AND METHODS Materials BSA (crystallized and lyophilized), PEG with average molecular weight of 1450 g/mol and 3350 g/mol (PEG 1450, PEG 3350) were obtained from Sigma Aldrich (St. Louis, MO, USA). PEG with average molecular weight of 2000 g/ mol and 4000 g/mol (PEG 2000, PEG 4000), dipotassium hydrogen orthophosphate (K2HPO4), potassium dihydrogen orthophosphate (KH2PO4), and sodium chloride (NaCl) were purchased from Merck (Hohenbrunn, Germany). Protein assay dye reagent concentrate was sourced from Bio-Rad Laboratories (California, US). All chemicals were of analytical grade. Protein partitioning studies The protein partitioning in the ATPS with a final mass of 10.0 g was performed in 15 mL graduated centrifuge tubes at room temperature by mixing appropriate amounts of 50% (w/w) PEG stock solution, 40% (w/w) phosphate buffer stock solution, 0.1% (w/w) BSA unless otherwise stated, and adequate distilled water according to the PEG-phosphate phase diagrams reported by Chow et al. (14). Stock solutions of 40% (w/w) K2HPO4 and KH2PO4 were mixed at appropriate ratio to prepare 40% (w/w) phosphate buffer stock solutions of varied pH. BSA was used without further purification to prepare a stock solution of 10% by mass. The mixture was mixed thoroughly using a vortex mixer (VelpScientificaZx3, Europe) and centrifuged at 4000 g for 5 min to achieve total phase separation. The volumes of both phases were measured and the VR was calculated as the ratio of volume in the top phase to the bottom phase. Samples from each phase were assayed separately for BSA concentration quantification. BSA concentration quantification The concentration of BSA in each of the phases of the ATPS was measured using the Bradford method (15). Five dilutions of a BSA standard were prepared for construction of the calibration curve. For each of the 10 mL samples pipetted into separate microtiter plate wells, 200 mL of diluted Coomassie brilliant blue G-250 dye reagent was added. These mixtures were mixed thoroughly using microplate mixer (Heidolph Instrument, Germany) and incubated at room temperature for 5 min. Samples from identical phase solution prepared in parallel without protein were used as blank to eliminate interference of phase-forming components. The absorbance was measured at 595 nm using ELISA microplate reader (Sunrise Tecan, Austria). The partitioning data was expressed as a mean of three independent readings. The K was calculated as the ratio of the concentration of BSA (mg/mL) partitioned at the top phase to the
bottom phase. The yield of the BSA in the top phase was calculated as YT ¼ 100/ [1 þ (1/(VR$K))]. Formulation of correlation between protein partitioning behavior and the effect of TLL The distribution of biomolecules between the top and interface can be expressed according to Luechau el at. (16) and Chow et al. (14) as pd2p ð1 cos qÞ $cðDPÞnþ1 2
ln G ¼
(1)
4kT
where DP is the difference in phase-forming component concentration between the top and bottom phases, pd2p is the surface area of the particle, q is the contact angle, c is a constant which relates DP to the interfacial tension, k is the Boltzmann constant and T is the absolute temperature (K). G ¼ N1/N2 is the partition ratio associating the number of biomolecules between one bulk phase, N1 and the interface, N2. For a polymer-salt ATPS with a small interface, n / 1, it can be deduced that 2
ln G1 ¼
pd2p ð1 cos qÞ $cðDP1 Þ2
(2)
4kT
and pd2p ð1 cos qÞ $cðDP2 Þ2 2
ln G2 ¼
(3)
4kT
where G1 ¼ NT/NI is the ratio of the amount of biomolecules in the top phase, NT, to the interface, NI; G2 ¼ NI/NB is the ratio of the amount of biomolecules at the interface, NI, to the bottom phase, NB. The DP1 and DP2 correspond to the difference in concentration between top and bottom phases for polymer and salt respectively. Since the particles in the fringe are moving as an entity (16), it can be safely assumed that there is a force holding the particles together. This implies that a surface tension exists on the surface of the fringe. For particles to enter or move out of this entity, the surface tension of this fringe must first be overcome. Thus, substituting a ¼ cpd2p ð1 cos qÞ2 =4kT and adding Eq. (2) to Eq. (3) yields ln G1 G2 ¼ aðDP1 Þ2 þ aðDP2 Þ2 :
(4)
Eqs. (1)e(4) are limited to ideal spherical biomolecules. To satisfactorily express the partition of real biomolecules, we propose N N N ln T I ¼ ln T ¼ aðDP1 Þ2 þ aðDP2 Þ2 þ b NI NB NB
(5)
where b is the error term and it is a constant. The term b accounts for the difference between the energy required to transfer ideal spherical particles and real biomolecules from one of the twoqaqueous phases to the interface or to the other ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi ffi DP 21 þ DP 22 and the K are given by K ¼ NT/NB ¼ CT/ aqueous phase. Since the TLL ¼ CB ¼ AeDE/kT according to the theory of Boltzmann distribution (11,16), Eq. (5) can be rewritten as ln K ¼ a ðTLLÞ2 þ b:
(6)
The term a is a constant which depends on the polymer molecular weight, biomolecules surface area, contact angle and temperature. From Eq. (6), it is expected that more energy is required for the transfer of biomolecules from one phase to another for the ATPS containing polymer with higher molecular weight. Since lnðKÞaðTLLÞ2
(7)
a plot of ln K against TLL2 is expected to give a linear relationship with slope a. Formulation of correlation between protein partitioning behavior and the effect of pH According to Albertsson (1), the partition of a charged protein in the ATPS, K can be expressed as: lnK ¼ lnK0 þ
ZF DF RT
(8)
where K0 is a non-electrostatic term which denotes the K of protein at the pH value corresponding to the protein isoelectric point, pI (Z ¼ 0 or DV ¼ 0). Z is the net protein charge, F is the Faraday constant (i.e., the charge for 1 mol of particles), and DV is the interfacial potential difference (i.e., the difference in the electrical potential between the top and bottom phases). To prove Eq. (8), we first consider the Boltzmann distribution DE
K ¼ Ae kT :
(9)
However, Eq. (9) does not include the electrostatic effect and thermodynamic activity of ions (i.e., the interfacial potential difference, DV). The difference in charge distribution between the two aqueous phases creates an electric field which drives the selective partition of charged proteins to the top phase. The total amount of energy required to move 1 mol of charged proteins to the top phase is given by ZFDV. Thus, to include the effect of DV, we introduce ZF DF RT
KP ¼ Beþ
(10)
Please cite this article in press as: Chow, Y. H., et al., Characterization of bovine serum albumin partitioning behaviors in polymer-salt aqueous two-phase systems, J. Biosci. Bioeng., (2014), http://dx.doi.org/10.1016/j.jbiosc.2014.11.021
VOL. xx, 2014
CHARACTERIZATION OF BSA PARTITIONING IN ATPS
where KP is the ratio of the number of charged proteins that partitioned to the top phase to the number of charged proteins in the bottom phase of the ATPS. Adding Eq. (10) to Eq. (9) we have DE
K ¼ Ae kT þ Be
ZF DF RT
:
(11)
Taking natural logarithm on both sides of Eq. (11) gives
DE
lnK ¼
kT
þ
ZF DF þ lnðABÞ RT
(12)
which agrees with Gerson (17). Substituting DE=kT ¼ pd2p cos q$cðDPÞnþ1 =kT (14,16) into Eq. (12) yields lnK ¼
pd2p cos q$cðDPÞnþ1 kT
þ
ZF DF ZF DF þ lnðABÞ ¼ lnK0 þ þ lnðABÞ RT RT
(13)
which has the same form as Eq. (8) where the first term on the right hand side (RHS) represents the contribution from concentration difference while the second term represents the contribution from electrostatic effect. The pH is defined as pH ¼ log½aHþ
(14)
where aþ H þ
¼ εC, ε is the activity coefficient, and C is the concentration of the hydrogen ions, H . From the Nernst equation (18), E ¼ Eo
2:303RT pH F
(15)
o
where E is the standard potential and E is the measured electrode potential. Taking DV ¼ gE where g is a constant, we have, from Eq. (13)
pd2p cos q$cðDPÞnþ1
ZF DF þ þ lnðABÞ RT o 2:303RT ZF g E F pH ¼ lnK0 þ þ lnðABÞ: RT
lnK ¼
kT
(16)
Eq. (16) can be simplified and the correlation between ln K and pH takes the form lnK ¼ a’ðpHÞ þ b’
a0
(17)
b0
where and are constant. We will thus expect a linear relationship between ln K and pH when the working pH of the ATPS is varied.
RESULTS AND DISCUSSION The effect of PEG molecular weight and TLL on the partition behavior of BSA Fig. 1 shows the effect of increasing TLL and PEG molecular weight on the partition behavior of BSA, ln K in the PEG-
3
phosphate ATPS with constant VR of one. The plot of ln K against the TLL2 exhibits good linear relationships for all the PEG molecular weights studied (see Fig. 1). The correlation results for the plot of ln K against TLL2 with Eq. (6): the slopes (a), intercepts (b), and correlation coefficients (r2) for the linear regression of the data points presented in Fig. 1 were shown in Table 1. All the r2 values reported in Table 1 exceeded 0.98. These results indicated that Eq. (6) provides good fits and description for the partition of BSA in the PEG-phosphate ATPS at pH 7.0 with TLL in the range of 25.0e50.0% (w/w). Results in Table 1 showed that the a and b of the linear regression for the partition of BSA in the PEG 1450-phosphate ATPS were higher as compared to that of the PEG 4000phosphate ATPS. This decreasing trend in a suggests that there exists a difference in protein-polymer hydrophobic interaction strength as a function of polymer chain length. This trend is in agreement with the fact that the partitioning behavior of protein with molecular weight above 50 kDa (i.e., molecular weight of BSA ¼ 66.7 kDa) is strongly affected by the molecular weight of the polymer used (19). It appeared that as the polymer chain length increases (i.e., PEG molecule of higher molecular weight), the increase in steric exclusion effect, tendency of PEG forming intramolecular bonds and the hike in interfacial tension between the phases will result in weaker protein-polymer hydrophobic interaction (20). Hence, the lowest ln K was observed from the PEG 4000 containing ATPS at all constant TLL2. It was observed from Fig. 1 that there exist gradual increment of ln K with TLL2 although the partition of BSA in the PEG-phosphate ATPS showed an overall bottom phase preference. This trend in ln K revealed that high TLL can help to enhance the interaction between PEG and BSA molecules as suggested by Eq. (6) (21). The increase in TLL corresponds to a higher amount of polymer and salt present in the top and bottom phases respectively. Thus, there is a higher amount of salt units available to dehydrate and increase the hydrophobicity of the proteins; and higher amount of PEG units to enhance the protein-polymer hydrophobic interaction (22,23). Furthermore, the net negative charge of BSA acquired at this working pH of the ATPS (i.e., pH 7.0) promotes additional protein affinity towards the polymer-rich top phase. Hence, the favorable protein-polymer hydrophobic interaction is expected to be considerably more expressive compared to the volume exclusion effect due to the increase in TLL. The result of positive values reported for the a in Table 1 affirmed this fact. The a would have been negative in value if the volume exclusion effect was the major contributing factor. Among the systems studied, better protein partitioning efficiency and YT (15.49%) was attained with PEG 1450phosphate ATPS at TLL of 49.5% (w/w) (i.e., an ATPS composed of 20.0% (w/w) PEG 1450 and 17.5% (w/w) phosphate at working pH of 7.0). The effect of VR on the partition behavior of BSA When the differences in the ATPS composition between top and bottom phases are high (i.e., high TLL), altering the VR will have an impact on the K of the targeted protein. The PEG 1450-phosphate ATPS with TLL of 49.5% (w/w) at pH 7.0 that exhibited maximum ln K value was selected to further investigate the effect of VR on the TABLE 1. Correlation results of ln K against TLL2 with Eq. (6) for the data presented in Fig. 1
a
ATPS
FIG. 1. The effect of tie-line length (TLL) and PEG molecular weight on the partition behavior of BSA. The initial concentration of BSA introduced was 0.1% (w/w). The ln K was calculated as the natural logarithm of the ratio of BSA concentration measured at the top phase (mg/mL) to the bottom phase (mg/mL) and plotted against the TLL2 according to Eq. (6).
PEG PEG PEG PEG
1450-phosphate 2000-phosphate 3350-phosphate 4000-phosphate
0.0021 0.0018 0.0016 0.0014
8.47 1.15 7.86 9.09
b
5
10 104 105 105
6.6258 6.8549 6.9334 6.9628
r2 0.1466 0.2046 0.1507 0.1704
0.995 0.988 0.993 0.987
The correlation constants, a and b of Eq. (6) for the partition of BSA in the PEG (molecular weight: 1450 g/mol, 2000 g/mol, 3350 g/mol and 4000 g/mol)-phosphate ATPS at pH 7.0.
Please cite this article in press as: Chow, Y. H., et al., Characterization of bovine serum albumin partitioning behaviors in polymer-salt aqueous two-phase systems, J. Biosci. Bioeng., (2014), http://dx.doi.org/10.1016/j.jbiosc.2014.11.021
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BSA partitioning. The effect of varying the VR from 0.34 to 5.13 along the TLL of 49.5% (w/w) on the K and YT of BSA was shown in Fig. 2. A K value of 0.19 but low YT of 6.17% was recorded at an extreme condition of low VR (i.e., 0.34). At this point, a third phase which was in equilibrium with the two aqueous phases was observed at the interface of the ATPS. This result and observation were in agreement with the fact that low VR leads to the loss of protein at the interface when the protein solubility limit in the top phase has been reached. The loss of protein at the interface could be minimized and the recovery of targeted protein to the top phase could be improved by increasing the VR. The K increased with VR above 3.50, attaining a maximum YT of 52.24% at VR ¼ 5.13, which corresponds to the PEG 1450-phosphate ATPS with TLL of 49.5% (w/w), containing 33.0% (w/w) PEG 1450 and 8.0% (w/w) phosphate at pH 7.0. This rise in K was attributed to the increase of free volume in the PEG-rich phase with VR to accommodate more targeted protein (24). The effect of protein load on the partition behavior of BSA To serve as a potential recovery step in bio-separation process, it is necessary for the ATPS to exhibit its capability in handling a substantially higher amount of protein load (25,26). The partition of BSA to the top phase of the 33.0% (w/w) PEG 1450e8.0% (w/w) phosphate ATPS at pH 7.0 was further optimized by increasing the amount of BSA load up to 0.5% (w/w) (see Fig. 3). In this study, the maximum capacity of a 10.0 g ATPS was found to be 0.3% (w/w) of BSA load with K ¼ 0.37 and YT ¼ 65.37%. Results showed that there were no significant improvement in K and YT with protein loading in excess of 0.3% (w/w). True partitioning behavior in the ATPS only occurs at a relatively low protein concentration (14). The relatively constant YT values recorded at BSA load greater than 0.3% (w/w) indicated that protein loss at the interface had occurred and the BSA solubility limit in the top phase had been exceeded (ca. 0.9 mg/ mL). The maximum protein concentration that can be solubilized in the top phase of an ATPS is determined by the protein-polymer hydrophobic interaction and salting-out effect (14). Since the concentrations of PEG and phosphate in the ATPS are constant, the amount of PEG and phosphate molecules present are therefore limited to promote further partition of BSA to the top phase once the protein saturation point has been reached. Hence,
FIG. 2. The effect of VR on the partition behavior of BSA. The partition behavior of BSA at increase of VR from 0.34 to 5.13 along the TLL of 49.5% (w/w) for the PEG 1450phosphate ATPS was studied.
FIG. 3. The effect of BSA load on the partition behavior of BSA. The partition of BSA in the 33.0% (w/w) PEG 1450e8.0% (w/w) phosphate ATPS at pH 7.0 with increasing protein load in the range of 0.05% (w/w) to 0.50% (w/w) was studied.
BSA loading of 0.3% (w/w) was the feasible concentration to further investigate the condition for maximal recovery of BSA in the ATPS. The effect of pH on the partition behavior of BSA Since the net charge of biomolecules will be altered at different pH values, the working pH of the ATPS is generally manipulated to steer the selective separation of biomolecules. The impact of pH on the partitioning of BSA in the 33.0% (w/w) PEG 1450e8.0% (w/w) phosphate ATPS with BSA load of 0.3% (w/w) was further studied. The pH of the ATPS was varied between 6.0 and 9.0 at an interval of 0.5 by carefully mixing 40% (w/w) K2HPO4 and KH2PO4 stock solutions at appropriate proportion. Based on the results in Fig. 4, it was shown that the ln K and YT increased dramatically with the increase of pH (pH > 6.0). The plot ln K against pH also exhibited a linear relationship between ln K and pH with a0 ¼ 0.633, b0 ¼ 5.475 and r2 ¼ 0.94. Hence, the ln K for the partition of BSA in the PEG-
FIG. 4. The effect of pH on the partition behavior of BSA. The working pH of the 33.0% (w/w) PEG 1450e8.0% (w/w) phosphate ATPS with BSA load of 0.3% (w/w) was varied between pH 6.0 and pH 9.0. The ln K was plotted against the pH according to Eq. (17).
Please cite this article in press as: Chow, Y. H., et al., Characterization of bovine serum albumin partitioning behaviors in polymer-salt aqueous two-phase systems, J. Biosci. Bioeng., (2014), http://dx.doi.org/10.1016/j.jbiosc.2014.11.021
VOL. xx, 2014 phosphate ATPS is linearly proportional to changes in pH as described by the correlation in Eq. (17). Within the requirement of electroneutrality at the interface, this significant increase in ln K with pH can be explained by the fact that the partition of slightly negatively charged BSA (pI ¼ 4.8) at the ATPS working pH above the pI of BSA tends to favor the electrostatic interaction with PEGrich top phase which has a higher positive charge density (27,28). This fact is further affirmed by the positive value of a0 reported here. Also, the increase in the concentration of phosphate ions in the bottom phase with pH repels the negatively charged BSA to the top phase. Therefore, the YT increased remarkably from 49.74% at pH 6.0 to a maximum of 84.32% at pH 9.0, attaining an optimum K of 1.08. Due to possible protein denaturation, operating the ATPS above pH 9.0 is not feasible (29). It is of interest to note that the Eq. (16) proposed here considerably resembles the relationship found by Gerson (17). The relationship found by Gerson (17) predicts a linear relationship between the logarithm of K, log K and either the difference in cell surface free energy between the phases, Dg, or the difference in electrical potential between phases, Dj, or a linear combination of both. Comparing both relationships, ln K for biomolecules partition between the two aqueous phases in this work is also a function of Dg, Dj and an activity coefficient. The effect of addition of neutral salt NaCl on the partition behavior of BSA Further investigation on the effect of addition of NaCl in the 33.0% (w/w) PEG 1450e8.0% (w/w) phosphate ATPS at pH 9.0 with BSA load of 0.3% (w/w) was carried out to examine ways of using NaCl in making the ATPS more selective. According to the results shown in Fig. 5, the K and YT decreased significantly to a minimum value of 0.01 and 2.23% respectively with an increase of NaCl concentration up to 2.0% (w/w). This phenomenon implies that the negatively charged BSA partition preferentially to the bottom phase. It appears that in the presence of 0.1%e2.0% (w/w) NaCl, the partition behavior of charged BSA in the ATPS depends solely on its surface net charge, with hydrophobicity effect to a lesser extent. However, above NaCl concentration of 2.0% (w/w), the BSA partition behavior is highly dependent on the hydrophobic interaction between the protein domain and PEG molecule (30). As a result, gradual increments in K and YT were
CHARACTERIZATION OF BSA PARTITIONING IN ATPS
5
observed when NaCl concentration increased from 2.0 to 8.5% (w/ w) (see Fig. 5). This trend was in agreement with the results reported for other proteins and soluble substances (27). Optimum recovery of BSA to the top phase was achieved with K of 2.40 and YT of 90.80% with addition of 8.5% (w/w) NaCl. These results and similar general linear trends in ln K against TLL and pH reported in literature for other proteins (31e36) suggest the possible applicability of the approach in Eq. (6) and Eq. (17) to elucidate and correlate the partition behavior of other biomolecules in the ATPS. The amount of experimental information required to obtain the relationship parameters is minimum and these straighteforward correlations are easy to apply in engineering scale-ups. Moreover the essential factors governing the protein partitioning behavior incorporated in Eq. (16) can serve as a good start for the understanding and the selection of suitable ATPS parameters to aid the design and optimization of the recovery of biomolecules. In particular, one can deduce from Eq. (16) that the recovery of targeted biomolecules in the top phase will improve with increase in phase-forming component compositions and pH. In this study, the effects of various system parameters such as PEG molecular weight, TLL, VR, protein load, pH and addition of neutral salt on the partitioning behavior of BSA in the PEGphosphate ATPS were investigated. An optimum BSA yield of 90.80% with K of 2.40 were achieved with 33.0% (w/w) PEG 1450e8.0% (w/w) phosphate ATPS at pH 9.0 and 8.5% (w/w) NaCl for a BSA loading of 0.3% (w/w). Relationships have been proposed to correlate the effect of TLL (Eq. (6)) and pH (Eq. (17)) with the partitioning behavior of protein in the ATPS. The experimental data for the partition of BSA in PEG (1450 g/mol, 2000 g/mol, 3350 g/ mol, and 4000 g/mol)-phosphate ATPS with TLL of 25.0e50.0% (w/ w) at pH 7.0 agreed with Eq. (6). The correlation constants also show correct dependence on the polymer molecular weights and TLL. For the partition of BSA in the ATPS containing 33.0% (w/w) PEG 1450 and 8.0% (w/w) phosphate with varied working pH between 6.0 and 9.0, the linear plot of ln K against pH exhibited a good fit to the Eq. (17) derived. These relationships, which incorporate the effects of polymer molecular weight, system temperature, protein size and surface net charge, describe the interaction between protein and the phase-forming components in the ATPS, thereby providing a better understanding of the underlying mechanics of the protein partitioning behavior in the ATPS. ACKNOWLEDGMENTS This work was supported by Fundamental Research Grant Scheme (FP005-2013B and FRGS/1/2013/SG05/UNIM/02/1), University Malaya Postgraduate Research Grant (PG037-2013A), and Ministry of Science, Technology and Innovation (MOSTI-02-02-12SF0256). References
FIG. 5. The effect of addition of neutral salt NaCl on the partition behavior of BSA. The partition behavior of BSA with the addition of NaCl up to 8.5% (w/w) in the 33.0% (w/w) PEG 1450e8.0% (w/w) phosphate ATPS at pH 9.0 with BSA load of 0.3% (w/w) was studied.
1. Albertsson, P. A.: Partition of cell particles and macromolecules, 3rd ed. Wiley, New York ( (1986). 2. Edahiro, J.-i., Yamada, M., Seike, S., Kakigi, Y., Miyanaga, K., Nakamura, M., Kanamori, T., and Seki, M.: Separation of cultured strawberry cells producing anthocyanins in aqueous two-phase system, J. Biosci. Bioeng., 100, 449e454 (2005). 3. Asenjo, J., Schmidt, A., Hachem, F., and Andrews, B.: Model for predicting the partition behaviour of proteins in aqueous two-phase systems, J. Chromatogr. A, 668, 47e54 (1994). 4. Brönsted, J. N.: Molekülgrösse und Phasenverteilung. I., Z, Phys. Chem. Ser. A (Leipzig, Bodenstein-Festband), 155, 257e266 (1931). 5. Zaslavsky, B. Y., Miheeva, L., Gasanova, G., and Mahmudov, A.: Effect of polymer composition on the relative hydrophobicity of the phases of the biphasic system aqueous dextran-poly (ethylene glycol), J. Chromatogr. A, 403, 123e130 (1987). 6. Diamond, A. D. and Hsu, J. T.: Correlation of protein partitioning in aqueous polymer two-phase systems, J. Chromatogr. A, 513, 137e143 (1990).
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7. Hartounian, H., Kaler, E. W., and Sandler, S. I.: Aqueous two-phase systems. 2. Protein partitioning, Ind. Eng. Chem. Res., 33, 2294e2300 (1994). 8. Forciniti, D., Hall, C., and Kula, M.: Protein partitioning at the isoelectric point: influence of polymer molecular weight and concentration and protein size, Biotechnol. Bioeng., 38, 986e994 (1991). 9. Lin, D.-Q., Wu, Y.-T., Mei, L.-H., Zhu, Z.-Q., and Yao, S.-J.: Modeling the protein partitioning in aqueous polymer two-phase systems: influence of polymer concentration and molecular weight, Chem. Eng. Sci., 58, 2963e2972 (2003). 10. Johansson, G.: Determination of ionic charge by liquiddliquid partition, J. Chromatogr. A, 322, 425e432 (1985). 11. Walter, H., Brooks, D. E., and Fisher, D.: Partitioning in aqueous twoephase system: theory, methods, uses, and applications to biotechnology. Academic Press Inc., Florida, USA (1985). 12. Eiteman, M. A.: Predicting partition coefficients of multi-charged solutes in aqueous two-phase systems, J. Chromatogr. A, 668, 21e30 (1994). 13. Zaslavsky, B. Y.: Aqueous two-phase partitioning: physical chemistry and bioanalytical applications. Marcel Dekker Inc., New York (1994). 14. Chow, Y. H., Yap, Y. J., Anuar, M. S., Tejo, B. A., Ariff, A., Show, P. L., Ng, E.-P., and Ling, T. C.: Interfacial partitioning behaviour of bovine serum albumin in polymer-salt aqueous two-phase system, J. Chromatogr. B, 934, 71e78 (2013). 15. Bradford, M. M.: A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding, Anal. Biochem., 72, 248e254 (1976). 16. Luechau, F., Ling, T. C., and Lyddiatt, A.: A descriptive model and methods for up-scaled process routes for interfacial partition of bioparticles in aqueous two-phase systems, Biochem. Eng. J., 50, 122e130 (2010). 17. Gerson, D. F.: Cell surface energy, contact angles and phase partition. I. Lymphocytic cell lines in biphasic aqueous mixtures, Biochim. Biophys. Acta, 602, 269e280 (1980). 18. Wahl, D.: A short history of electrochemistry-part 2, Galvanotechtnik, 96, 1820e1828 (2005). 19. Lebreton, B., Huddleston, J., and Lyddiatt, A.: Polymereprotein interactions in aqueous two phase systems: fluorescent studies of the partition behaviour of human serum albumin, J. Chromatogr. B, 711, 69e79 (1998). 20. Johansson, H.-O., Karlström, G., Tjerneld, F., and Haynes, C. A.: Driving forces for phase separation and partitioning in aqueous two-phase systems, J. Chromatogr. B, 711, 3e17 (1998). 21. Selvakumar, P., Ling, T. C., Walker, S., and Lyddiatt, A.: Partitioning of haemoglobin and bovine serum albumin from whole bovine blood using aqueous two-phase systems, Sep. Purif. Technol., 90, 182e188 (2012). 22. Chen, J. and Sun, Y.: Modeling of the salt effects on hydrophobic adsorption equilibrium of protein, J. Chromatogr. A, 992, 29e40 (2003). 23. da Silva, C. A. S., Coimbra, J. S. R., Rojas, E. E. G., and Teixeira, J. A. C.: Partitioning of glycomacropeptide in aqueous two-phase systems, Process Biochem., 44, 1213e1216 (2009).
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Please cite this article in press as: Chow, Y. H., et al., Characterization of bovine serum albumin partitioning behaviors in polymer-salt aqueous two-phase systems, J. Biosci. Bioeng., (2014), http://dx.doi.org/10.1016/j.jbiosc.2014.11.021
Characterization of bovine serum albumin partitioning behaviors in polymer-salt aqueous two-phase systems Yin Hui Chow,1 Yee Jiun Yap,2 Chin Ping Tan,3 Mohd Shamsul Anuar,1 Bimo Ario Tejo,4 Pau Loke Show,5 Arbakariya Bin Ariff,6 Eng-Poh Ng,7 and Tau Chuan Ling8, * Department of Process and Food Engineering, Faculty of Engineering, Universiti Putra Malaysia, UPM Serdang 43400, Selangor, Malaysia,1 Department of Applied Mathematics, Faculty of Engineering, University of Nottingham Malaysia Campus, Jalan Broga, Semenyih 43500, Selangor Darul Ehsan, Malaysia,2 Department of Food Technology, Faculty of Food Science and Technology, Universiti Putra Malaysia, UPM Serdang 43400, Selangor, Malaysia,3 Center for Infectious Diseases Research, Surya University, JI. Scientia Boulevard Block U/ 7, Gading Serpong, Tangerang 15810, Banten, Indonesia,4 Manufacturing and Industrial Processes Division, Faculty of Engineering, Centre for Food and Bioproduct Processing, University of Nottingham Malaysia Campus, Jalan Broga, Semenyih 43500, Selangor Darul Ehsan, Malaysia,5 Department of Bioprocess Technology, Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia, UPM Serdang 43400, Selangor, Malaysia,6 School of Chemical Sciences, Universiti Sains Malaysia, Minden 11800, Malaysia,7 and Institute of Biological Sciences, Faculty of Science, University of Malaya, Kuala Lumpur 50603, Malaysia8 Received 26 September 2014; accepted 26 November 2014 Available online xxx
In this paper, a linear relationship is proposed relating the natural logarithm of partition coefficient, ln K for protein partitioning in poly (ethylene glycol) (PEG)-phosphate aqueous two-phase system (ATPS) to the square of tie-line length (TLL2). This relationship provides good fits (r2 > 0.98) to the partition of bovine serum albumin (BSA) in PEG (1450 g/mol, 2000 g/mol, 3350 g/mol, and 4000 g/mol)-phosphate ATPS with TLL of 25.0e50.0% (w/w) at pH 7.0. Results also showed that the plot of ln K against pH for BSA partitioning in the ATPS containing 33.0% (w/w) PEG1450 and 8.0% (w/w) phosphate with varied working pH between 6.0 and 9.0 exhibited a linear relationship which is in good agreement (r2 [ 0.94) with the proposed relationship, ln K [ a0 pH D b0 . These results suggested that both the relationships proposed could be applied to correlate and elucidate the partition behavior of biomolecules in the polymer-salt ATPS. The influence of other system parameters on the partition behavior of BSA was also investigated. An optimum BSA yield of 90.80% in the top phase and K of 2.40 was achieved in an ATPS constituted with 33.0% (w/w) PEG 1450 and 8.0% (w/w) phosphate in the presence of 8.5% (w/w) sodium chloride (NaCl) at pH 9.0 for 0.3% (w/w) BSA load. Ó 2014, The Society for Biotechnology, Japan. All rights reserved. [Key words: Aqueous two-phase system; Statistical mechanics; Bioseparation; Protein recovery; Purification; Bovine serum albumin]
Aqueous two-phase systems (ATPS), a liquideliquid extraction technique, have drawn the interest of the biotechnological industry for its potential in separation and purification of biomolecules (1). This system of two mutually immiscible phases is built by combining the aqueous solution of polymerepolymer or polymersalt above a specific concentration. With each of these components concentrated in one of the phases, a selective environment favoring the isolation and concentration of target biomolecules into one of the phases is generated. This technique has been extensively applied for the purification and recovery of biomolecules such as cells, proteins, nucleic acids and antibodies. The high water content (80e85%) and low interfacial tension of an ATPS create a relatively mild environment that ensures the biological activity of biomolecules is preserved during the purification process (2). As compared to conventional multi-step biomolecules purification techniques, the ATPS is easier to be scaled-up, economically viable, and provides a platform for faster biomolecules recovery since simultaneous recovery of target biomolecule and removal of contaminants can be integrated into one step. Hence, the ATPS offers a better alternative to serve as a primary purification step.
* Corresponding author. Tel.: þ60 3 79674104; fax: þ60 3 79674178. E-mail address: [email protected] (T.C. Ling).
The partition of biomolecules in the ATPS is a complex phenomenon. The selective partition of biomolecule is influenced by the physico-chemical properties of the biomolecules (e.g., surface hydrophobicity, isoelectric point, molecular size and mass) and the system parameters of the ATPS (e.g., polymer molecular weight, tieline length (TLL), volume ratio (VR), pH and the presence of neutral salt). To elucidate the thermodynamics of biomolecules partitioning in the ATPS, several models have been derived. Empirical results show that the partition coefficient, K, can be expressed as a function of hydrophobicity, electrochemical, biospecificity, size and conformation of the biomolecules (1,3). The complexity of this type of model, difficulty in measurement of the parameters involved and the amount of information required have restricted its application to predict biomolecules partitioning in engineering scale-up especially. Among all the parameters which will affect the partitioning of protein in the ATPS, the effect of phase-forming component composition, in particular, is most obvious and should be firstly taken into consideration. Models that relate the biomolecules partitioning to the concentration difference of one of the phase-forming components between the two phases have been established by Brönsted (4), Zaslavsky et al. (5) and Diamond and Hsu (6). In fact, the selective partition of biomolecules in the ATPS involves its interaction with both phase-forming components. Thus, these models are incomplete since the contribution of second
1389-1723/$ e see front matter Ó 2014, The Society for Biotechnology, Japan. All rights reserved. http://dx.doi.org/10.1016/j.jbiosc.2014.11.021
Please cite this article in press as: Chow, Y. H., et al., Characterization of bovine serum albumin partitioning behaviors in polymer-salt aqueous two-phase systems, J. Biosci. Bioeng., (2014), http://dx.doi.org/10.1016/j.jbiosc.2014.11.021
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CHOW ET AL.
J. BIOSCI. BIOENG.,
phase-forming component is not considered simultaneously. Hartounian et al. (7) showed that the K can be described as a function of TLL, but this model is only limited to the polymerepolymer ATPS with low salt and protein concentrations. Furthermore, Forciniti et al. (8) suggested that the effect of polymer molecular weight and size of the biomolecules should not be isolated from polymer concentration. Thus, Lin et al. (9) developed a model which takes into account the simultaneous effect of these parameters, but this model is yet to be supported experimentally. The working pH of the ATPS is usually manipulated to further enhance the selective partition of charged biomolecules. In this case, an additional term which accounts for the contribution from electrostatic effect is necessary in the model. Based on experimental studies, Johansson (10) deduced that K can be expressed as a function of the net charge of the biomolecules. Models similar to this kind of approach can be difficult to realize in practice since the determination of electrostatic dependence of biomolecules surface on the cell surface free energy difference has been proven to be problematic (11). Eiteman (12) developed a mathematical model to predict the partition of charged biomolecules as a function of the pH. However, the assumption of the phases’ properties remain unaffected when the pH is varied is flawed since it has been shown that phase composition changes with the variation in pH (13). The present work is aimed at the development of relationships to correlate the effect of the TLL to K and the effect of pH to K. Building on the work in Chow et al. (14), we further extended the model to elucidate the K of protein partitioning in the ATPS by evaluating the effect of TLL and pH. We have attempted to apply the derived relationships to elucidate and correlate the protein partitioning behavior of a model protein, bovine serum albumin (BSA), in the polymer-salt ATPS. The dependence of the partition of BSA in the PEG-salt ATPS on other system parameters such as PEG molecular weight, VR, protein load, and the presence of neutral salt to achieve favorable conditions for the ATPS protein extraction was also investigated. MATERIALS AND METHODS Materials BSA (crystallized and lyophilized), PEG with average molecular weight of 1450 g/mol and 3350 g/mol (PEG 1450, PEG 3350) were obtained from Sigma Aldrich (St. Louis, MO, USA). PEG with average molecular weight of 2000 g/ mol and 4000 g/mol (PEG 2000, PEG 4000), dipotassium hydrogen orthophosphate (K2HPO4), potassium dihydrogen orthophosphate (KH2PO4), and sodium chloride (NaCl) were purchased from Merck (Hohenbrunn, Germany). Protein assay dye reagent concentrate was sourced from Bio-Rad Laboratories (California, US). All chemicals were of analytical grade. Protein partitioning studies The protein partitioning in the ATPS with a final mass of 10.0 g was performed in 15 mL graduated centrifuge tubes at room temperature by mixing appropriate amounts of 50% (w/w) PEG stock solution, 40% (w/w) phosphate buffer stock solution, 0.1% (w/w) BSA unless otherwise stated, and adequate distilled water according to the PEG-phosphate phase diagrams reported by Chow et al. (14). Stock solutions of 40% (w/w) K2HPO4 and KH2PO4 were mixed at appropriate ratio to prepare 40% (w/w) phosphate buffer stock solutions of varied pH. BSA was used without further purification to prepare a stock solution of 10% by mass. The mixture was mixed thoroughly using a vortex mixer (VelpScientificaZx3, Europe) and centrifuged at 4000 g for 5 min to achieve total phase separation. The volumes of both phases were measured and the VR was calculated as the ratio of volume in the top phase to the bottom phase. Samples from each phase were assayed separately for BSA concentration quantification. BSA concentration quantification The concentration of BSA in each of the phases of the ATPS was measured using the Bradford method (15). Five dilutions of a BSA standard were prepared for construction of the calibration curve. For each of the 10 mL samples pipetted into separate microtiter plate wells, 200 mL of diluted Coomassie brilliant blue G-250 dye reagent was added. These mixtures were mixed thoroughly using microplate mixer (Heidolph Instrument, Germany) and incubated at room temperature for 5 min. Samples from identical phase solution prepared in parallel without protein were used as blank to eliminate interference of phase-forming components. The absorbance was measured at 595 nm using ELISA microplate reader (Sunrise Tecan, Austria). The partitioning data was expressed as a mean of three independent readings. The K was calculated as the ratio of the concentration of BSA (mg/mL) partitioned at the top phase to the
bottom phase. The yield of the BSA in the top phase was calculated as YT ¼ 100/ [1 þ (1/(VR$K))]. Formulation of correlation between protein partitioning behavior and the effect of TLL The distribution of biomolecules between the top and interface can be expressed according to Luechau el at. (16) and Chow et al. (14) as pd2p ð1 cos qÞ $cðDPÞnþ1 2
ln G ¼
(1)
4kT
where DP is the difference in phase-forming component concentration between the top and bottom phases, pd2p is the surface area of the particle, q is the contact angle, c is a constant which relates DP to the interfacial tension, k is the Boltzmann constant and T is the absolute temperature (K). G ¼ N1/N2 is the partition ratio associating the number of biomolecules between one bulk phase, N1 and the interface, N2. For a polymer-salt ATPS with a small interface, n / 1, it can be deduced that 2
ln G1 ¼
pd2p ð1 cos qÞ $cðDP1 Þ2
(2)
4kT
and pd2p ð1 cos qÞ $cðDP2 Þ2 2
ln G2 ¼
(3)
4kT
where G1 ¼ NT/NI is the ratio of the amount of biomolecules in the top phase, NT, to the interface, NI; G2 ¼ NI/NB is the ratio of the amount of biomolecules at the interface, NI, to the bottom phase, NB. The DP1 and DP2 correspond to the difference in concentration between top and bottom phases for polymer and salt respectively. Since the particles in the fringe are moving as an entity (16), it can be safely assumed that there is a force holding the particles together. This implies that a surface tension exists on the surface of the fringe. For particles to enter or move out of this entity, the surface tension of this fringe must first be overcome. Thus, substituting a ¼ cpd2p ð1 cos qÞ2 =4kT and adding Eq. (2) to Eq. (3) yields ln G1 G2 ¼ aðDP1 Þ2 þ aðDP2 Þ2 :
(4)
Eqs. (1)e(4) are limited to ideal spherical biomolecules. To satisfactorily express the partition of real biomolecules, we propose N N N ln T I ¼ ln T ¼ aðDP1 Þ2 þ aðDP2 Þ2 þ b NI NB NB
(5)
where b is the error term and it is a constant. The term b accounts for the difference between the energy required to transfer ideal spherical particles and real biomolecules from one of the twoqaqueous phases to the interface or to the other ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi ffi DP 21 þ DP 22 and the K are given by K ¼ NT/NB ¼ CT/ aqueous phase. Since the TLL ¼ CB ¼ AeDE/kT according to the theory of Boltzmann distribution (11,16), Eq. (5) can be rewritten as ln K ¼ a ðTLLÞ2 þ b:
(6)
The term a is a constant which depends on the polymer molecular weight, biomolecules surface area, contact angle and temperature. From Eq. (6), it is expected that more energy is required for the transfer of biomolecules from one phase to another for the ATPS containing polymer with higher molecular weight. Since lnðKÞaðTLLÞ2
(7)
a plot of ln K against TLL2 is expected to give a linear relationship with slope a. Formulation of correlation between protein partitioning behavior and the effect of pH According to Albertsson (1), the partition of a charged protein in the ATPS, K can be expressed as: lnK ¼ lnK0 þ
ZF DF RT
(8)
where K0 is a non-electrostatic term which denotes the K of protein at the pH value corresponding to the protein isoelectric point, pI (Z ¼ 0 or DV ¼ 0). Z is the net protein charge, F is the Faraday constant (i.e., the charge for 1 mol of particles), and DV is the interfacial potential difference (i.e., the difference in the electrical potential between the top and bottom phases). To prove Eq. (8), we first consider the Boltzmann distribution DE
K ¼ Ae kT :
(9)
However, Eq. (9) does not include the electrostatic effect and thermodynamic activity of ions (i.e., the interfacial potential difference, DV). The difference in charge distribution between the two aqueous phases creates an electric field which drives the selective partition of charged proteins to the top phase. The total amount of energy required to move 1 mol of charged proteins to the top phase is given by ZFDV. Thus, to include the effect of DV, we introduce ZF DF RT
KP ¼ Beþ
(10)
Please cite this article in press as: Chow, Y. H., et al., Characterization of bovine serum albumin partitioning behaviors in polymer-salt aqueous two-phase systems, J. Biosci. Bioeng., (2014), http://dx.doi.org/10.1016/j.jbiosc.2014.11.021
VOL. xx, 2014
CHARACTERIZATION OF BSA PARTITIONING IN ATPS
where KP is the ratio of the number of charged proteins that partitioned to the top phase to the number of charged proteins in the bottom phase of the ATPS. Adding Eq. (10) to Eq. (9) we have DE
K ¼ Ae kT þ Be
ZF DF RT
:
(11)
Taking natural logarithm on both sides of Eq. (11) gives
DE
lnK ¼
kT
þ
ZF DF þ lnðABÞ RT
(12)
which agrees with Gerson (17). Substituting DE=kT ¼ pd2p cos q$cðDPÞnþ1 =kT (14,16) into Eq. (12) yields lnK ¼
pd2p cos q$cðDPÞnþ1 kT
þ
ZF DF ZF DF þ lnðABÞ ¼ lnK0 þ þ lnðABÞ RT RT
(13)
which has the same form as Eq. (8) where the first term on the right hand side (RHS) represents the contribution from concentration difference while the second term represents the contribution from electrostatic effect. The pH is defined as pH ¼ log½aHþ
(14)
where aþ H þ
¼ εC, ε is the activity coefficient, and C is the concentration of the hydrogen ions, H . From the Nernst equation (18), E ¼ Eo
2:303RT pH F
(15)
o
where E is the standard potential and E is the measured electrode potential. Taking DV ¼ gE where g is a constant, we have, from Eq. (13)
pd2p cos q$cðDPÞnþ1
ZF DF þ þ lnðABÞ RT o 2:303RT ZF g E F pH ¼ lnK0 þ þ lnðABÞ: RT
lnK ¼
kT
(16)
Eq. (16) can be simplified and the correlation between ln K and pH takes the form lnK ¼ a’ðpHÞ þ b’
a0
(17)
b0
where and are constant. We will thus expect a linear relationship between ln K and pH when the working pH of the ATPS is varied.
RESULTS AND DISCUSSION The effect of PEG molecular weight and TLL on the partition behavior of BSA Fig. 1 shows the effect of increasing TLL and PEG molecular weight on the partition behavior of BSA, ln K in the PEG-
3
phosphate ATPS with constant VR of one. The plot of ln K against the TLL2 exhibits good linear relationships for all the PEG molecular weights studied (see Fig. 1). The correlation results for the plot of ln K against TLL2 with Eq. (6): the slopes (a), intercepts (b), and correlation coefficients (r2) for the linear regression of the data points presented in Fig. 1 were shown in Table 1. All the r2 values reported in Table 1 exceeded 0.98. These results indicated that Eq. (6) provides good fits and description for the partition of BSA in the PEG-phosphate ATPS at pH 7.0 with TLL in the range of 25.0e50.0% (w/w). Results in Table 1 showed that the a and b of the linear regression for the partition of BSA in the PEG 1450-phosphate ATPS were higher as compared to that of the PEG 4000phosphate ATPS. This decreasing trend in a suggests that there exists a difference in protein-polymer hydrophobic interaction strength as a function of polymer chain length. This trend is in agreement with the fact that the partitioning behavior of protein with molecular weight above 50 kDa (i.e., molecular weight of BSA ¼ 66.7 kDa) is strongly affected by the molecular weight of the polymer used (19). It appeared that as the polymer chain length increases (i.e., PEG molecule of higher molecular weight), the increase in steric exclusion effect, tendency of PEG forming intramolecular bonds and the hike in interfacial tension between the phases will result in weaker protein-polymer hydrophobic interaction (20). Hence, the lowest ln K was observed from the PEG 4000 containing ATPS at all constant TLL2. It was observed from Fig. 1 that there exist gradual increment of ln K with TLL2 although the partition of BSA in the PEG-phosphate ATPS showed an overall bottom phase preference. This trend in ln K revealed that high TLL can help to enhance the interaction between PEG and BSA molecules as suggested by Eq. (6) (21). The increase in TLL corresponds to a higher amount of polymer and salt present in the top and bottom phases respectively. Thus, there is a higher amount of salt units available to dehydrate and increase the hydrophobicity of the proteins; and higher amount of PEG units to enhance the protein-polymer hydrophobic interaction (22,23). Furthermore, the net negative charge of BSA acquired at this working pH of the ATPS (i.e., pH 7.0) promotes additional protein affinity towards the polymer-rich top phase. Hence, the favorable protein-polymer hydrophobic interaction is expected to be considerably more expressive compared to the volume exclusion effect due to the increase in TLL. The result of positive values reported for the a in Table 1 affirmed this fact. The a would have been negative in value if the volume exclusion effect was the major contributing factor. Among the systems studied, better protein partitioning efficiency and YT (15.49%) was attained with PEG 1450phosphate ATPS at TLL of 49.5% (w/w) (i.e., an ATPS composed of 20.0% (w/w) PEG 1450 and 17.5% (w/w) phosphate at working pH of 7.0). The effect of VR on the partition behavior of BSA When the differences in the ATPS composition between top and bottom phases are high (i.e., high TLL), altering the VR will have an impact on the K of the targeted protein. The PEG 1450-phosphate ATPS with TLL of 49.5% (w/w) at pH 7.0 that exhibited maximum ln K value was selected to further investigate the effect of VR on the TABLE 1. Correlation results of ln K against TLL2 with Eq. (6) for the data presented in Fig. 1
a
ATPS
FIG. 1. The effect of tie-line length (TLL) and PEG molecular weight on the partition behavior of BSA. The initial concentration of BSA introduced was 0.1% (w/w). The ln K was calculated as the natural logarithm of the ratio of BSA concentration measured at the top phase (mg/mL) to the bottom phase (mg/mL) and plotted against the TLL2 according to Eq. (6).
PEG PEG PEG PEG
1450-phosphate 2000-phosphate 3350-phosphate 4000-phosphate
0.0021 0.0018 0.0016 0.0014
8.47 1.15 7.86 9.09
b
5
10 104 105 105
6.6258 6.8549 6.9334 6.9628
r2 0.1466 0.2046 0.1507 0.1704
0.995 0.988 0.993 0.987
The correlation constants, a and b of Eq. (6) for the partition of BSA in the PEG (molecular weight: 1450 g/mol, 2000 g/mol, 3350 g/mol and 4000 g/mol)-phosphate ATPS at pH 7.0.
Please cite this article in press as: Chow, Y. H., et al., Characterization of bovine serum albumin partitioning behaviors in polymer-salt aqueous two-phase systems, J. Biosci. Bioeng., (2014), http://dx.doi.org/10.1016/j.jbiosc.2014.11.021
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BSA partitioning. The effect of varying the VR from 0.34 to 5.13 along the TLL of 49.5% (w/w) on the K and YT of BSA was shown in Fig. 2. A K value of 0.19 but low YT of 6.17% was recorded at an extreme condition of low VR (i.e., 0.34). At this point, a third phase which was in equilibrium with the two aqueous phases was observed at the interface of the ATPS. This result and observation were in agreement with the fact that low VR leads to the loss of protein at the interface when the protein solubility limit in the top phase has been reached. The loss of protein at the interface could be minimized and the recovery of targeted protein to the top phase could be improved by increasing the VR. The K increased with VR above 3.50, attaining a maximum YT of 52.24% at VR ¼ 5.13, which corresponds to the PEG 1450-phosphate ATPS with TLL of 49.5% (w/w), containing 33.0% (w/w) PEG 1450 and 8.0% (w/w) phosphate at pH 7.0. This rise in K was attributed to the increase of free volume in the PEG-rich phase with VR to accommodate more targeted protein (24). The effect of protein load on the partition behavior of BSA To serve as a potential recovery step in bio-separation process, it is necessary for the ATPS to exhibit its capability in handling a substantially higher amount of protein load (25,26). The partition of BSA to the top phase of the 33.0% (w/w) PEG 1450e8.0% (w/w) phosphate ATPS at pH 7.0 was further optimized by increasing the amount of BSA load up to 0.5% (w/w) (see Fig. 3). In this study, the maximum capacity of a 10.0 g ATPS was found to be 0.3% (w/w) of BSA load with K ¼ 0.37 and YT ¼ 65.37%. Results showed that there were no significant improvement in K and YT with protein loading in excess of 0.3% (w/w). True partitioning behavior in the ATPS only occurs at a relatively low protein concentration (14). The relatively constant YT values recorded at BSA load greater than 0.3% (w/w) indicated that protein loss at the interface had occurred and the BSA solubility limit in the top phase had been exceeded (ca. 0.9 mg/ mL). The maximum protein concentration that can be solubilized in the top phase of an ATPS is determined by the protein-polymer hydrophobic interaction and salting-out effect (14). Since the concentrations of PEG and phosphate in the ATPS are constant, the amount of PEG and phosphate molecules present are therefore limited to promote further partition of BSA to the top phase once the protein saturation point has been reached. Hence,
FIG. 2. The effect of VR on the partition behavior of BSA. The partition behavior of BSA at increase of VR from 0.34 to 5.13 along the TLL of 49.5% (w/w) for the PEG 1450phosphate ATPS was studied.
FIG. 3. The effect of BSA load on the partition behavior of BSA. The partition of BSA in the 33.0% (w/w) PEG 1450e8.0% (w/w) phosphate ATPS at pH 7.0 with increasing protein load in the range of 0.05% (w/w) to 0.50% (w/w) was studied.
BSA loading of 0.3% (w/w) was the feasible concentration to further investigate the condition for maximal recovery of BSA in the ATPS. The effect of pH on the partition behavior of BSA Since the net charge of biomolecules will be altered at different pH values, the working pH of the ATPS is generally manipulated to steer the selective separation of biomolecules. The impact of pH on the partitioning of BSA in the 33.0% (w/w) PEG 1450e8.0% (w/w) phosphate ATPS with BSA load of 0.3% (w/w) was further studied. The pH of the ATPS was varied between 6.0 and 9.0 at an interval of 0.5 by carefully mixing 40% (w/w) K2HPO4 and KH2PO4 stock solutions at appropriate proportion. Based on the results in Fig. 4, it was shown that the ln K and YT increased dramatically with the increase of pH (pH > 6.0). The plot ln K against pH also exhibited a linear relationship between ln K and pH with a0 ¼ 0.633, b0 ¼ 5.475 and r2 ¼ 0.94. Hence, the ln K for the partition of BSA in the PEG-
FIG. 4. The effect of pH on the partition behavior of BSA. The working pH of the 33.0% (w/w) PEG 1450e8.0% (w/w) phosphate ATPS with BSA load of 0.3% (w/w) was varied between pH 6.0 and pH 9.0. The ln K was plotted against the pH according to Eq. (17).
Please cite this article in press as: Chow, Y. H., et al., Characterization of bovine serum albumin partitioning behaviors in polymer-salt aqueous two-phase systems, J. Biosci. Bioeng., (2014), http://dx.doi.org/10.1016/j.jbiosc.2014.11.021
VOL. xx, 2014 phosphate ATPS is linearly proportional to changes in pH as described by the correlation in Eq. (17). Within the requirement of electroneutrality at the interface, this significant increase in ln K with pH can be explained by the fact that the partition of slightly negatively charged BSA (pI ¼ 4.8) at the ATPS working pH above the pI of BSA tends to favor the electrostatic interaction with PEGrich top phase which has a higher positive charge density (27,28). This fact is further affirmed by the positive value of a0 reported here. Also, the increase in the concentration of phosphate ions in the bottom phase with pH repels the negatively charged BSA to the top phase. Therefore, the YT increased remarkably from 49.74% at pH 6.0 to a maximum of 84.32% at pH 9.0, attaining an optimum K of 1.08. Due to possible protein denaturation, operating the ATPS above pH 9.0 is not feasible (29). It is of interest to note that the Eq. (16) proposed here considerably resembles the relationship found by Gerson (17). The relationship found by Gerson (17) predicts a linear relationship between the logarithm of K, log K and either the difference in cell surface free energy between the phases, Dg, or the difference in electrical potential between phases, Dj, or a linear combination of both. Comparing both relationships, ln K for biomolecules partition between the two aqueous phases in this work is also a function of Dg, Dj and an activity coefficient. The effect of addition of neutral salt NaCl on the partition behavior of BSA Further investigation on the effect of addition of NaCl in the 33.0% (w/w) PEG 1450e8.0% (w/w) phosphate ATPS at pH 9.0 with BSA load of 0.3% (w/w) was carried out to examine ways of using NaCl in making the ATPS more selective. According to the results shown in Fig. 5, the K and YT decreased significantly to a minimum value of 0.01 and 2.23% respectively with an increase of NaCl concentration up to 2.0% (w/w). This phenomenon implies that the negatively charged BSA partition preferentially to the bottom phase. It appears that in the presence of 0.1%e2.0% (w/w) NaCl, the partition behavior of charged BSA in the ATPS depends solely on its surface net charge, with hydrophobicity effect to a lesser extent. However, above NaCl concentration of 2.0% (w/w), the BSA partition behavior is highly dependent on the hydrophobic interaction between the protein domain and PEG molecule (30). As a result, gradual increments in K and YT were
CHARACTERIZATION OF BSA PARTITIONING IN ATPS
5
observed when NaCl concentration increased from 2.0 to 8.5% (w/ w) (see Fig. 5). This trend was in agreement with the results reported for other proteins and soluble substances (27). Optimum recovery of BSA to the top phase was achieved with K of 2.40 and YT of 90.80% with addition of 8.5% (w/w) NaCl. These results and similar general linear trends in ln K against TLL and pH reported in literature for other proteins (31e36) suggest the possible applicability of the approach in Eq. (6) and Eq. (17) to elucidate and correlate the partition behavior of other biomolecules in the ATPS. The amount of experimental information required to obtain the relationship parameters is minimum and these straighteforward correlations are easy to apply in engineering scale-ups. Moreover the essential factors governing the protein partitioning behavior incorporated in Eq. (16) can serve as a good start for the understanding and the selection of suitable ATPS parameters to aid the design and optimization of the recovery of biomolecules. In particular, one can deduce from Eq. (16) that the recovery of targeted biomolecules in the top phase will improve with increase in phase-forming component compositions and pH. In this study, the effects of various system parameters such as PEG molecular weight, TLL, VR, protein load, pH and addition of neutral salt on the partitioning behavior of BSA in the PEGphosphate ATPS were investigated. An optimum BSA yield of 90.80% with K of 2.40 were achieved with 33.0% (w/w) PEG 1450e8.0% (w/w) phosphate ATPS at pH 9.0 and 8.5% (w/w) NaCl for a BSA loading of 0.3% (w/w). Relationships have been proposed to correlate the effect of TLL (Eq. (6)) and pH (Eq. (17)) with the partitioning behavior of protein in the ATPS. The experimental data for the partition of BSA in PEG (1450 g/mol, 2000 g/mol, 3350 g/ mol, and 4000 g/mol)-phosphate ATPS with TLL of 25.0e50.0% (w/ w) at pH 7.0 agreed with Eq. (6). The correlation constants also show correct dependence on the polymer molecular weights and TLL. For the partition of BSA in the ATPS containing 33.0% (w/w) PEG 1450 and 8.0% (w/w) phosphate with varied working pH between 6.0 and 9.0, the linear plot of ln K against pH exhibited a good fit to the Eq. (17) derived. These relationships, which incorporate the effects of polymer molecular weight, system temperature, protein size and surface net charge, describe the interaction between protein and the phase-forming components in the ATPS, thereby providing a better understanding of the underlying mechanics of the protein partitioning behavior in the ATPS. ACKNOWLEDGMENTS This work was supported by Fundamental Research Grant Scheme (FP005-2013B and FRGS/1/2013/SG05/UNIM/02/1), University Malaya Postgraduate Research Grant (PG037-2013A), and Ministry of Science, Technology and Innovation (MOSTI-02-02-12SF0256). References
FIG. 5. The effect of addition of neutral salt NaCl on the partition behavior of BSA. The partition behavior of BSA with the addition of NaCl up to 8.5% (w/w) in the 33.0% (w/w) PEG 1450e8.0% (w/w) phosphate ATPS at pH 9.0 with BSA load of 0.3% (w/w) was studied.
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