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Peptide Hydrophobicity and Partitioning in Poly(ethy1ene glycol)/ Magnesium Sulfate Aqueous Two-Phase Systems Mark A. Eiteman and John L. Gainer* Center for Bioprocess and Product Development, Department of Chemical Engineering, Thornton Hall, University of Virginia, Charlottesville, Virginia 22903-2442
Several amino acids and peptides were partitioned in poly(ethy1ene glycol) (PEG)/ magnesium sulfate (MgSO4) aqueous two-phase systems. The partition coefficients measured for amino acids and peptides were proportional to the difference in PEG concentration between the phases. The partitioning data were used to calculate the relative hydrophobicities of individual amino acids, which were then used to estimate the hydrophobicities of peptides. The partition coefficients of several dipeptides were predicted from these estimated hydrophobicities. A series of peptide fragments that compose the pentapeptide leucine enkephalin was also partitioned in the PEG/ MgSO, system. Again, the partitioning depended upon the hydrophobicities of the individual exposed amino acids.
Introduction Aqueous two-phase systems occur when two mutually incompatible components, such as poly(ethy1ene glycol) (PEG) and dextran or PEG and magnesium sulfate (MgSOd), are dissolved together in water. As shown by Albertsson (1986), at concentrations above those defined by a phase boundary, two liquid phases will form, with each dissolved component predominating in one or the other phase. The phases are termed “aqueous”because over 80 weight percent of each is composed of water. Solutes partition between these phases, and this can be quantified by a partition coefficient, K , which is usually defined as the solute concentration in the upper phase divided by the solute concentration in the lower phase. Many types of solutes have been shown to partition in aqueous twophase systems, for example, small organic molecules (Mattiasson et al., 1982; Eiteman and Gainer, 1988) and salts (Johansson, 1970, 1974), but the most interest has been for protein (Albertsson, 1958, 1959; Sasakawa and Walter, 1972) partitioning. Numerous investigators have suggested that these systems can provide an innocuous environment for the purification of biomaterials (Albertsson, 1986; Veide et al., 1983; Walter et al., 1985; Hustedt et al., 1985; Mattiasson and Kaul, 1986). A general theory for predicting partitioning of proteins is currently not available. Ideally, one should be able to predict such behavior for a specific protein, a priori, from a set of general heuristics. This might prove difficult, due to the diversity and complexity of proteins and their interactions with phase system components. Moreover, the phase systems themselves are complex. Many factors are thought to influence partitioning, including concentrations of phase-forming components,molecular weight of polymers, temperature, pH, solute charge, and hydrophobicity (Albertsson, 1986; Ballard et al., 1979). The few models developed to describe aqueous two-phase behavior and partitioning have been derived from classical polymer solution thermodynamics. These include recent derivations using the Flory-Huggins lattice model (Diamond and Hsu, 1989a,b), a modified lattice model (Baskir et al., 19871, UNIQUAC (Kang and Sandler, 1987, 19881, a model based on statistical mechanics (Cabezas and Szlag, 1989), and an extension of the osmotic virial expansion (King et al., 1988). Although each of these 8756-7938/90/3006-0479$02.50/0
models has provided numerous insights into the behavior of aqueous two-phase systems, none has yet yielded relationships that permit selection of optimal systems for a specific protein separation. In order to understand complex protein behavior in aqueous two-phase systems, it may be useful first to examine the behavior of amino acids and peptides in such systems. For example, if the protein surface in contact with the phase system is the primary influence on partitioning, then the specific amino acid residues on the surface may determine the partitioning. Thus, an improved understanding of amino acid and peptide partitioning behavior may lead to a better understanding of protein partitioning. There are previous reports of the partitioning of peptides in aqueous two-phase systems. Sasakawa and Walter (1974) examined several amino acids and peptides. Most of the amino acids they examined partitioned essentially equally between the phases. However, they noted that hydrophobic and hydrophilic amino acids partitioned differently if sodium chloride or sodium sulfate was added to the PEG/dextran. They further noted that triglycine (Gly-Gly-Gly)had a partition coefficient slightly lower than Gly-Gly, which, in turn, was lower than the partition coefficient for glycine itself. More recently, Diamond and Hsu (1989b) partitioned a series of dipeptides, noting that increasing the number of methylene groups (-CHz-) on one of the residues increases the partition coefficient. For example, Gly-Gly has a partition coefficient less than GlyAla. The dipeptide Gly-Nval showed the largest partition coefficient in PEG/dextran, although all four dipeptides examined partitioned below unity. Addition of a methylene group appears to have the opposite effect of an addition of a Gly [as observed by Sasakawa and Walter (1974)]. This indicates that for these small molecules the partition coefficients might not be simply related to molecular weight or size. Other studies have more explicitly noted the importance of hydrophobicity in aqueous two-phase systems. Shanbhag and Axelsson (1975) suggested measuring hydrophobic interactions of proteins in aqueous environments by using aqueous two-phase partitioning. Zaslavsky et al.
0 1990 American Chemical Society and American Institute of Chemical Engineers
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(1978, 1981, 1984) suggested t h a t t h e hydrophobic properties of aqueous two-phase systems could be quantified by the use of a AGCH2value (Davis et al., 1972), which is the free energy of methylene group transfer between the phases. This free energy is a measure of the difference in partition coefficients in a homologous series of compounds between solutes differing in one chain length (-CH2-). Both previous studies of peptide partitioning used the PEG/dextran system with an added buffer. Although shown t o be innocuous for biochemical partitioning (Mattiasson et al., 1982), these systems generally have a low free energy, AGCHz (Zaslavsky et al., 1981). Therefore, differences between the partition coefficients of solutes varying only slightly in hydrophobicity may be difficult to distinguish. In order to magnify the effect of side groups on amino acid residues in peptides, a two-phase system should be selected in which the phase-forming components are more dissimilar. The PEG/magnesium sulfate aqueous two-phase system is a good choice for a system to use to study peptide partitioning because (1)the two phase-formingcomponents are dissimilar, resulting in a high AGCH2, and (2) the salt is itself mildly buffering and therefore produces solutions of different concentrations varying only slightly in pH. Thus, the pH is constant without the need for additional salts. The study reported here focuses on the partition of amino acids that do not carry a charged side chain and peptides comprising these amino acids in the PEG/ magnesium sulfate aqueous two-phase system, and the peptides selected cover a large range of hydrophobic character. Thus any effect of hydrophobicity observed with this system should be applicable to other aqueous two-phase systems.
from the phase diagram, or more conveniently, the concentration difference of one of the phase-forming components between the phases:
Mathematical Model
R T In K = ( a p+ Af)Aw2
Zaslavsky noted (1981, 1984, 1987) that in a twophase system of constant composition, the partition coefficients of a homologous series of compounds vary linearly:
The relative hydrophobicity should first be normalized by selecting a solute that is arbitrarily assigned a value of Af = 0. Actual partition coefficient data for this solute may then be used to calculate cyp from eq 6. Since the relative solute hydrophobicity, Af, is related only to the free energy of transfer, its value has additiveconstitutive properties. That is, the hydrophobicity for a dimer, for example, should be calculable from the hydrophobicity for the monomer minus the free energy lost by the removal of atoms by dimerization and also that lost by any self-interaction. Peptides, of course, are composed of amino acids linked by amide bonds. The hydrophobicity of a peptide containing m amino acids (4d should therefore be determined from the hydrophobicity of the individual amino acids:
R T In K = RTC + AGCH2n (1) where R is the gas constant, T i s the absolute temperature, C is a parameter that depends on the particular series of homologous compounds, and n is the number of -CH2groups. A change in the composition of the phases (Le., the location of the solution on the phase diagram) alters the values of C and AGcH2n. In eq 1,the value of AGCHz indicates the hydrophobicity of the side chain of that solute in the phase system, while RTC normalizes the partition coefficients for the particular homologous series of solutes in that phase system. Thus, RTC is related t o the hydrophobicity of the remaining fragment without -CHzgroups. The value of RTC may therefore be divided into two parts: RTC = RTc + AGR (2) AGR is the theoretical relative free energy of transfer of the fragment without -CH2- groups, and depends on the solute. RTc is that portion independent of the solute. The values of the parameters in eqs 1and 2 are valid only for a specific phase system composition. It would be preferable to express the partition coefficient in terms independent of the composition. Several previous studies (Albertsson, 1986;Diamond and Hsu, 1989a,b; King e t al., 1988) have noted that the partition coefficient is proportional to the tie-line length
In K = KoAwz (3) Here, Aw2 is the concentration (wt ?6 / w t 76)difference between the phases of the component that predominates the upper phase (taken to be component 2). KOis a proportionality constant that does not depend on the phase system composition. Equations 1-3 suggest that a more general relationship may be found for the partition coefficient in aqueous twophase systems. Specifically, eq 3 indicates that the partition coefficient may be expressed as the product of the two terms: one that depends on the solute and the general phase system selected and a second term that depends on the concentration of the components in the phase system. Thus the following may be defined: AfAw, = AGR + AGCH2n
(4) such that Af depends on the solute and the phase system but not on the concentrations of the components in each phase. For a system of specific concentration (Awp constant), Af is proportional to the theoretical free energy of transfer of the solute and thus is interpreted as being a relative measure of the solute hydrophobicity. The parameter c may also be related to the concentration difference between the phases: apAw2 = c (5) Here, a p would depend on the phase system but, as before, be independent of the concentrations of the phaseforming components. From eqs 1,4, and 5 (6)
m
Af = Af, = x A f i - ( m - l)AfH,-, - Af*
(7)
i=l
Aft is the relative hydrophobicity of the ith amino acid, AfHzo is the relative hydrophobicity lost by each condensation, and Af* is the total free energy (in terms of relative hydrophobicity) lost by interactions between amino acids on the same peptide. Equation 7 assumes that each peptide bond formation, with subsequent removal of water, is accompanied by an identical loss of hydrophobicity. In a large peptide or protein, the interaction term dominates the calculation of hydrophobicity. However, if the peptide is sufficiently short, the interaction term may be assumed to be negligible. In order to use eqs 6 and 7, a reference amino acid must be chosen. Since glycine has no side chains, this molecule
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is chosen to have a Af = 0. Partitioning glycine in a twophase system at different concentrations (different values of A W Z will ) permit the calculation of a value for a p from eq 6. Partitioning the dimer, Gly-Gly, and trimer, GlyGly-Gly, etc., should then result in a value for AfHzofrom eq 7, assuming Af* equal to zero. (Since glycine does not have any side-chain group, peptides containing only this amino acid are the most likely of all peptides to have a negligible interaction term, Af*.) In the study reported here, a series of amino acids was selected for partitioning in the PEG/MgS04 aqueous twophase system. After calculation of the value for ap,from the partition coefficients of amino acids, values may be obtained by eq 6 for the hydrophobicity, Afi, of each amino acid. With these values, eqs 6 and 7 may then be used to predict the partition coefficient of peptides in this phase system, assuming interactions between amino acids on a single peptide are negligible.
Materials and Methods Phase Systems. Poly(ethy1ene glycol) of molecular weight 8000 (PEG-8000) (Lot 49F-0383) and all peptides used in the partitioning experiments were purchased from Sigma Chemical Co., St. Louis, MO. Magnesium sulfate (MgSOg7HzO) (Lot 746031) was obtained from Fisher Scientific Co., Pittsburgh, PA. A series of solutions, each having a volume of 10.0 mL and containing 0.60-1.40 M MgS04, was prepared from a stock solution of 2.00 M MgS04 with distilled deionized water. To each of these solutions, 1.30 g of PEG-8000 was added. The resulting two-phase systems had pH values of 5.4-5.9. For density measurements, PEG concentration measurements, and partitioning experiments, the phases were thoroughly mixed and equilibrated at 25 "C (f0.05 "C) in a constanttemperature bath (Brinkmann Instruments Inc.) for about 4 days. A 10-mL pycnometer was used to measure density at 25 "C. The concentration of PEG was found by freezedrying each phase and then extracting PEG from the residue with warm acetone. Partitioning Experiments. A single peptide (5-30 mg) was added to 10.0 mL of separate two-phase solutions, and each system was equilibrated for about 4 days. The phases were then carefully separated with glass Pasteur pipets. Liquid chromatography was employed to determine the solute concentration in each phase. The partition coefficients for small molecules were found to be independent of solute concentration for the range of dilute solutions prepared. The HPLC system comprised a Waters gradient controller, UV detector, Model 481, and pumps, Model 510, with a Gilson Model 231 sample injector and Model 121 fluorometer and a Hewlett-Packard 3392A integrator. Amino acids and peptides with strong UV absorbances(e.g., Trp-Trp and Trp-Phe) were analyzed by reverse-phase chromatography with a Whatman 5-pm partisphere c8 column. Amino acids and peptides with poor UV absorbances (e.g., Gly-Gly and Leu) were analyzed by a derivatization method (Schmidt and McClain, 1987) using a Waters 5-pm Resolve Radial-Pak (28 column and the fluorometer. The standard error of the mean from the analyses amounted to 2 4 % with 4-6 injections per sample.
Results and Discussion Five solutions having different MgS04 and PEG concentrations were used throughout these experiments, and these are listed in Table I. The prime refers to the upper phase and the double prime to the lower phase. Several amino acids without side-chain charge were selected
Table 1. Summary of PEG/MgSOp7HzO Two-Phase System at 25 "C
PEG-8000, MgSOp7H20, wt%
wt%
0.109 0.107 0.105 0.103 0.101
0.138 0.181 0.221 0.260 0.299
p',
g/mL 1.0676 1.0664 1.0686 1.0718 1.0756
P",
g/mL 1.0987 1.1244 1.1466 1.1684 1.1900
AWZ,g/g of solvent 0.206 0.349 0.485 0.602 0.680
for partitioning studies, and the results are shown in Figure 1. The slope of each of these lines should be equal to the term ap + Af from eq 6, where ap is assumed to be constant for the phase system. Glycine was selected as having a Af value of zero. Therefore, from the glycine partitioning data, the value of ap can be calculated to be -2310 cal/ mol. This value indicates that, in this phase system, solutes with a relative hydrophobicity ( A f )greater than +2310 cal/ mol will result in partition coefficients greater than unity, while solutes with a relative hydrophobicityless than +2310 cal/mol will have partition coefficients less than unity. By using this value of ap,values of the relative hydrophobicity ( A f , for other amino acids may be calculated from their partition coefficients; these results are shown in Table 11. The correlation coefficients listed are calculated by a linear least-squares fit of the experimental data, by a line forced through a partition coefficient equal to unity at zero concentration difference. (Correlation coefficients will be lowest for compounds with partition coefficients near one, where a small error in measurement will cause a relatively large deviation from the theoretical line forced through this intercept.) The calculated values for the relative hydrophobicities indicate that tryptophan is the most hydrophobic of the amino acids studied. These hydrophobicities may be compared to other amino acid hydrophobicity scales. For example, Nozaki and Tanford (1971) constructed an amino acid hydrophobicity scale from solubility measurements. Figure 2 shows a comparison of their hydrophobicity scale and the hydrophobicitiesobtained from partitioning in the PEG/MgS04 aqueous two-phase system. General agreement exists between the two scales, although the scale of Nozaki and Tanford placed phenylalanine slightly above tyrosine in hydrophobicity, while the opposite was observed in the partitioning experiment. For the calculation of hydrophobicities for peptides (Af,), according to eq 7, a value is required for the loss in effective hydrophobicity due to the condensation of the amino acids. The value for AfH20 was determined by partitioning Gly-Gly and Gly-Gly-Gly (results shown in Figure 3). By using eq 7 and assuming Af* = 0, from the slope of diglycine data, the value of AfH20 was calculated to be +440 cal/mol, while from triglycine data, the calculated value was +515 cal/mol. Assuming an average value for AfHzo of +480 cal/mol, the partition coefficients of other peptides can be predicted by using eqs 6 and 7. A series of tryptophan-, tyrosine-, and phenylalanine-containing dipeptides were selected initially, and these results are shown in Figures 4, 5, and 6, respectively. (The relative hydrophobicities observed from the slopes of the lines, and the corresponding correlation coefficients, are shown in Table 111, as are the relative hydrophobicities calculated by using eqs 6 and 7.) The dotted lines on each of these figures indicate the partition coefficients that are predicted from eqs 6 and 7. In general, the observed partition coefficients agree with the predictions. The model consistently overpredicts the behavior of the alanine-containing dipeptides, suggesting that, if the model is correct, alanine interacts the most (of the amino acids studied) with the large adjacent residues.
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0.3- A
@
1 1 4
B
Bl
0
A
0.2 -
8
0 A
K
*
0.1 -
0
0 0
0.05 -
A I
0.2
I
I
I
0.03.-
I
0.3 0.4 0.5 0.6 0.7
Figure 1. Measured partition Coefficients ( K ) of amino acids versus the PEG concentration difference between the phases (Awd in the PEG/MgSOd system: tryptophan, O ; tyrosine, 0,phenylalanine, A, leucine, . ;alanine, .; glycine, A. Table 11. Observed Relative Amino Acid Hydrophobicity and Linear Least-Squares Correlation Coefficients in the PEG/MgSOd Two-Phase System at 25 O C obsd amino hydrophobicity, correlation acid cal/mol coefficient GlY 00 0.996 Ala 670 0.999 Leu 1760 0.965 Phe 2210 0.831 TYr 2490 0.773 TrP 3340 0.948
3oool
I
I
I
I
0
0
A
o
, Q,
Figure 3. Measured partition Coefficients ( K )of glycine, 0,diglycine (Gly-Gly),0,and triglycine (Gly-Gly-Gly),A,versus PEG concentration difference between the phases ( A m ) in the PEG/ MgS04 system.
50
t
20 -
K
10 -
5-
Set to zero.
0
2-
IC
0
2500
0
I
I
I
0.2 0.3 0.4
I
0.5
I
I
0.6 0.7
2000 -
0 1500 -
Figure 4. Measured partition coefficients ( K ) of tryptophancontaining dipeptides versus the PEG concentration difference between the phases (Awz) in the PEG/MgSO4 system: model tryptophan-tryptophan, W; tryptophan-phenylprediction, alanine, 0;tryptophan-alanine, A; tryptophan-glycine, 0. -a;
1000-
Af, Figure 2. Comparison of relative amino acid hydrophobicity (4i, cal/mol) with hydrophobicity scale (Aft, cal/mol) of Nozaki and Tanford (1971).
As Table I11 indicates, each tryptophan-containing dipeptide was more hydrophobic than the corresponding tyrosine-containing dipeptide, which in turn was more hydrophobic than the corresponding phenylalaninecontaining dipeptide. Moreover, as Figures 4-6 show, within each group of dipeptides, the observed partition
coefficients followed the single amino acid hydrophobicity scale: Gly < Ala < Leu C Phe < Tyr C Trp. The relative hydrophobicities calculated by eq 7 from single amino acid data varied only slightly from the observed hydrophobicities. The deviation between the observed and calculated partition coefficients might be taken to be a measure of self-interaction on the peptide chain. These interaction energies, determined by the difference between the calculated and observed partition coefficients, are shown in Table IV. The next step was to examine the validity of the same model for larger peptides. Fragments that compose the peptide leucine enkephalin were selected for these experiments. Figure 7a shows the results for three tripeptides, while Figure 7b shows results for two tetrapeptides and a pentapeptide. The dotted lines again indicate
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10
5
K 2
1
0.7 I
I
I
I
0.2 0.3 0.4 0.5
I
I
0.6 0.7
aw2 Figure 5. Measured partition coefficients ( K ) of tyrosinecontaining dipeptides versus the PEG concentration difference between the phases (Awz) in the PEG/MgS04 system: model prediction, tyrosine-tyrosine, 8;tyrosine-phenylalanine, 0;tyrosine-alanine, A;tyrosine-glycine, 0.
.
-e;
I
_.
I
Table 111. Calculated and Observed Relative Peptide Hydrophobicity and Linear Least-Squares Correlation Coefficients of the Observed Hydrophobicity in the PEG/ MgSOa Two-Phase System at 25 OC calcd hydrophoobsd bicity, (ZAfifi)- hydropho(m- l)Af~,o, bicity, Afm, correlation peptide cal/mol cal/mol coefficient Gly-GlyO -440 -440 0.993 Gly-Gly-Glp -1030 -1030 0.900 2860 2980 0.939 Trp-Gly 3530 3060 0.989 Trp-Ala 5070 4940 0.995 Trp-Phe 6200 6010 0.954 Trp-Trp 2010 2000 0.944 Ty-Gly 2680 2050 0.880 Tyr-Ala Tyr-Phe 4220 4140 0.990 4500 4230 0.989 Tyr-Tyr 1730 1870 0.920 Phe-Gly 2400 1940 0.653 Phe-Ala 3490 3160 0.986 Phe-Leu 3940 3900 0.996 Phe-Phe 1530 1640 0.920 Tyr-Gly-Gly 0.998 Tyr-Gly-Gly-Phe 3260 4170 0.950 Tyr-Gly-Gly-Phe-Leu 4540 4640 0.853 Gly-Phe-Leu 3010 2920 0.974 Gly-Gly-Phe 1250 1620 2530 2990 0.886 Gly-Gly-Phe-Leu 0 Calculated and observed relative hydrophobicities equated to determine A f ~ , o .
Table IV. Calculated Interaction of Side Chains of Peptides in the PEG/MgSOd Two-Phase System at 25 OC interaction, interaction, At*, cal/mol peptide Af*, cal/mol peptide Phe-Gly -80 Gly-Gly P Phe-Ala +460 Gly-Gly-Gly 0" Phe-Leu +330 Trp-Gly -120 Phe-Phe +40 Trp-Ala +470 Tyr-Gly-Gly -110 Trp-Phe +130 Tyr-Gly-Gly-Phe -910 Trp-Trp +190 Tyr-Gly +10 Tyr-Gly-Gly-Phe-Leu -100 Gly-Phe-Leu +90 Tyr-Ala +630 -370 Tyr-Phe +80 Gly-Gly-Phe Tyr-Tyr +270 Gly-Gly-Phe-Leu -460 Set to zero. I
I:
a
2
15
Figure 6. Measured partition coefficients ( K ) of phenylalaninecontaining dipeptides versus the PEG concentration difference between the phases (Awz) in the PEG/MgSO4 system: model prediction, phenylalanine-phenylalanine, 8; phenylalanineleucine, 0;phenylalanine-alanine, A;phenylalanine-glycine, 0.
0
1'
e-;
the model predictions. The model predicts the partition coefficients well for both relatively hydrophobic and hydrophilic compounds. Interestingly, the tetrapeptide TyrGly-Gly-Phe (Figure 7b, 0)is poorly predicted by the model. However, the model prediction for Tyr-Phe (dashed line in Figure 7b) appears to coincide with the observationsfor Tyr-Gly-Gly-Phe. Thus, it is possible that the presence of two large hydrophobic residues on the ends of this tetrapeptide effectively removes the influence of the two intermediate glycine residues.
Conclusions The partition coefficients of amino acids and peptides in the PEG/MgS04 aqueous two-phase system may be correlated with the hydrophobicity of the solute. The
K
Figure 7. Measured partition coefficients ( K ) of leucine enkephalin fragments versus the PEG concentration difference between the phases (Awz) in the PEG/MgS04 system. Model prediction, (a) Tyrosine-glycine-glycine, 0;glycinephenylalanine-leucine, 0;glycine-glycine-phenylalanine, A. (b) Tyrosine-glycine-glycine-phenylalanine-leucine,0;tyrosineglycine-glycine-phenylalanine, 0;glycine-glycine-phenylalanineleucine, A; prediction for tyrosine-phenylalanine, - - -. a-.
partition coefficients for amino acids were used-to calculate their relative hydrophobicities, A f , assuming glycine to have a hydrophobicity of zero. These calculated hydropho-
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bicities show agreement with amino acid hydrophobicities determined previously. These calculated hydrophobicities were incorporated into the model to predict partition coefficients for peptides, with good agreement.
Acknowledgment We are grateful to the National Science Foundation Graduate Fellowship Program and the Virginia Center for Innovative Technology for support for this research.
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