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Partitioning behavior of amino acids in aqueous two-phase systems containing polyethylene glycol and phosphate buffer
Fluid Phase Equilibria 219 (2004) 195–203
Partitioning behavior of amino acids in aqueous two-phase systems containing polyethylene glycol and phosphate buffer Q.K. Shang a,b , W. Li a , Q. Jia a , D.Q. Li a,∗ a
Key Laboratory of Rare Earths Chemistry and Physics, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun 130022, PR China b College of Chemistry, Northeast Normal University, Changchun 130024, PR China Received 6 May 2003; received in revised form 29 January 2004; accepted 30 January 2004
Abstract The partitioning behavior of four amino acids, cysteine, phenylalanine, methionine, and lysine in 15 aqueous two-phase systems (ATPSs) with different polyethylene glycol (PEG) molecular weights and phosphate buffers has been studied in the present paper. The phase diagrams of the systems are investigated together with the effect of the PEG molecular weight and pH of the phosphate solutions. The composition of these systems and some parameters such as density and refractive index are determined. The influences of salts in ATPSs, side chain structure of the amino acids, pH of ATPSs, and the PEG molecular weight on the distribution ratios of the amino acids have been studied. This work is useful for the purification of amino acids and the separation of some proteins whose main surface exposed amino acid residues are these four amino acids, respectively. © 2004 Elsevier B.V. All rights reserved. Keywords: Aqueous two-phase system; Partitioning; Amino acid
1. Introduction When two polymers or one polymer and one salt are mixed in water above certain concentration, two immiscible aqueous phases are formed. One phase is rich in polymer and the other phase is rich in the other polymer or salt, this type of systems are called aqueous two-phase systems (ATPSs). ATPSs were discovered by Beijerinck [1] and were first used for extraction and separation purposes by Albertsson [2]. Zaslavsky [3] has published a monograph covering most topics in the area of aqueous two-phase partitioning. In the second half of the 20th century, there has been increasing attention focused on ATPSs in laboratory scale, especially for the separation of proteins, cells and other biological materials which require mild aqueous environments [4–10]. The most common ATPSs used for the separation of biomolecules are polyethylene glycol (PEG)/dextran or PEG/salt systems because of low-cost, rapid mass transfer, and phase equilibrium. The processes scale-up easily can be used as continu∗ Corresponding author. Tel.: +86-431-5262036; fax: +86-431-5698041. E-mail address: [email protected] (D.Q. Li).
0378-3812/$ – see front matter © 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.fluid.2004.01.032
ous process and are more economical than other separation processes. Some aqueous two-phase systems have been designed for scale-up of downstream processes for biomaterial separation [11–14]. The surface of different proteins has common features in terms of hydrophobicity/hydrophilicity properties even though the primary sequence and overall fold of the proteins differ. However, the occurrence of individual types of residues on the protein surface differs and the surface properties of a protein are often crucial for recognition and interaction with other proteins. Partitioning of amino acids residues in ATPSs has the potential to be used as a rapid and simple method to study and characterize the protein surface, since the partitioning of a protein depends on its surface properties [15]. It has been proved that amino acid residues determine the protein surface properties by some authors [16–18]. It is interesting to move towards a deeper understanding of the influence of the surface exposed amino acid residues on partitioning in aqueous two-phase systems. During the past decades, there have been many papers about the partitioning behavior of amino acids or low molecular mass peptides in ATPSs [6,16,19–22]. Some aqueous two-phase systems composed of polyethylene glycol/salts
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or dextran have been used, e.g., dextran and PEG 8000 systems [6], PEG 8000 and magnesium sulfate systems [16], PEG 2000, PEG 4000, and recyclable volatile salt systems [19], PEG 3400 and potassium phosphate systems [20], PEG 8000 and potassium phosphate systems [21], PEG 6000, 35000, and dextran 500 systems [22]. Partitioning of more than 20 amino acids in these aqueous two-phase systems has been studied. For instance, Zaslavsky et al. [6] investigated the partitioning of glycine, lysine and aspartic acid and their oligopeptides in an aqueous dextran-polyethylene glycol 8000 two-phase system. Van Berlo et al. [19] studied the partitioning behavior of seven amino acids (l-serine, glycine, l-alanine, l-valine, l-methionine, l-isoleucine, and l-phenylalanine) in PEG 2000, PEG 4000 with aqueous recyclable volatile salt two-phase systems. However, PEG 5000, PEG 6000, and PEG 10000 with aqueous phosphates two-phase systems for the partitioning of amino acids have not been studied so far. The effects of polymer molecular weight, the composition and some properties of the systems on the distribution ratios of amino acids have also not been investigated in detail. In this work, the partitioning behavior of 4 amino acids with different side chains, cysteine, phenylalanine, methionine, and lysine in 15 PEG/phosphate aqueous two-phase systems has been studied. The phase diagrams of the systems in PEG 5000, 6000, and 10000 with different K2 HPO4 –KH2 PO4 compositions are determined together with the effects of polymer molecular weight and pH of phosphate buffers on the aqueous two-phase systems. The physical parameters of the systems such as density and refractive index are also determined. Factors affecting the partitioning of amino acids, such as salts, side chain structures of amino acids, pH of ATPSs and PEG molecular weight have been studied. The results may be used to predict the separation of some proteins whose main surface exposed amino acid residues are these four amino acids.
to the desired pH (about 6–8) values. The phosphate solutions, in pH 6.51, 6.71, 7.24, 7.71, and 7.93, were composed of the mixed solution of K2 HPO4 (3.5 mol/l) and KH2 PO4 (1.0 mol/l) in the ratio 1:3, 1:2, 1:1, 2:1, and 3:1, respectively. The binodal curves were determined by a titration method at a controlled temperature of 298 ± 1 K. PEG, phosphate solutions, and water with the total weight of about 60 g were added into vessels. The vessels were vibrated for 15 min and then placed at a controlled temperature of 298 ± 1 K for 20 h at least to ensure complete equilibration. The concentrations of phosphates in both phases were determined by a titration method. The concentrations of PEG in both phases were calculated from the refractive indexes considering the effect of the concentration of salts [23]. The relation between the refractive index, n, and the mass fractions of salt, ws , and PEG, wp can be expressed as follows n = 1.342780 + 0.000864wp + 0.001138ws (top phase)
(1)
n = 1.336427 + 0.000426wp + 0.001562ws (bottom phase)
(2)
The precision of the mass fraction of PEG achieved using Eqs. (1) and (2) was better than 0.005 and 0.002, respectively. Three milligram amino acids and 3 ml top phase and bottom phase, respectively, were placed in special glass tubes and centrifuged for 15 min at 66.6 r/s. The mixtures were allowed settle for 15 h at 298 ± 1 K. The concentrations of amino acids in both phases were then determined. The distribution ratios of amino acids have been tested five times in parallel and other experiments have been repeated three times. The standard deviations for the distribution ratios varied between 0.049 and 0.0011. 2.3. Analyses of samples
2. Experimental 2.1. Chemicals Cysteine, methionine, phenylalanine, and lysine were obtained from Shanghai Institute of Chemical Reagent. PEG 5000 (molecular weight M = 4900 ∼ 5100), PEG 6000, (M = 5500 ∼ 6500), and PEG 10000 (M = 9000 ∼ 11000), all analytical grade, were purchased from Guangzhou Chemical Reagent Corporation. Potassium dihydrogen phosphate and dipotassium hydrogen phosphate and other chemical reagents were of analytical reagent grade. Water was distilled and deionized.
The density was determined using a Pomeishi density meter (Shanghai, China). The density meter was calibrated against air and water at 298 ± 1 K before every set of measurements. The pH value was measured with a SA 720 pH meter (Orion, USA). The refractive index was determined using a 2WAJ Abbe refractive meter (Shanghai, China). The concentration of amino acid was measured by a UV-VIS-NIR recording spectrophotometer of Model UV-365 (Shimadzu, Japan) at 570 nm after reacting with ninhydrin. 3. Results
2.2. Experimental procedures
3.1. Effects of pH of phosphate solutions on the phase diagrams
The aqueous two-phase systems were prepared from stock solutions of PEG in water and phosphate solutions according
The phase diagrams of the systems of PEG 5000, 6000, and 10000 in a pH range of 6.51∼7.93 phosphate solutions
Q.K. Shang et al. / Fluid Phase Equilibria 219 (2004) 195–203
197
40
% PEG 5000(w/w)
35 30 25 20 15 10 5 0 4
6
8
10
12
14
16
18
20
% Salt (w/w)
(a)
45 40
% PEG 6000 (w/w)
35 30 25 20 15 10 5 0
4
6
8
10
12
14
16
% Salt (w/w)
(b)
40
% PEG 10000 (w/w)
35 30 25 20 15 10 5 0 4
(c)
6
8
10
12
14
16
18
% Salt (w/w)
Fig. 1. Effect of pH on the binodal of PEG-phosphate systems for (a) PEG 5000: (䊐) pH 6.51, (䊊) pH 6.71, () pH 7.24, (䉫) pH 7.71, () pH 7.93; (b) PEG 6000: (䊐) pH 6.51, (䊊) pH 6.71, () pH 7.24, (䉫) pH 7.71, () pH 7.93; (c) PEG 10000: (䊐) pH 6.51, (䊊) pH 6.71, () pH 7.24, (䉫) pH 7.71, () pH 7.93.
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Fig. 2. Effect of PEG molecular weight on the bimodal of systems at (a) pH 6.51: (䊐) PEG 5000, (䊊) PEG 6000, () PEG 10000, (+) Tie line of system A, (䉫) Tie line of system B, () Tie line of system C (see Table 1); (b) pH 6.71: (䊐) PEG 5000, (䊊) PEG 6000, () PEG 10000, (+) Tie line of system A, (䉫) Tie line of system B, () Tie line of system C (see Table 1); (c) pH 7.24: (䊐) PEG 5000, (䊊) PEG 6000, () PEG 10000, (+) Tie line of system A, (䉫) Tie line of system B, () Tie line of system C (see Table 1); (d) pH 7.71: (䊐) PEG 5000, (䊊) PEG 6000, () PEG 10000, (+) Tie line of system A, (䉫) Tie line of system B, () Tie line of system C (see Table 1); (e) pH 7.93: (䊐) PEG 5000, (䊊) PEG 6000, () PEG 10000, (+) Tie line of system A, (䉫) Tie line of system B, () Tie line of system C (see Table 1).
Table 1 Compositions and physical properties of PEG-phosphates systems
PEG (%, w/w)
Top phase
Bottom phase
Salt Water PEG Salt Water Density (%, w/w) (%, w/w) (%, w/w) (%, w/w) (%, w/w) (g/cm3 )
Refractive index
pH
PEG Salt Water Density (%, w/w) (%, w/w) (%, w/w) (g/cm3 )
Refractive index
pH
Volume ratio (Vtop /Vbottom)
Phase separation times(s)
PEG 5000-K2 HPO4 –KH2 PO4 A 10.8 10.44 D 14.58 10.53 G 14.28 10.39 J 17.05 11.77 M 19.02 10.57
78.76 74.89 75.33 71.18 70.41
23.24 22.72 30.38 41.25 39.41
7.42 8.32 6.45 5.3 5.81
69.34 68.96 63.17 53.45 54.78
1.087 1.088 1.087 1.09 1.087
1.37476 1.37024 1.35833 1.39135 1.38734
6.90 6.93 7.54 8.10 8.28
3.54 4.58 5.23 4.56 3.66
12.52 13.31 12.55 15.76 14.24
83.94 82.11 82.22 79.68 82.1
1.146 1.142 1.162 1.197 1.179
1.35766 1.35585 1.35967 1.36589 1.36269
6.70 6.92 7.39 7.85 7.98
1.04 1.08 1.07 1.00 1.45
323 294 253 242 225
PEG 6000-K2 HPO4 –KH2 PO4 B 11.31 11.13 E 11.31 11.18 H 13.45 9.54 K 15.37 11.97 N 16.98 9.71
77.56 77.51 77.01 72.66 73.31
23.23 23.87 27.57 41.36 36.76
7.76 7.64 6.13 4.85 5.32
69.01 68.49 66.30 53.79 57.92
1.102 1.093 1.089 1.089 1.089
1.36923 1.37175 1.37828 1.38683 1.38030
6.70 6.95 7.48 7.78 7.90
2.96 3.22 4.71 3.70 3.66
13.45 13.24 11.86 15.63 12.92
83.59 83.54 83.43 80.67 83.42
1.144 1.141 1.16 1.188 1.156
1.35766 1.35766 1.35917 1.3632 1.35967
6.70 6.90 7.34 7.72 7.91
1.08 1.10 1.10 0.93 1.63
341 315 261 250 235
PEG 10000- K2 HPO4 –KH2 PO4 C 11.54 11.98 76.48 H 13.62 9.29 77.09 I 10.77 9.65 79.58 L 16.33 9.43 74.24 O 17.1 9.11 73.79
23.17 21.85 25.06 34.07 36.53
9.52 7.01 5.63 4.75 4.82
67.31 71.14 69.31 61.18 58.65
1.085 1.083 1.084 1.084 1.084
1.37426 1.37326 1.38130 1.35983 1.38369
6.76 7.02 7.56 7.99 8.10
4.03 4.44 4.12 3.58 3.31
13.78 11.61 11.45 13.00 12.4
82.19 83.95 84.43 83.42 84.29
1.134 1.134 1.15 1.16 1.158
1.35015 1.35436 1.35766 1.36018 1.35902
6.79 6.94 7.41 7.79 7.96
0.98 1.51 0.70 0.91 1.27
364 346 331 273 264
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Total system
A∼O represent TLL as shown in Fig. 2.
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with ionic strength from 2.98 to 6.73 are shown in Fig. 1. It can be seen that the acidity shows similar effects on the binodals despite different PEG molecular weights. The binodal curve shifts toward lower PEG and salt concentration with increasing pH values of the systems, which results in an increase of the region of phase separation. The two-phase region expands greatly when pH is lower than 7.00 while it remains constant at a pH value larger than 7.00. A possible explanation may be that the concentration of K2 HPO4 increases with increasing pH values. The two phases are more prone to form when the concentration of small multivalent anions in the systems is higher because of the increased ionic strength. 3.2. Effects of PEG molecular weights on the phase diagrams The effect of PEG molecular weight (5000, 6000, and 10000) on the phase diagrams at different pH values is shown in Fig. 2 where the lines A. . . O represent the tie-lines of the systems investigated. It can be seen that the binodals have similar shapes for each system [24,25]. However, the two-phase region decreases with decreasing PEG molecular weight at the experimental acidities considered. 3.3. Composition and characteristics of aqueous two-phase systems The composition of both phases and the determined data for aqueous two-phase systems such as density, volume ratio of top phase and bottom phase, and phase separation times are listed in Table 1. It is evident that the top phase is rich in PEG while the bottom phase is rich in salts. It can be seen that the densities of the systems, as expected, are close to 1.0 g/cm3 because of the high water content [26]. The PEG molecular weight does not influence the densities of both the top and the bottom phases. The density of the bottom phase increases a little with increasing pH but the density of the top phase does not change. It can be seen from Table 1 that the longest phase separation time is 364 seconds and that at larger pH values of the system, the phase separation time is shorter. The separation time is important for normal gravitational operations. A short operation time for phase separation is an important factor for low-cost applications of aqueous two-phase systems. Based on the results presented in Table 1, all systems can be used for normal gravitational operations in practical applications. It can also be seen from Table 1 that the phase volume ratios of the systems E, J and O are much larger than those of the other systems. Correspondingly, the separation time of systems E, J, and O is shortest for PEG 5000, 6000, and 10000 systems, respectively. In other words, the separation time of the two phases decreases with increasing phase volume ratio of the top phase and the bottom phase. The pH values of top and bottom phase have a little difference presented in Table 1. That may due to the big molecules
of polyethylene glycol producing the bigger obstruction for H+ ions than the small salt molecules. So the pH values of top phase are larger than those of bottom phase. This result is same as the [22]. 3.4. Partitioning behavior of amino acids in aqueous two-phase systems Amino acids are amphoteric substances and exist as anions, cations, or neutral molecules depending on the pH of the solution. When the pH is lower than the isoelectric point, amino acids exist as cations at higher pH as anions. Around the isoelectric point both functional groups are charged and the main chain carries no net charge. Some polar or nonpolar groups incorporated in the side chain of the amino acids cause a different hydrophobicity or hydrophilicity. So the partition behavior of amino acids in ATPSs is decided by both the ATPS and the structures of the amino acids. Table 2 shows the distribution ratios of four amino acids in 15 ATPSs (from A to O). 3.4.1. Effect of salts on partitioning Salts can change the electrostatic charge of ATPSs and influence the distribution ratios of charged amino acids. Lysine exists as a cation in the pH range considered (6.5∼8.0) because of its high isoelectric point, 9.74. Other amino acids are anions at the experimental condition. Table 2 shows that the distribution ratios of lysine are the lowest among the four amino acids. That means the electrostatic interaction between lysine cation and salt anion is the biggest so that lysine prefers to be in the salt-rich phase. Amino acids with negative charges prefer to be in the polymer-rich phase that may be because of the repulsion caused by the salt anions. Table 2 Distribution ratios of four amino acids in ATPSs Systems
Distribution ratios Dphe
DCys
Dlys
Dmet
PEG 5000
A D G J M
0.75 0.90 1.14 1.73 1.92
0.72 0.78 0.69 0.94 1.24
0.0084 0.043 0.32 0.60 0.58
0.56 0.74 0.73 0.71 0.98
PEG 6000
B E H K N
0.53 0.82 1.09 1.52 1.83
0.92 1.01 1.14 1.16 1.96
0.0052 0.076 0.56 0.60 0.79
0.52 0.54 0.45 0.72 1.06
PEG 10000
C F I L O
0.62 0.71 1.22 2.15 2.59
0.94 1.00 1.34 1.81 2.53
0.30 0.40 0.69 0.77 0.96
0.41 0.64 0.83 1.11 1.19
Note: Systems A–O are same as Fig. 2.
2.8
2.4
Distribution ratios (D)
2.0 1.6 1.2 0.8 0.4 0.0 6.4
6.6
6.8
7.0
7.2
7.4
7.6
7.8
1.6
1.2
0.8
0.4
8.0
pH
(a)
2.0
6.4
6.6
6.8
7.0
7.2
7.4
7.6
7.8
8.0
pH
(b)
1.0 1.2 0.8
Distribution ratios (D)
Distribution ratios (D)
1.0 0.6
0.4
0.2
0.8
0.6
Q.K. Shang et al. / Fluid Phase Equilibria 219 (2004) 195–203
Distribution ratios (D)
2.4
0.4
0.0 0.2 6.4
(c)
6.8
7.2
pH
7.6
8.0
6.4
(d)
6.8
7.2
7.6
8.0
pH
Fig. 3. Effect of pH and PEG molecular weight on the distribution ratios of phenylalanine (a): (䊐) PEG 5000, (䊊) PEG 6000, () PEG 10000; Effect of pH and PEG molecular weight on the distribution ratios of cysteine (b): (䊐) PEG 5000, (䊊) PEG 6000, () PEG 10000; Effect of pH and PEG molecular weight on the distribution ratios of lysine (c): (䊐) PEG 5000, (䊊) PEG 6000, () PEG 10000; Effect of pH and PEG molecular weight on the distribution ratios of mehtionine (d): (䊐) PEG 5000, (䊊) PEG 6000, () PEG 10000. 201
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3.4.2. Effect of the side chain of amino acids on partitioning It is shown in Table 2 that the distribution ratios of phenylalanine are higher than those of the other three amino acids at the same condition, which may be explained by the hydrophobicity of amino acids. Methionine has a nonpolar aliphatic side chain while phenylalanine has a nonpolar aromatic side group, lysine a polar side chain with positive charge and cysteine a polar side chain without charge. A nonpolar side chain causes increasing hydrophobicity, resulting in amino acids preferring the polymer-rich phase. The hydrophobicity of phenylalanine is larger than that of methionine because of its aromatic group so that the distribution ratio of methionine is smaller than that of phenylalanine. Their distribution ratios at systems M, N, L, and O are also larger than those in the literature [19]. The order of distribution ratios of lysine and phenylalanine is same as that in Berggren t al., work [17]. The experimental results show that the structure of the side chains of amino acids has a significant influence on the partitioning in ATPSs.
agrams of the PEG/phosphates buffer are presented given which show that the two-phase region expands with increasing PEG molecular weights and acidities of the phosphates. The influence of salts, side chain structures of amino acids, pH values, and PEG molecular weights of ATPSs on partitioning behavior of the amino acids are investigated. Electrostatic interaction and hydrophobicity are two main factors affecting the partitioning of amino acids in ATPSs. Salts in ATPSs can change the electrostatic charge of systems and cause amino acids with positive charge to prefer the salt-rich phase. Amino acids with a nonpolar side chain prefer the polymer-rich phase because of their stronger hydrophobicity. Increasing pH and PEG molecular weight of the ATPSs results in an increase of the distribution ratios of four amino acids due to the change of electrostatic interaction and the hydrophobicity. These results may be used to predict the separation of some proteins whose surface residues contain these four amino acids.
3.4.3. Effect of pH on partitioning For different PEG molecular weights considered systems with five phosphates of different pH values have been studied. The distribution ratios of the four amino acids increase more or less with increasing pH (see Fig. 3), which may be caused by the electrostatic interaction between amino acids and ATPSs. When the pH of the solutions increases, i.e. the concentration of OH− increases, the lysine cations will be neutralized partly, which causes decreasing electrostatic interaction. At the same time the increase of the HPO4 2− concentration causes increasing repulsion with phenylalanine, methionine and cysteine anions. Therefore, the largest distribution ratios of the four amino acids are obtained at the largest pH values, as shown in the systems M, N, and O in Table 2.
Acknowledgements
3.4.4. Effect of PEG molecular weights on partitioning It can be found from Fig. 3 that distribution ratios of four amino acids increase as the increasing of PEG molecular weight in most experimental conditions especially at a higher pH value [27]. That may be caused by the hydrophobicity of amino acids. The bigger the PEG molecular weight, the more hydrophobicity the top phase. It is one of the important influences for amino acids preferring the polymer-rich phase. The largest distribution ratios of the four amino acids obtained at the largest pH value in the ATPSs with the biggest PEG molecular weight may due to the hydrophobicity acting together with the electrostatic interaction.
4. Conclusions Aqueous two-phase systems with different polyethylene glycol molecular weight and phosphate buffers have been used to study the partitioning behavior of four amino acids, cysteine, phenylalanine, methionine and lysine. Phase di-
This project is supported by State Key Project of Fundamental Research (G1998061301), National “863” Project (2002AA647070) and the National Natural Science Foundation of China (29771028, 29801004).
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Partitioning behavior of amino acids in aqueous two-phase systems containing polyethylene glycol and phosphate buffer Q.K. Shang a,b , W. Li a , Q. Jia a , D.Q. Li a,∗ a
Key Laboratory of Rare Earths Chemistry and Physics, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun 130022, PR China b College of Chemistry, Northeast Normal University, Changchun 130024, PR China Received 6 May 2003; received in revised form 29 January 2004; accepted 30 January 2004
Abstract The partitioning behavior of four amino acids, cysteine, phenylalanine, methionine, and lysine in 15 aqueous two-phase systems (ATPSs) with different polyethylene glycol (PEG) molecular weights and phosphate buffers has been studied in the present paper. The phase diagrams of the systems are investigated together with the effect of the PEG molecular weight and pH of the phosphate solutions. The composition of these systems and some parameters such as density and refractive index are determined. The influences of salts in ATPSs, side chain structure of the amino acids, pH of ATPSs, and the PEG molecular weight on the distribution ratios of the amino acids have been studied. This work is useful for the purification of amino acids and the separation of some proteins whose main surface exposed amino acid residues are these four amino acids, respectively. © 2004 Elsevier B.V. All rights reserved. Keywords: Aqueous two-phase system; Partitioning; Amino acid
1. Introduction When two polymers or one polymer and one salt are mixed in water above certain concentration, two immiscible aqueous phases are formed. One phase is rich in polymer and the other phase is rich in the other polymer or salt, this type of systems are called aqueous two-phase systems (ATPSs). ATPSs were discovered by Beijerinck [1] and were first used for extraction and separation purposes by Albertsson [2]. Zaslavsky [3] has published a monograph covering most topics in the area of aqueous two-phase partitioning. In the second half of the 20th century, there has been increasing attention focused on ATPSs in laboratory scale, especially for the separation of proteins, cells and other biological materials which require mild aqueous environments [4–10]. The most common ATPSs used for the separation of biomolecules are polyethylene glycol (PEG)/dextran or PEG/salt systems because of low-cost, rapid mass transfer, and phase equilibrium. The processes scale-up easily can be used as continu∗ Corresponding author. Tel.: +86-431-5262036; fax: +86-431-5698041. E-mail address: [email protected] (D.Q. Li).
0378-3812/$ – see front matter © 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.fluid.2004.01.032
ous process and are more economical than other separation processes. Some aqueous two-phase systems have been designed for scale-up of downstream processes for biomaterial separation [11–14]. The surface of different proteins has common features in terms of hydrophobicity/hydrophilicity properties even though the primary sequence and overall fold of the proteins differ. However, the occurrence of individual types of residues on the protein surface differs and the surface properties of a protein are often crucial for recognition and interaction with other proteins. Partitioning of amino acids residues in ATPSs has the potential to be used as a rapid and simple method to study and characterize the protein surface, since the partitioning of a protein depends on its surface properties [15]. It has been proved that amino acid residues determine the protein surface properties by some authors [16–18]. It is interesting to move towards a deeper understanding of the influence of the surface exposed amino acid residues on partitioning in aqueous two-phase systems. During the past decades, there have been many papers about the partitioning behavior of amino acids or low molecular mass peptides in ATPSs [6,16,19–22]. Some aqueous two-phase systems composed of polyethylene glycol/salts
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or dextran have been used, e.g., dextran and PEG 8000 systems [6], PEG 8000 and magnesium sulfate systems [16], PEG 2000, PEG 4000, and recyclable volatile salt systems [19], PEG 3400 and potassium phosphate systems [20], PEG 8000 and potassium phosphate systems [21], PEG 6000, 35000, and dextran 500 systems [22]. Partitioning of more than 20 amino acids in these aqueous two-phase systems has been studied. For instance, Zaslavsky et al. [6] investigated the partitioning of glycine, lysine and aspartic acid and their oligopeptides in an aqueous dextran-polyethylene glycol 8000 two-phase system. Van Berlo et al. [19] studied the partitioning behavior of seven amino acids (l-serine, glycine, l-alanine, l-valine, l-methionine, l-isoleucine, and l-phenylalanine) in PEG 2000, PEG 4000 with aqueous recyclable volatile salt two-phase systems. However, PEG 5000, PEG 6000, and PEG 10000 with aqueous phosphates two-phase systems for the partitioning of amino acids have not been studied so far. The effects of polymer molecular weight, the composition and some properties of the systems on the distribution ratios of amino acids have also not been investigated in detail. In this work, the partitioning behavior of 4 amino acids with different side chains, cysteine, phenylalanine, methionine, and lysine in 15 PEG/phosphate aqueous two-phase systems has been studied. The phase diagrams of the systems in PEG 5000, 6000, and 10000 with different K2 HPO4 –KH2 PO4 compositions are determined together with the effects of polymer molecular weight and pH of phosphate buffers on the aqueous two-phase systems. The physical parameters of the systems such as density and refractive index are also determined. Factors affecting the partitioning of amino acids, such as salts, side chain structures of amino acids, pH of ATPSs and PEG molecular weight have been studied. The results may be used to predict the separation of some proteins whose main surface exposed amino acid residues are these four amino acids.
to the desired pH (about 6–8) values. The phosphate solutions, in pH 6.51, 6.71, 7.24, 7.71, and 7.93, were composed of the mixed solution of K2 HPO4 (3.5 mol/l) and KH2 PO4 (1.0 mol/l) in the ratio 1:3, 1:2, 1:1, 2:1, and 3:1, respectively. The binodal curves were determined by a titration method at a controlled temperature of 298 ± 1 K. PEG, phosphate solutions, and water with the total weight of about 60 g were added into vessels. The vessels were vibrated for 15 min and then placed at a controlled temperature of 298 ± 1 K for 20 h at least to ensure complete equilibration. The concentrations of phosphates in both phases were determined by a titration method. The concentrations of PEG in both phases were calculated from the refractive indexes considering the effect of the concentration of salts [23]. The relation between the refractive index, n, and the mass fractions of salt, ws , and PEG, wp can be expressed as follows n = 1.342780 + 0.000864wp + 0.001138ws (top phase)
(1)
n = 1.336427 + 0.000426wp + 0.001562ws (bottom phase)
(2)
The precision of the mass fraction of PEG achieved using Eqs. (1) and (2) was better than 0.005 and 0.002, respectively. Three milligram amino acids and 3 ml top phase and bottom phase, respectively, were placed in special glass tubes and centrifuged for 15 min at 66.6 r/s. The mixtures were allowed settle for 15 h at 298 ± 1 K. The concentrations of amino acids in both phases were then determined. The distribution ratios of amino acids have been tested five times in parallel and other experiments have been repeated three times. The standard deviations for the distribution ratios varied between 0.049 and 0.0011. 2.3. Analyses of samples
2. Experimental 2.1. Chemicals Cysteine, methionine, phenylalanine, and lysine were obtained from Shanghai Institute of Chemical Reagent. PEG 5000 (molecular weight M = 4900 ∼ 5100), PEG 6000, (M = 5500 ∼ 6500), and PEG 10000 (M = 9000 ∼ 11000), all analytical grade, were purchased from Guangzhou Chemical Reagent Corporation. Potassium dihydrogen phosphate and dipotassium hydrogen phosphate and other chemical reagents were of analytical reagent grade. Water was distilled and deionized.
The density was determined using a Pomeishi density meter (Shanghai, China). The density meter was calibrated against air and water at 298 ± 1 K before every set of measurements. The pH value was measured with a SA 720 pH meter (Orion, USA). The refractive index was determined using a 2WAJ Abbe refractive meter (Shanghai, China). The concentration of amino acid was measured by a UV-VIS-NIR recording spectrophotometer of Model UV-365 (Shimadzu, Japan) at 570 nm after reacting with ninhydrin. 3. Results
2.2. Experimental procedures
3.1. Effects of pH of phosphate solutions on the phase diagrams
The aqueous two-phase systems were prepared from stock solutions of PEG in water and phosphate solutions according
The phase diagrams of the systems of PEG 5000, 6000, and 10000 in a pH range of 6.51∼7.93 phosphate solutions
Q.K. Shang et al. / Fluid Phase Equilibria 219 (2004) 195–203
197
40
% PEG 5000(w/w)
35 30 25 20 15 10 5 0 4
6
8
10
12
14
16
18
20
% Salt (w/w)
(a)
45 40
% PEG 6000 (w/w)
35 30 25 20 15 10 5 0
4
6
8
10
12
14
16
% Salt (w/w)
(b)
40
% PEG 10000 (w/w)
35 30 25 20 15 10 5 0 4
(c)
6
8
10
12
14
16
18
% Salt (w/w)
Fig. 1. Effect of pH on the binodal of PEG-phosphate systems for (a) PEG 5000: (䊐) pH 6.51, (䊊) pH 6.71, () pH 7.24, (䉫) pH 7.71, () pH 7.93; (b) PEG 6000: (䊐) pH 6.51, (䊊) pH 6.71, () pH 7.24, (䉫) pH 7.71, () pH 7.93; (c) PEG 10000: (䊐) pH 6.51, (䊊) pH 6.71, () pH 7.24, (䉫) pH 7.71, () pH 7.93.
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Fig. 2. Effect of PEG molecular weight on the bimodal of systems at (a) pH 6.51: (䊐) PEG 5000, (䊊) PEG 6000, () PEG 10000, (+) Tie line of system A, (䉫) Tie line of system B, () Tie line of system C (see Table 1); (b) pH 6.71: (䊐) PEG 5000, (䊊) PEG 6000, () PEG 10000, (+) Tie line of system A, (䉫) Tie line of system B, () Tie line of system C (see Table 1); (c) pH 7.24: (䊐) PEG 5000, (䊊) PEG 6000, () PEG 10000, (+) Tie line of system A, (䉫) Tie line of system B, () Tie line of system C (see Table 1); (d) pH 7.71: (䊐) PEG 5000, (䊊) PEG 6000, () PEG 10000, (+) Tie line of system A, (䉫) Tie line of system B, () Tie line of system C (see Table 1); (e) pH 7.93: (䊐) PEG 5000, (䊊) PEG 6000, () PEG 10000, (+) Tie line of system A, (䉫) Tie line of system B, () Tie line of system C (see Table 1).
Table 1 Compositions and physical properties of PEG-phosphates systems
PEG (%, w/w)
Top phase
Bottom phase
Salt Water PEG Salt Water Density (%, w/w) (%, w/w) (%, w/w) (%, w/w) (%, w/w) (g/cm3 )
Refractive index
pH
PEG Salt Water Density (%, w/w) (%, w/w) (%, w/w) (g/cm3 )
Refractive index
pH
Volume ratio (Vtop /Vbottom)
Phase separation times(s)
PEG 5000-K2 HPO4 –KH2 PO4 A 10.8 10.44 D 14.58 10.53 G 14.28 10.39 J 17.05 11.77 M 19.02 10.57
78.76 74.89 75.33 71.18 70.41
23.24 22.72 30.38 41.25 39.41
7.42 8.32 6.45 5.3 5.81
69.34 68.96 63.17 53.45 54.78
1.087 1.088 1.087 1.09 1.087
1.37476 1.37024 1.35833 1.39135 1.38734
6.90 6.93 7.54 8.10 8.28
3.54 4.58 5.23 4.56 3.66
12.52 13.31 12.55 15.76 14.24
83.94 82.11 82.22 79.68 82.1
1.146 1.142 1.162 1.197 1.179
1.35766 1.35585 1.35967 1.36589 1.36269
6.70 6.92 7.39 7.85 7.98
1.04 1.08 1.07 1.00 1.45
323 294 253 242 225
PEG 6000-K2 HPO4 –KH2 PO4 B 11.31 11.13 E 11.31 11.18 H 13.45 9.54 K 15.37 11.97 N 16.98 9.71
77.56 77.51 77.01 72.66 73.31
23.23 23.87 27.57 41.36 36.76
7.76 7.64 6.13 4.85 5.32
69.01 68.49 66.30 53.79 57.92
1.102 1.093 1.089 1.089 1.089
1.36923 1.37175 1.37828 1.38683 1.38030
6.70 6.95 7.48 7.78 7.90
2.96 3.22 4.71 3.70 3.66
13.45 13.24 11.86 15.63 12.92
83.59 83.54 83.43 80.67 83.42
1.144 1.141 1.16 1.188 1.156
1.35766 1.35766 1.35917 1.3632 1.35967
6.70 6.90 7.34 7.72 7.91
1.08 1.10 1.10 0.93 1.63
341 315 261 250 235
PEG 10000- K2 HPO4 –KH2 PO4 C 11.54 11.98 76.48 H 13.62 9.29 77.09 I 10.77 9.65 79.58 L 16.33 9.43 74.24 O 17.1 9.11 73.79
23.17 21.85 25.06 34.07 36.53
9.52 7.01 5.63 4.75 4.82
67.31 71.14 69.31 61.18 58.65
1.085 1.083 1.084 1.084 1.084
1.37426 1.37326 1.38130 1.35983 1.38369
6.76 7.02 7.56 7.99 8.10
4.03 4.44 4.12 3.58 3.31
13.78 11.61 11.45 13.00 12.4
82.19 83.95 84.43 83.42 84.29
1.134 1.134 1.15 1.16 1.158
1.35015 1.35436 1.35766 1.36018 1.35902
6.79 6.94 7.41 7.79 7.96
0.98 1.51 0.70 0.91 1.27
364 346 331 273 264
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Total system
A∼O represent TLL as shown in Fig. 2.
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with ionic strength from 2.98 to 6.73 are shown in Fig. 1. It can be seen that the acidity shows similar effects on the binodals despite different PEG molecular weights. The binodal curve shifts toward lower PEG and salt concentration with increasing pH values of the systems, which results in an increase of the region of phase separation. The two-phase region expands greatly when pH is lower than 7.00 while it remains constant at a pH value larger than 7.00. A possible explanation may be that the concentration of K2 HPO4 increases with increasing pH values. The two phases are more prone to form when the concentration of small multivalent anions in the systems is higher because of the increased ionic strength. 3.2. Effects of PEG molecular weights on the phase diagrams The effect of PEG molecular weight (5000, 6000, and 10000) on the phase diagrams at different pH values is shown in Fig. 2 where the lines A. . . O represent the tie-lines of the systems investigated. It can be seen that the binodals have similar shapes for each system [24,25]. However, the two-phase region decreases with decreasing PEG molecular weight at the experimental acidities considered. 3.3. Composition and characteristics of aqueous two-phase systems The composition of both phases and the determined data for aqueous two-phase systems such as density, volume ratio of top phase and bottom phase, and phase separation times are listed in Table 1. It is evident that the top phase is rich in PEG while the bottom phase is rich in salts. It can be seen that the densities of the systems, as expected, are close to 1.0 g/cm3 because of the high water content [26]. The PEG molecular weight does not influence the densities of both the top and the bottom phases. The density of the bottom phase increases a little with increasing pH but the density of the top phase does not change. It can be seen from Table 1 that the longest phase separation time is 364 seconds and that at larger pH values of the system, the phase separation time is shorter. The separation time is important for normal gravitational operations. A short operation time for phase separation is an important factor for low-cost applications of aqueous two-phase systems. Based on the results presented in Table 1, all systems can be used for normal gravitational operations in practical applications. It can also be seen from Table 1 that the phase volume ratios of the systems E, J and O are much larger than those of the other systems. Correspondingly, the separation time of systems E, J, and O is shortest for PEG 5000, 6000, and 10000 systems, respectively. In other words, the separation time of the two phases decreases with increasing phase volume ratio of the top phase and the bottom phase. The pH values of top and bottom phase have a little difference presented in Table 1. That may due to the big molecules
of polyethylene glycol producing the bigger obstruction for H+ ions than the small salt molecules. So the pH values of top phase are larger than those of bottom phase. This result is same as the [22]. 3.4. Partitioning behavior of amino acids in aqueous two-phase systems Amino acids are amphoteric substances and exist as anions, cations, or neutral molecules depending on the pH of the solution. When the pH is lower than the isoelectric point, amino acids exist as cations at higher pH as anions. Around the isoelectric point both functional groups are charged and the main chain carries no net charge. Some polar or nonpolar groups incorporated in the side chain of the amino acids cause a different hydrophobicity or hydrophilicity. So the partition behavior of amino acids in ATPSs is decided by both the ATPS and the structures of the amino acids. Table 2 shows the distribution ratios of four amino acids in 15 ATPSs (from A to O). 3.4.1. Effect of salts on partitioning Salts can change the electrostatic charge of ATPSs and influence the distribution ratios of charged amino acids. Lysine exists as a cation in the pH range considered (6.5∼8.0) because of its high isoelectric point, 9.74. Other amino acids are anions at the experimental condition. Table 2 shows that the distribution ratios of lysine are the lowest among the four amino acids. That means the electrostatic interaction between lysine cation and salt anion is the biggest so that lysine prefers to be in the salt-rich phase. Amino acids with negative charges prefer to be in the polymer-rich phase that may be because of the repulsion caused by the salt anions. Table 2 Distribution ratios of four amino acids in ATPSs Systems
Distribution ratios Dphe
DCys
Dlys
Dmet
PEG 5000
A D G J M
0.75 0.90 1.14 1.73 1.92
0.72 0.78 0.69 0.94 1.24
0.0084 0.043 0.32 0.60 0.58
0.56 0.74 0.73 0.71 0.98
PEG 6000
B E H K N
0.53 0.82 1.09 1.52 1.83
0.92 1.01 1.14 1.16 1.96
0.0052 0.076 0.56 0.60 0.79
0.52 0.54 0.45 0.72 1.06
PEG 10000
C F I L O
0.62 0.71 1.22 2.15 2.59
0.94 1.00 1.34 1.81 2.53
0.30 0.40 0.69 0.77 0.96
0.41 0.64 0.83 1.11 1.19
Note: Systems A–O are same as Fig. 2.
2.8
2.4
Distribution ratios (D)
2.0 1.6 1.2 0.8 0.4 0.0 6.4
6.6
6.8
7.0
7.2
7.4
7.6
7.8
1.6
1.2
0.8
0.4
8.0
pH
(a)
2.0
6.4
6.6
6.8
7.0
7.2
7.4
7.6
7.8
8.0
pH
(b)
1.0 1.2 0.8
Distribution ratios (D)
Distribution ratios (D)
1.0 0.6
0.4
0.2
0.8
0.6
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Distribution ratios (D)
2.4
0.4
0.0 0.2 6.4
(c)
6.8
7.2
pH
7.6
8.0
6.4
(d)
6.8
7.2
7.6
8.0
pH
Fig. 3. Effect of pH and PEG molecular weight on the distribution ratios of phenylalanine (a): (䊐) PEG 5000, (䊊) PEG 6000, () PEG 10000; Effect of pH and PEG molecular weight on the distribution ratios of cysteine (b): (䊐) PEG 5000, (䊊) PEG 6000, () PEG 10000; Effect of pH and PEG molecular weight on the distribution ratios of lysine (c): (䊐) PEG 5000, (䊊) PEG 6000, () PEG 10000; Effect of pH and PEG molecular weight on the distribution ratios of mehtionine (d): (䊐) PEG 5000, (䊊) PEG 6000, () PEG 10000. 201
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3.4.2. Effect of the side chain of amino acids on partitioning It is shown in Table 2 that the distribution ratios of phenylalanine are higher than those of the other three amino acids at the same condition, which may be explained by the hydrophobicity of amino acids. Methionine has a nonpolar aliphatic side chain while phenylalanine has a nonpolar aromatic side group, lysine a polar side chain with positive charge and cysteine a polar side chain without charge. A nonpolar side chain causes increasing hydrophobicity, resulting in amino acids preferring the polymer-rich phase. The hydrophobicity of phenylalanine is larger than that of methionine because of its aromatic group so that the distribution ratio of methionine is smaller than that of phenylalanine. Their distribution ratios at systems M, N, L, and O are also larger than those in the literature [19]. The order of distribution ratios of lysine and phenylalanine is same as that in Berggren t al., work [17]. The experimental results show that the structure of the side chains of amino acids has a significant influence on the partitioning in ATPSs.
agrams of the PEG/phosphates buffer are presented given which show that the two-phase region expands with increasing PEG molecular weights and acidities of the phosphates. The influence of salts, side chain structures of amino acids, pH values, and PEG molecular weights of ATPSs on partitioning behavior of the amino acids are investigated. Electrostatic interaction and hydrophobicity are two main factors affecting the partitioning of amino acids in ATPSs. Salts in ATPSs can change the electrostatic charge of systems and cause amino acids with positive charge to prefer the salt-rich phase. Amino acids with a nonpolar side chain prefer the polymer-rich phase because of their stronger hydrophobicity. Increasing pH and PEG molecular weight of the ATPSs results in an increase of the distribution ratios of four amino acids due to the change of electrostatic interaction and the hydrophobicity. These results may be used to predict the separation of some proteins whose surface residues contain these four amino acids.
3.4.3. Effect of pH on partitioning For different PEG molecular weights considered systems with five phosphates of different pH values have been studied. The distribution ratios of the four amino acids increase more or less with increasing pH (see Fig. 3), which may be caused by the electrostatic interaction between amino acids and ATPSs. When the pH of the solutions increases, i.e. the concentration of OH− increases, the lysine cations will be neutralized partly, which causes decreasing electrostatic interaction. At the same time the increase of the HPO4 2− concentration causes increasing repulsion with phenylalanine, methionine and cysteine anions. Therefore, the largest distribution ratios of the four amino acids are obtained at the largest pH values, as shown in the systems M, N, and O in Table 2.
Acknowledgements
3.4.4. Effect of PEG molecular weights on partitioning It can be found from Fig. 3 that distribution ratios of four amino acids increase as the increasing of PEG molecular weight in most experimental conditions especially at a higher pH value [27]. That may be caused by the hydrophobicity of amino acids. The bigger the PEG molecular weight, the more hydrophobicity the top phase. It is one of the important influences for amino acids preferring the polymer-rich phase. The largest distribution ratios of the four amino acids obtained at the largest pH value in the ATPSs with the biggest PEG molecular weight may due to the hydrophobicity acting together with the electrostatic interaction.
4. Conclusions Aqueous two-phase systems with different polyethylene glycol molecular weight and phosphate buffers have been used to study the partitioning behavior of four amino acids, cysteine, phenylalanine, methionine and lysine. Phase di-
This project is supported by State Key Project of Fundamental Research (G1998061301), National “863” Project (2002AA647070) and the National Natural Science Foundation of China (29771028, 29801004).
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