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Hydrophobic partitioning of proteins in aqueous two-phase systems
Hydrophobic partitioning of proteins in aqueous two-phase systems F. Hachem, B. A. Andrews, Biochemical
Engineering
and J. A. Asenjo
Laboratory,
University of Reading, Reading, United Kingdom
The hydrophobicity of five proteins was estimated by reversed-phase chromatography (RPC). hydrophobicinteraction chromatography (HIC), and precipitation with ammonium sulfate. These data were correlated to partition behavior in aqueous two-phase systems. A parameter (I/m*) that was derived from the precipitation curves is based on the solubility of proteins in an electrolyte solution. The correlation between l/m* and the retention times in HIC and RPC was poor due to interaction effects with the chromatography matrices and probably partial unfolding of the proteins: however, this parameter (I/m*) was expected to be a measure of hydrophobicity of proteins that relates better than the chromatographic data to experiments where the hydrophobic behavior of proteins in an aqueous solution is used for their separation. The partition behavior of the five proteins in aqueous two-phase systems (ATPS) in the absence and presence of NaCl was investigated. A poor correlation was found between log K (K is the partition coefficient) in ATPS and the hydrophobicity values measured by RPC and HIC; however, a very good correlation was found between log I/m* which is a measure of protein hydrophobicity based on the solubility of the protein during precipitation and log K, particularly in PEG/PO, systems with added NaCl. The parameter (I/m*) also demonstrated a good correlation with log K in PEG/dextran systems. A simple correlation for the prediction of partitioning in specific ATPS based on this parameter has been evaluated. An expression describing its resolution power, R, and a parameter describing the hydrophobic& of the system, P, was determined making the correlation potentially predictive for other proteins in the ATPSs used. Hydrophobicity of proteins was better exploited in PEG/PO, 0 1996 by systems than in PEG/dextran ones as a much higher resolution (R) is obtained in the former. Elsevier Science Inc.
Keywords: Hydrophobicity;
chromatography;
solubility:
aqueous two-phase
Introduction Evaluation
of hydrophobicity
Methods used to quantify the hydrophobicity of proteins can be divided into two groups. The first group measures the total overall hydrophobicity while the second measures the surface hydrophobicity or the functional hydrophobicity of a protein. In the first category, the amino acids which make
Address reprint requests to Professor J. A. Asenjo, Department of Chemical Engineering, Universidad de Chile, Centre for Biochemical Engineering and Biotechnology, Beauchef 861, Santiago, Chile The present address of B. A. Andrews is the Centre for Biochemical Engineering and Biotechnology, Department of Chemical Engineering, University of Chile, Beauchef 861, Santiago, Chile Received 5 October 1995; revised 12 November 1995; accepted 10 January 1996
Enzyme and Microbial Technology 19:507-517,1996 Q 1996 by Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010
partitioning;
correlation
up the protein are given hydrophobic values that stand, in most cases, for the free energy of transfer of the side chain from an aqueous solution representing the surface of a protein to an organic solution (ethanol, octanol, hexane) that mimicks the interior of the protein. The total hydrophobicity of the protein is calculated by simple addition of the hydrophobic values of each constituent amino acid’ or an average hydrophobicity is calculated by dividing this sum by the number of amino acid residues in a protein to give what is called the Bigelow number.’ With the discovery by Lee and Richards3 that the surface of globular proteins also contained nonpolar amino acids, interest in the hydrophobic interactions between a protein and its environment increased which resulted in the emergence of methods that quantify the surface hydrophobicity rather than the total overall hydrophobicity. Of the methods that measure surface hydrophobicity, there are the hydrophobic probe meth-
0141-0229/96/$15.00 PII SU141-0229(96)00062-2
Papers
Materials and Methods
ods, the hydrophobic binding methods, reversed-phase chromatography (RPC), and hydrophobic interaction chromatography (HIC). With the advent of chromatographic systems such as HPLC and F’PLC which reduce the time involved in specific measurements, chromatography as a method for measuring the surface hydrophobicity of proteins became popular.
Partitioning
P-lactoglobulin A, a-lactalbumin, bovine serum albumin, oonalbumin, and hen egg lysozyme were purchased from Sigma (Poole, England). Lysozyme was a gift from STC Laboratories Inc., Winnipeg, Canada. Table I shows the isoelectric points and molecular weights of the proteins. Acetonitrile was HPLC grade and trifluoroacetic acid (TFA) was SpectrosoL grade. All solvents were filtered through a 0.45 pm filter and degassed prior to use. All other reagents used were the highest purity available.
in aqueous two-phase systems
The partition coefficient of a protein in an aqueous twophase system (ATPS), K, is defined as C& where C, and C, represent the equilibrium concentrations of the partitioned protein in the top and bottom phases, respectively. It is possible, therefore, to separate a mixture of proteins by choosing a system where the respective K values of the proteins are widely different. The partitioning of molecules between the two phases is a complex phenomenon because of the involvement of many factors in the interactions between the solute and the phase-forming components like hydrogen bonding, charge interactions, van der Waals forces, hydrophobic interactions, and steric effects. This makes the molecular weight and chemical properties of the polymer and the size and the chemical properties of the partitioned solute of extreme importance. Since the surface of a protein is made of different types of amino acids, two properties of the protein surface, namely its charge and hydrophobicity, play important roles in the partition of proteins in ATPS. If systems are chosen where the effect of the potential difference on the partitioning of proteins is nullified, factors other than charge dominate, especially the hydrophobic effect. To make such systems, neutral salts are usually added at very low concentration to a PEG/dextran system. Under such conditions, Johansson and Hartman found that most proteins maintain the same K value independently of pH. Due to the complexity of the partitioning phenomenon, it is difficult to predict protein behavior and select separation conditions for the rational planning of experiments. Models that can predict separation a priori are, therefore, very useful; however, most of the models available to date involve a large number of parameters to be calculated which hinders their utilization in bioseparations. The model of Eiteman and Gainer5 is promising since it relates the partition coefficient to two parameters, the hydrophobicity of the solute and the weight difference of one of the polymers between the phases. Both can be easily measured. The first of these parameters, namely the protein hydrophobicity, however, might pose a problem because there are many methods normally used for measuring it and in many cases the values obtained correlate poorly. In this paper, we compare two modes of chromatography widely used to measure the functional hydrophobicity of proteins, RPC and HIC, and also introduce a parameter for measuring this property (l/m*) that differs from the former ones in that it is a measure of functional hydrophobicity in solution without the aid of a matrix. These methods are then evaluated as a means of estimating the hydrophobic behavior of proteins in ATPS. 508
Materials
Enzyme Microb. Technol.,
Equipment
and methods
The HPLC system employed for RPC consisted of a Perkin Elmer Binary LC pump 250 (Norwalk, CT) and a Rheodyne Model 7 125 valve equipped with a 20 ~1 sample loop. The experiments were performed on a Nucleosil 300-7 Cl8 reversed phase column (length, 25 cm; i.d., 4.6 mm; Hichrom, Reading, UK). It had a pore size of 30 nm and a particle size of 5.7 + 1.5 pm. The effluent of the column was monitored at 280 nm using a UV-VIS Sepectroflow 757 spectrophotometer with a Kratos analytical detector. The chromatograms were obtained under gradient elution at a flow rate of 1 ml min-’ and at ambient temperature. After each run, the column was equilibrated for a period of approximately 20 min. with 10% eluant A. The profile of the gradient was as follows: 3 min at 10% A after which the sample was injected; 30 min from 10% to 100% A; and 3 min from 100% to 10% A where A was acetonitrile + 0.1% TFA (v/v) and B was water + 0.1% TFA (v/v). Proteins were dissolved in solvent B at a concentration of 1 mg ml-’ except for lysozyme which was dissolved at a concentration of 0.5 mg ml-‘. All samples were filtered through a 0.2 pm filter before application to the column. Reported retention times are the average of four replicates. Values obtained were within * 5%. Fast protein liquid chromatography (FPLC, Pharmacia) was used for HIC. The column HR 5/5 (1 ml) was packed with lowsubstituted, fast-flow Phenyl Sepharose (Pharmacia). Detection was carried out at 280 nm for all proteins and the gradient employed was: 3 min at 100% 1.5 M ammonium sulfate in 0.05 M sodium phosphate buffer pH 7.0; 30 min linear gradient from 1.5 M to 0.0 M ammonium sulfate in the same buffer; and 3 min at 100% sodium phosphate buffer. Proteins were dissolved in buffer and filtered (0.2 km filter) before elution at a flow rate of 1 ml mini. The column was considered equilibrated when a stable baseline was obtained. Retention time is reported as the average of 4-5 runs. For calculating the average capacity factors of the proteins, the method of Quarry et ~1.~ was followed which involved eluting the proteins using the same column and elution conditions under two different gradient run times. In the RPC experiments, the gradient run times employed were 30 and 17 min whereas in the HIC experiments, they were 30 and 15 min. The value of t,, (dead retention time) was found from the spike at which the change in
Table 1 Molecular the proteins
weights (MW) and isoelectric points (pl) of
Protein a-Lactalbumin P-Lactoglobulin A Bovine serum albumin Conalbumin Lysozyme
1996, vol. 19, November
15
MW (Da) 14,000 37,100 66,500 77,000 14,300
PI 4.2-4.5 5.13 4.9 5.88 10.3
Hydrophobic the mobile front occurred, and t, (delay time before the system starts the gradient) was found by running a gradient with a UVabsorbing solvent (acetone) as one of the two mobile phase solvents. For the HPLC-RP system, to and t,, were 3.40 and 3.35 min, respectively. For the FPLC-HIC system, the values were 1.2 and 0.6 min, respectively.
Precipitation
of proteins
Proteins were dissolved in 0.05 M sodium phosphate buffer pH 7.0 at a concentration of 2 mg ml-‘. To a measured volume of each solution, solid ammonium sulfate was added slowly and with continuous stirring to reach a certain saturation according to Scopes.’ After all of the salt had dissolved, the solutions were left to equilibrate for I5 min in a water bath at 24°C. After centrifugation (25,000 g for 20 min), an aliquot of the supematant was assayed for protein concentration. To the rest of the supematant, more ammonium sulfate was added, thus making the concentration higher. Again, the solution was left to equilibrate, was centrifuged, and the new supematant was assayed for protein after dilution. A blank was prepared for each level of saturation and diluted in the same way as the solution containing the protein.
Protein determination Protein was measured by a modified Bradford dye-binding assay.8 Standard curves were prepared for each protein. Blank systems were made to account for the interference of ammonium sulfate. For the PEG/dextran systems, absorbance of the diluted top and bottom phases were measured at 280 nm against a blank. Concentrations of the proteins were calculated from standard curves in which absorbance at 280 nm was plotted against increasing concentration of the same protein.
Phase systems PEG of molecular weight 8000 was purchased from Sigma while analytical grade K,HPO, - 3H,O and NaH,PO, * 2H,O were purchased from BDH Phase systems with compositions of 8% (w/w) PEG 8,000 and 12% (w/w) phosphate were prepared from stock solution of PEG (50% w/w) and phosphate solution (40% w/w). The phosphate solution was pH 7.0. Sodium chloride was added to attain the following final concentrations in the system: 0,0.48,4.8, 9.6, and 17.6% (w/w). In the 17.6% (w/w) system, the salt was added as a solid. The protein concentration in the initial stock solution was 2 mg/ml-‘. Low speed centrifugation was used (1,200 g, for 5 min) to accelerate the phase separation after gentle and thorough mixing of the system. Samples from the top and bottom phases were assayed for protein concentration. All partition experiments were done at room temperature. For the experiments on the effect of molecular weight on the separation of proteins, two PEG/phosphate systems were chosen. One contained 4.8% (w/w) sodium chloride and the other contained 9.6% (w/w) sodium chloride. Additional proteins used include immunoglobulin G (human, 150,000 Da), ol-amylase (Bacillus subrilis, 55,000 Da), ovalbumin (chicken egg, 45,000 Da), trypsin (bovine pancreas, 23,800 Da), transfenin (human, 77,000 Da), and glutathione reductase (Bakers’ yeast, 100,000 Da). All proteins were purchased from Sigma. PEG/dextran systems were prepared containing 4% PEG, 5% dextran. 0.15 M (0.73% w/w) NaCl, and 0.01 M sodium phosphate buffer.
Results and discussion Measuring
the hydrophobic@
of proteins
Hydrophobicity by chromatography. Five globular teins
of different
molecular
weights
and isoelectric
propoints
partitioning
of proteins:
F. Hachem
et al.
(Table 1) were eluted in RPC and HIC. In addition to being a powerful separating and purifying technique, RPC has been used as a method to determine the hydrophobicity of
peptides and proteins. g*lo More hydrophobic proteins will have higher retention times; the retention time is lower with higher concentrations of the modifier. In the case of HIC, the high concentration of the salt in the initial buffer may cause a small salting-out of the protein from solution but also an increase in the hydrophobic interactions with the hydrophobic tails of the matrix in the chromatography column causing adsorption; therefore. solutes that are hydrophobic bind strongly to the matrix and elute at lower concentrations of the salt when a decreasing gradient is used. In both RPC and HIC, the way to quantify the hydrophobic character of the solute is by reporting their retention times (or volumes) or their respective capacity factors. The capacity factor is the standardized retention quantity which normalizes the effects of the dimensions of the column usually used for isocratic elution. In the case of gradient elution which is often used for proteins, an average capacity factor, k”, can be used. This factor can be calculated from retention times obtained using two different gradients.h.” Table 2 shows the retention times (tR) and average capacity factors (K’) for RPC and HIC for the five proteins. From the elution profiles (Table 2), it is observed that the order of hydrophobicity of the proteins is different when comparing RPC values of retention times to those of HIC, e.g., BSA, which elutes second in RPC, elutes last in HIC. A similar behavior was observed for values of the capacity factors, K’. This type of behavior has been obtained by Fausnaugh et al. I2 who compared the retention of 12 proteins on a phenyl-acetyl column CHIC) and a Synchropack RP-8 column (RPC) and found that proteins which were more hydrophilic on the HIC column (e.g., cytochrome c and myoglobin) were retained by RPC (less hydrophilic). On the other hand, P-glucosidase. a protein that adsorbed to the phenyl-acetyl column, was only weakly retained by the RP-8 column. Disaccordance in the elution order has also been observed when milk proteins were chromatographed using a phenyl-Superose column.” a C-8 column,14 and a C-6 column. I5 The difference in elution behavior of a protein between RPC and HIC can be explained by the different conformational changes which proteins undergo when interacting with the matrices and the mobile phase. Although the two
Table 2 The retention times (tR) average capacity factors (I’) of the different proteins on RPC-Cl8 and I-W Phenyl Sepharose columns during a 30 min gradient RPC Protein a-Lactalbumin @-Lactoglobulin A Bovine serum albumin Conalbumin Lysozyme
Enzyme Microb. Technol.,
HIC
fR (min)
K
tR (min)
K
20.5 21.7 19.8 20.0 19.4
1.53 2.48 2.23 1.67 1.77
14.0 10.3 16.3 8.6 10.4
4.83 5.43 4.58 3.78 6.69
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509
Papers
,
-21 Concentration
A
3
2
1
I
4
of ammonium
Concentration
sulphate
084 7
2
3
of ammonium
4
sulphate
084 -
02 -
o,o-
h
c 29 ul
4
C
-0,2
-
-0,4
-
-0,6
-
-033
3
2‘4
Concentration
2.6
2‘0
of ammonium
Concentration
3,2
3,4
Concentration
4
of ammonium
sulphate
modes of chromatography exploit the hydrophobicity of the proteins, they may have different selectivity. In RPC, the harsh conditions employed (organic modifkrs and a very hydrophobic and dense stationary phase) lead to the partial denaturation of the protein structure. In contrast in the HIC mode, the mild hydrophobic surface and the high antichaotropic salt concentration that stabilizes the protein structure result in the separation of the proteins in their native forms. Hydrophobicity by precipitationkolubility. In the process of precipitating proteins by the addition of electrolytes, a small increase in the concentration of salt transfers the proteins from being relatively soluble to being largely precipitated; l6 therefore, it is essential to know the concentration of salt at which a protein starts to precipitate which is done by plotting the logarithm of solubility against the concentration of salt. 570
Enzyme Microb. Technol.,
2
3,6
sulphate
3
2 E
3,0
4
3
of ammonium
sulphate
Figure 1 Protein solubility curves (log S) as a function of concentration (MI of ammonium sulfate at 25°C. Conalbumin, A; lysozyme, B; BSA, C; p-lactoglobulin, D; and a-lactalbumin, E
It should be noted that the shape of the solubility curve is determined by the interaction between the surface of the protein and the salt used. The salubility curves of the five proteins used in this study consist of two segments (Figure I). The first corresponds to a region where protein solubility changes very little with the concentration of salt. This behavior is observed at low concentrations of protein (e.g., l-2 g 1-l) such as the work reported in this paper. At high concentrations of protein, a salting-in region is observed at low salt concentrations where protein solubility increases with salt concentration.9 The second segment corresponds to the salting-out region in a way consistent with theory. The concentration of salt at which the discontinuity occurs (point at which the protein starts precipitating given a fixed initial concentration, e.g., 1 g 1-l) serves as a good reference for measuring the solubility or hydrophilicity of a protein; therefore, the inverse of the concentration of salt at
1996, vol. 19, November
15
Hydrophobic
partitioning
of proteins:
F. Hachem
et al.
showed how the slope of the solubility curve changed sharply at a certain concentration. Values of dS/dM are obtained as follows: ‘I log S = p - K, M d(log S)/d(M)
(2)
= - K,
(3)
since d(ln S) = d(S)/S and d(ln S) = 2.303 d(log S) (4) then 0.2
I
I
I
I
16
17
18
19
time
(min)
0.2 _I-____/ 6
8
10
Retention
12
time
14
16
(min)
Figure 2 Correlation between the corrected retention times in RPC and HIC and the experimental value of l/m*for the proteins studied. The abbreviations are: Lac, a-lactalbumin; Lag, 8-lactoglobulin; BSA, bovine serum albumin; Conal, conalbumin; and Lys, lysozyme. RPC using a C-18 column, (A); and HIC using Phenyl Sepharose (B)
this discontinuity point could be used as a good indicator of the protein hydrophobicity in solution. We have called the concentration of the salt at the point of discontinuity, m*, after Przybycien and Bailey. I7 The value is used as a comparative measure of the solubility or hydrophilicity; its inverse is a measure of the hydrophobicity of the proteins in solution (l/m *). Precipitation of proteins occurs at relatively high salt concentrations (above 1.0 M). At such concentrations, charge interactions of proteins are neutralized. Przybycien and Bailey17 found the value of m* on the solubility curve by the following expression: m* = (p - In S,)/ K,
S)
(5)
and
! 15
Retention
d(log S) = d(S)/(2.303
(1)
where S, is the initial concentration of the protein and p and K, are parameters from the equation of solubility in the salting-out region. To find the value of m* experimentally, the solubility data on the five proteins was fitted into two equations; one for the region to the left of point m* which corresponds to the region reflecting a very small effect of the salt on the solubility of the protein and one for the saltingout region. The point of intersection of the two curves determines the salt concentration, m* (Figure I). By differentiating the solubility curve (&Y&4), Stanley I8
d(S)/d(M) = -2.303 S K, (6) when d(S)ld(M) is plotted as a function of the molarity (M), a good value of the discontinuity m* is thus obtained.’ ‘.I9 In Table 3, the parameters derived from the solubility curves for the five different proteins are shown. There is very good agreement between the values of m * as calculated from the salting-out curve by equating S to the initial concentration of the protein in solution (m*_,) and as derived when the two segments of the solubility curve are fitted to two separate curves (m*,,J. Also, the values of m* derived from the differential graphs using d(S)/d(M) (m*& are very close to the values determined experimentally and by calculation. In most cases, m*diff is closer to m*,_. The value of the parameter m* can be easily determined from experimental data and thus becomes a useful parameter for determining the solubility (and thus hydrophilicity) of a protein in salt solutions and, as already discussed, we will use l/m* as a measure of the protein hydrophobicity in solution. The parameter l/m* was compared with the hydrophobicity values for the same proteins obtained from RPC and HIC as shown in Figure 2. These Figures show that there was little or no correlation between the hydrophobicity values of the proteins determined by RPC and the values of the parameter l/m* for the five proteins under study. In the cases of HIC, the hydrophobicity values of the proteins also did not correlate with those of I/m*. The retention values from an HIC column reflect the interaction between the salt, the matrix, and the protein while the value of l/m* reflects the interaction between the salt and the protein only. Using the normalized retention data, i.e., the capacity factors, a small improvement in the correlation between K’ and I/m* was observed in the case of RPC but not in the case of HIC” (data not shown). The correlation of the hydrophobicity measured by RPC, Table 3 The salting-out constant (KS), the extrapolated solubility to zero molarity of ammonium sulfate (6), and the point of discontinuity between the two segments of the solubility curve determined experimentally (m*,,,,), calculated (IT-I’,,,), and derived from the differential graphs (m*&
KS
P
mxmp
m*cal
m*dirr
Protein
M-’
g I-’
M
M
M
a-Lactalbumin 8-Lactoglobulin A Bovine serum albumin Conalbumin Lysozyme
1.82 2.48 2.09 1.68 1.14
5.34 8.79 6.87 4.79 2.89
2.83 3.48 3.21 2.90 2.39
2.77 3.42 3.14 2.67 2.26
2.84 3.49 3.20 2.59 2.33
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511
Papers HIC, and precipitation with the hydrophobic behavior proteins in ATPS is studied in the next section.
Partitioning in aqueous two-phase evaluation of hydrophobicity
centrations >4% (w/w) added salt (NaCl) and a-lactalbumin prefers the upper phase when the salt concentration is approximately 14% (w/w) while BSA, B-lactoglobulin A. and conalbumin prefer the lower salt-rich phase at all concentrations of NaCl. Johanssonz3 has noted that in PEG-salt systems, most proteins strongly favor the lower salt-rich phase. This is especially true when the molecular weight (MW) of the polymer is high because with increasing MW, the concentration of the protein and the salt will increase in the “free” solvent in the polymer-rich phase causing a strong partition of the protein to the lower phase.24 With the use of PEG 8,000 in the polymer phase, it is not surprising that all of the proteins resided in the lower salt-rich phase at 0% (w/w) NaCl. The same result was obtained by Cascone el &.*”
of
systems and
Previous publications have shown that the partition coefficients of certain proteins can be increased quite dramatically when high concentrations of NaCl are added to ATPS”)-‘* whereas others are not much affected by this. This effect has been attributed to possible hydrophobic interactions. Hence, ATPSs with different levels of NaCl in the range O-17.6% were chosen. Figure 3 shows that proteins behave differently when the concentration of NaCl is increased in the system. Lysozyme prefers the upper PEG-rich phase at con-
1
“II 2-
1
0
x
X
4
4
-1
-1 -
-2d
-3,
Concentration
A
0
20
of NaCl (Ohwh)
10
Concentration
B
X
X
4
3 -2 -
-22
-31
-31
of N&l
20
(OAw/w)
1
0
10
Concentration
C
20
of NaCl (% w/w)
0
D
10
Concentration
20
of NaCl (Oh w/w)
‘I
0
X
4 - .
lb
0 E
512
Concentration
Enzyme Microb.
2b
of NaCl (% wb)
Technol.,
Figure 3 The effect of NaCl concentration on the partition coefficient of proteins in a system containing 8% PEG 8,000 and 12% PO, of pH 7.0. Each point on the curve represents the average of 3-4 experiments cariied out in duplicate ‘(results were within f 5%). Lysozyme (A); BSA (B); 4actalbumin (C); P-lactoglobulin (D); and conalbumin (E)
1996, vol. 19, November
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Hydrophobic when partitioning thaumatin in PEG 6,OOO/PO, systems. In the first instance with no NaCl added, thaumatin preferred the lower phase; however, increasing the NaCl concentration resulted in a drastic increase in its partition coefficient. To investigate the effect of the hydrophobicity of proteins, the logarithms of the partition coefficients for the five proteins in the different PEG/PO, systems used were plotted against the logarithms of the volumetric fractions of organic solvent at which these proteins eluted from a RP-C 18 column (log p). the logarithms of the inverse of the molar salt fractions at which they eluted from an HIC phenyl Sepharose column (as a measure of hydrophobicity), and the logarithms of the parameter proposed to measure hydrophobicity in solution (I/m*). The results are shown in Figures 4, 5, and 6. Volumetric fractions of organic solvent in RPC will be referred to as hydrophobicity since more hydrophobic proteins will elute at higher solvent concentrations in gradient elution. Similarly, the inverse of the molar fractions of salt in HIC will be used as a measurement of hydrophobicity since more hydrophobic proteins will elute at lower salt concentrations in a (decreasing) gradient elution. Figure 4 shows that a good correlation does not exist between log p and log K in the PEG/PO, systems used. This indicates that the hydrophobicity determined by RP-HPLC does not correlate with the protein partitioning behavior in the ATPS used for the five proteins under study. To verify whether or not the method used to determine hydrophobicity was the cause of the poor correlation, HIC using phenyl Sepharose as the hydrophobic matrix and a decreasing gradient of (NH&SO, was used. The results are shown in Figure 5. In these Figures, the hydrophobicity of the proteins is taken as the inverse of the molar fraction of salt needed for their elution with the initial salt concentration of 1.5 M representing 100%. This method for measuring hydrophobicity, which was successful for evaluating the hydrophobicity of a family of chemically modified proteins with different hydrophobicities’” but with the same charge and MW, did not improve the correlation between hydrophobicity of the proteins and the partition coefficient (Figure 5) for the five proteins studied. The poor correlation between the hydrophobicity measured by RP-HPLC and K is not totally surprising in view of the fact that the use of organic solvents in the former leads to at least partial denaturation of the native proteins which affects the opening up of the molecule and the exposure of the hydrophobic residues that were previously hidden in the core of the protein. In the case of HIC, the proteins are not denatured but hydrophobicity depends on the interaction between the matrix used and the hydrophobic patches on specific areas on the protein. Hence, distribution of localized hydrophobic patches on the surface may be playing a role. Figure 6 shows the correlation between log (l/m*), which corresponds to the evaluation of hydrophobicity in solution by precipitation, and log K. There exists a good linear relationship between the hydrophobicity of the proteins measured as a function of their solubility and their partitioning coefficient in systems of PEG/salt with a high concentration of NaCl for the five proteins studied. This strongly suggests that the ATPSs of PEG and salt with a high concentration of NaCl principally exploit hydrophobic
partitioning
of proteins:
F. Hachem
et al.
O-
x
Y
*Lys
he m
Q
-1 -
W
Cond El -2 -
q
%&
-3)
-0.24
-0.23
-0.22
-0.21
-0.20
-0.19
-0.18
1% p
A I
X
4
.Lys ImC
0
-1 -
0 BSA 0 0
-2 -
-3-m -0.24
-0.23
-0.22
2 1
4
Q
-0.21
-0.20
-0.19
-0.18
-0.20
-0.19
-0.18
log p
B
X
w
Cord
Ws 0 i
I
0-l -1
Cond
1
-0.24
0
-0.23
C
-0.22
La q
-0.21
1% p
Figure 4 The relationship between log K of the five proteins in a two-phase system (8% PEG and 12% PO,, at pH 7.0) and hydrophobicity (log p) in RPC-HPLC. Abbreyiations same as in Figure 2. 0% (w/w) NaCl (A); 0.48% (w/w) NaCl (B); and 9.8% (w/w) NaCl (C)
differences for partitioning and that the parameter m *, originally proposed as a measurement of protein solubility, is also a good estimator of protein hydrophilicity/hydrophobicity in solution. On the other hand, the results show that the correlation is very sensitive to ahanges in the system. An increase of as little as 0.48% (w/w) of NaCl in the PEG/phosphate system increased the correlation coefficient between log (l/m*) and log K from 0.457 to 0.837. The results show that the concentration of added NaCl need not be high in order to promote hydrophobic interactions
Enzyme Microb. Technol.,
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Papers Table 4 The calculated values of f? and the intrinsic hydrophobicity, log PO, of the ATPSs at 20°C Two-phase 8% 8% 8% 8% 8%
-31
0.3
0.2
0.1
0.4
log (wu
A
PEG, PEG, PEG, PEG, PEG,
system (wt%)
12% 12% 12% 12% 12%
PO, PO, PO, PO, PO,
+ + + +
0.48% NaCl 4.8% NaCl 9.6% NaCl 17.6% NaCl
R 5.4 8.3 14.9 22.4 22.8
log
po
-0.23 -0.29 -0.38 -0.45 -0.45
show that by adding NaCl to a PEG/phosphate system, the hydrophobic difference between the phases is increased and NaCl may also increase the hydrophobic interaction between the protein and the PEG.
A correlation
x 4
A correlation for predicting the partition coefficient of proteins in ATPS that takes into account the effect of hydrophobicity has been proposed”,25J6
Conal Q
-2
0
log K = R log(P/P,)
(7)
log K = R log P - R log PO
(8)
or -31 0.2
0.1 B
0.3 log
(
0.4
l/M)
2G l0
24 w 0
-1
-
-2
1
hC
Cond m
a
BSA ID
w 0
1 -3 C
! 0.1
I 02
I 0.3 log
I 0.4
(1/M)
Figure 5 The relationship between log K of the five proteins in a two-phase system (8% PEG and 12% P04, at pH 7.0) and hydrophobicity in HIC, log (l/molar fraction of ammonium sulfate) at which the proteins eluted from a Phenyl Sepharose column. Abbreviations same as in Figure 2. 0% (w/w) NaCl (A); 0.48% (w/w) NaCl (B); 9.6% (w/w) NaCl VIZ)
between PEG and the proteins which is confirmed by an almost doubling of the correlation coefficient between O-0.48% (w/w) NaCl for the five proteins studied in PEG/ phosphate systems. Since the values of the other correlation coefficients fluctuate around 0.813 at 4.8% salt, 0.915 at 9.6%, and 0.857 at 17.6%, it might well be that the small addition of 0.48% (w/w) NaCl brings the unstructured water zone to the concentration of salt where all the protein is salted-out. It can be seen in Table 4 that the resolution for hydrophobicity of the ATPSs, R, increases with increasing NaCl concentration between 0.48-9.6%. These results 514
Enzyme Microb. Technol.,
where P = (l/m*) is the protein hydrophobicity in solution measured by precipitation and log POrepresents the intrinsic hydrophobicity of the given ATPS. Log PO = log P when K = 1, thus with more negative values of log P,, the proteins tend to partition more favorably to the top phase. R represents the hydrophobic resolution which is the ability of the system to discriminate between solutes with different hydrophobicities. Table 4 shows the calculated values of R and the intrinsic hydrophobicity, log PO, of the ATPS used at 20°C. Clearly, the systems with higher concentrations of NaCl give a higher resolution to exploit the protein hydrophobicity in partitioning which is given by the value of the slope, R, in Table 4. Also, with increasing concentration of NaCl, log P,, has more negative values and hence the proteins will partition more favorably to the top phase.
PEG/dextran systems In order to compare the hydrophobic behavior of proteins in PEG/salt systems to that in PEG/dextran, the model proteins were partitioned in a PEG/dextran system at their respective isoelectric points and in another PEG/dextran system having the same concentrations of the polymers at pH 7.0 to which 0.15 M NaCl was added. In both systems, the effect of charge is minimized. In the first, because partitioning takes place at the p1 and in the second because the interfacial potential is made close to zero by the addition of a neutral salt at a low concentration. 27,28When log K was plotted as a function of hydrophobicity measured by chromatography (RP-HPLC or HIC), no trend was observed” (data not shown); however, a trend was observed when log K was plotted against log(l/m*) as shown in Figure 7. The values of R in these systems, however, are considerably smaller than in PEG/phosphate systems (Table 5).
1996, vol. 19, November
15
Hydrophobic
partitioning
of proteins:
F. Hachem
et al.
1
0
X
4
_2f
X
-1
-3L-----l -0.6
A
-0s
v
-0.4
-0.3
log l/m*
-0.6
-0.5
-1
-0.4
log l/m*
A
2I1
-I
I
J
x
X
4
I?
w
O-
Lyz
kc
-
p
CL.1
0 BcJ*
-1 -
-4 -I -0.6
I -0.5 log
B
I -0.4
J -0.3
I/m*
I
-2
-(
log l/m*
B
4
r
-0.4
-0.5
-0.6
1 Figure 7 The relationship between log K and hydrophobicity determined by precipitation and log (l/m*) in a two-phase system of 4% PEG and 5% dextran. Abbreviations same as in Figure 2. Points on the curve represent the average of five experiments carrier out in duplicate at a pH equal to the pl of the proteins (A) and at pH 7 with 0.15 M NaCl (B)
X
4 shown in Figure 8. It can be seen that the proteins do not exhibit a clear correlation between their MW and partition coefficients in the PEG/PO, system as has been demonstrated for PEG/dextran systems2g.30 where the value of K -0.6
C
-0.5
-0.4
-0.3 3 ,
log l/m*
2Figure 6 The relationship between log K and hydrophobicity determined by precipitation, log (7/m*) in a two-phase system (8% PEG and 12% PO,, at pH 7.0). Abbreviations same as in Figure 2. 0% (w/w) NaCl (A); 0.48% (w/w) NaCl (8); 9.6% (w/w) NaCl (C)
y
Amy1 o
l-
* x
O-
Y
-1-m
. hc
Oval ~
-2 Molecular
weight of proteins
-3 -
In order to evaluate whether the MW of the protein has an important effect on partitioning in the PEG/salt systems used in this work, proteins with MWs varying between 14,000-150,000 Da were partitioned in two systems, one that showed the highest correlation coefficient between hydrophobicity and partition coefficient, i.e., a system to which 4.8% (w/w) NaCl was added and another which contained 9.6% (w/w) NaCl (where hydrophobic resolution was the highest). The results for the system with 9.6% NaCl are
Glut o
G
Conal P
W 0
m~htut BSA
Izg
-I
-4:
0
50000
100000
150000
200000
MW (Da) Figure 8 The relationship between the MW of proteins and log Kin 8% PEG/12% PO., systems at pH 7 with 9.6% NaCI. Abbreviations same as in Figure 2, plus IgG, lmmunoglobulin G; Amyl, cY-amylase; Oval, ovalbumin; Try, trypsin; Tran, transferrin; Glut, glutathione reductase.
Enzyme Microb. Technol.,
1996, vol. 19, November
15
515
Papers Table 5 The calculated values of Rand the intrinsic bicity, log P,,, of the PEG/dextran systems
hydropho-
References I.
Two-phase
systems
(wt%)
R
log p0 7.
4% PEG, 5% dextran, 4% PEG, 5% dextran,
pH = pl 0.15 M NaCI, pH 7.0
2.2 2.4
-0.38 -0.40 3. 4.
tends to decrease with increasing protein MW. Clearly, the results reported in this paper regarding the effect of hydrophobicity are not affected by protein MW.
5.
Conclusions
6.
A parameter for measuring the hydrophobicity of proteins (Z/m*) has been derived from precipitation curves by addition of a salt such as (NH,),SO,. The correlation between the proposed parameter l/m* based on the solubility of proteins and retention times in RPC and HIC for the five proteins was rather poor. Hydrophobicity, measured by the volumetric fraction of organic solvents from RPC or the inverse of the molar fraction of salts from HIC columns needed to elute proteins or their capacity factors, does not correlate with partition coefficients in the PEG/PO, systems with or without added NaCl. Measuring the hydrophobicity of proteins in solution using precipitation curves from which the parameter log(I/m*) was derived shows a linear correlation with partition coefficient (log K) in the PEG/PO, systems, particularly in systems with NaCl. The parameter log (l/m*) also shows a good correlation with log K in PEG/dextran systems that exploit the hydrophobicity of the solutes, i.e., systems at the p1 of the native proteins and a system with neutralized electric potential difference of approximately zero between the phases. Hydrophobicity of proteins is better exploited in PEG/ PO, systems than in PEG/dextran systems since in the former systems, a much higher resolution R, can be obtained. The addition of NaCl to PEG/PO, systems increases the hydrophobic difference between the phases and promotes hydrophobic interaction between the proteins and PEG. The MW of proteins do not contribute to their partitioning in PEG/PO, systems confirming that partitioning is due to attractive interactions of a hydrophobic nature between the proteins and the polymer. A simple correlation for the prediction of partitioning in a specific ATPS based on the parameter (I/m*) has been used. It describes the hydrophobic resolution of the system, R, which is a measure of the separation power. R was higher in PEG/PO, systems with higher concentrations of NaCl.
Acknowledgments
Enzyme Microb. Technol.,
8.
9.
10.
11.
12.
13. 14. 15. 16. 17.
18.
19. 20.
21.
22.
23.
24.
Financial assistance from the European Community, the Hariri Foundation, and the Agricultural and Food Research Council (AFRC) are gratefully acknowledged.
516
I.
25.
1996, vol. 19, November
Tanford. C. Contribution of hydrophobic interactions to the stability of the globular conformation of proteins. J. Am. Chem. Sac,. 1962, 84,42404247 Bigelow. C. On the average hydrophobicity of proteins and the relation between it and protein structure. J. Theor. Bio/. 1967, 16, 187-211 Lee. B. and Richards, F. M. The interpretation of protein structures: Estimation of static accessibility. J. Mol. Biol. 1971, 55, 379-400 Johansson, G. and Hartman, A. Partitioning of proteins in two-phase systems containing charged poly(ethylene glycol). In: Proceedings of the International Solvent Extmction Conference Vol. 1 (Thornton, J. D., Naylor. A.. McKay. H. A. C., and Jeffreys, G. V. Eds.). Society of Chemistry Industry, London, 1974 Eiteman, M. A. and Gainer, J. L. A model for the prediction of partition coefficients in aqueous two-phase systems. Bioseparation 1989, 2, 3141 Quarry, M. A., Grab, R. L., and Snyder, L. R. Prediction of precise isocratic retention data from two or more gradient elution runs. Analysis of some associated errors. Anal. Chem. 1986, 58,907-9 I7 Scopes, R. K. Protein Purification: Principles and Practice. Springer-Verlag. New York, 1982 Sedmak, J. J. and Grossberg, S. E. A rapid, sensitive, and versatile assay for protein using Coomassie Brilliant Blue G2.50. Anal. Biothem. 1977, 79, 544-552 Melander, W. and Horvath, C. Salt effects on hydrophobic interactions in precipitation and chromatography of proteins: An interpretation of the lyotropic series. Arch. Biochem. Biophys. 1977, 183, 200-2 15 El Rassi, Z., Lee, A. L. and Horvath, C. Reversed-phase and hydrophobic interaction chromatography of peptides and proteins. In: Separation Processes in Biotechnology (Asenjo. J. A.. Ed.). Marcel Dekker, New York, 1990, 447494 Hachem, F. Hydrophobic behaviour of five model proteins in chromatography, precipitation, and aqueous two-phase systems. Ph.D. Thesis, University of Reading, UK, 1992 Fausnaugh, J. L., Kennedy, L. A. and Regnier, F. E. Comparison of hydrophobic-interaction and reversed-phase chromatography of proteins. J. Chromatogr. 1984, 317, 141-155 Chaplin, L. C. Hydrophobic interaction fast protein liquid chromatography of milk proteins. J. Chromatogr. 1986, 363, 329-335 Diosady, L. L. and Bergen, I. High performance liquid chromatography of whey proteins. Milchwissenschaft 1980, 35(1 l), 67 l-674 Pearce, R. R. Analysis of whey proteins by high performance liquid chromatography. Aust. J. Dairy Technol. 1983, 38, 114-l 17 Green, A. A. Studies in the physical chemistry of the proteins. J. Biol. Chem. 1931, XC111 (2), 495-516 Przybycien, T. M. and Bailey, J. E. Solubility-activity relationships in the inorganic salt-induced precipitation of a-chymotrypsin. Enzytne Microb. Technol. 1989, 11, 264-276 Stanley, P. G. Salting-out of multi-component systems with and without the use of constant final volumes. Biochim. Biophys. Acta 1963. 75,442444 Dixon, M. and Webb, E. C. Enzyme fractionation by salting-out: A theoretical note. Adv. Prot. Chem. 1961, 16, 197-219 Cascone, 0.. Andrews, B.A. and Asenjo, J. A. Partitioning and purification of thaumatin in aqueous two-phase systems. Enzyme Microb. Technol. 1991, 13, 629-635 Schmidt, A. S., Ventom, A. M. and Asenjo, J. A. Partitioning and purification of cY-amylase in aqueous two-phase systems. Enzyme Microb. Technoi. 1994, 16, 131-142 Hodgson, C. Two-phase aqueous systems for the separation and purification of tPA (tissue plasminogen activator). Ph.D. thesis. University of Reading, UK, 1992 Johansson, G. Partitioning of proteins. In: Partitioning in Aqueous Two-Phase Systems: Theory. Methods, Uses and Applications in Biotechnology (Walter, H.. Brooks, D. E., and Fisher, D.) Academic Press, London, 1985, 161-226 Baskir, J. N., Hatton, T. A., and Suter, U. W. Protein partitioning in two-phase aqueous polymer systems. Biotechnol. Bioeng. 1989,34, 541-558 France, T. T., Andrews, A. T., and Asenjo, J. A. Use of chemically modified proteins to study the effect of a single protein property on
15
Hydrophobic
26.
27.
partitioning in aqueous two-phase systems: Effect of surface hydrophobicity. BiotechnoL Bioeng. 1996, 49, 3OC308 Asenjo, J. A.. Schmidt, A. S., Hachem, F., and Andrew% B. A. A model for predicting the partition behaviour of proteins in aqueous two-phase systems. J. Chromat. 1994, 668, 47-54 Reitherman, R., Flanagan, S. D., and Barondes, S. H. Electromotive phenomena in partition of erythrocytes in aqueous polymer twophase systems. Biochem. Biophys. Acta 1973, 297, 193-202
28.
29. 30.
partitioning
of proteins:
F. Hachem
et al.
Gaiscone, P. S. and Fisher, D. The dependence of cell partition in two-polymer aqueous phase systems on the electrostatic potential between the phases. Biochem. Sot. Truns. 1984. 12(6), 1085-1086 Albertsson, P. A. Partition of proteins in liquid polymer-polymer two-phase systems. Nature 1958. 182, 709-71 I Sasakawa. S. and Walter. H. Partition behaviour of native proteins in aqueous dextran-poly(ethylene glycol) phase systems. Biochrmistry 1972, 11, 2760-2765
Enzyme Microb. Technol.,
1996, vol. 19, November
15
517
Engineering
and J. A. Asenjo
Laboratory,
University of Reading, Reading, United Kingdom
The hydrophobicity of five proteins was estimated by reversed-phase chromatography (RPC). hydrophobicinteraction chromatography (HIC), and precipitation with ammonium sulfate. These data were correlated to partition behavior in aqueous two-phase systems. A parameter (I/m*) that was derived from the precipitation curves is based on the solubility of proteins in an electrolyte solution. The correlation between l/m* and the retention times in HIC and RPC was poor due to interaction effects with the chromatography matrices and probably partial unfolding of the proteins: however, this parameter (I/m*) was expected to be a measure of hydrophobicity of proteins that relates better than the chromatographic data to experiments where the hydrophobic behavior of proteins in an aqueous solution is used for their separation. The partition behavior of the five proteins in aqueous two-phase systems (ATPS) in the absence and presence of NaCl was investigated. A poor correlation was found between log K (K is the partition coefficient) in ATPS and the hydrophobicity values measured by RPC and HIC; however, a very good correlation was found between log I/m* which is a measure of protein hydrophobicity based on the solubility of the protein during precipitation and log K, particularly in PEG/PO, systems with added NaCl. The parameter (I/m*) also demonstrated a good correlation with log K in PEG/dextran systems. A simple correlation for the prediction of partitioning in specific ATPS based on this parameter has been evaluated. An expression describing its resolution power, R, and a parameter describing the hydrophobic& of the system, P, was determined making the correlation potentially predictive for other proteins in the ATPSs used. Hydrophobicity of proteins was better exploited in PEG/PO, 0 1996 by systems than in PEG/dextran ones as a much higher resolution (R) is obtained in the former. Elsevier Science Inc.
Keywords: Hydrophobicity;
chromatography;
solubility:
aqueous two-phase
Introduction Evaluation
of hydrophobicity
Methods used to quantify the hydrophobicity of proteins can be divided into two groups. The first group measures the total overall hydrophobicity while the second measures the surface hydrophobicity or the functional hydrophobicity of a protein. In the first category, the amino acids which make
Address reprint requests to Professor J. A. Asenjo, Department of Chemical Engineering, Universidad de Chile, Centre for Biochemical Engineering and Biotechnology, Beauchef 861, Santiago, Chile The present address of B. A. Andrews is the Centre for Biochemical Engineering and Biotechnology, Department of Chemical Engineering, University of Chile, Beauchef 861, Santiago, Chile Received 5 October 1995; revised 12 November 1995; accepted 10 January 1996
Enzyme and Microbial Technology 19:507-517,1996 Q 1996 by Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010
partitioning;
correlation
up the protein are given hydrophobic values that stand, in most cases, for the free energy of transfer of the side chain from an aqueous solution representing the surface of a protein to an organic solution (ethanol, octanol, hexane) that mimicks the interior of the protein. The total hydrophobicity of the protein is calculated by simple addition of the hydrophobic values of each constituent amino acid’ or an average hydrophobicity is calculated by dividing this sum by the number of amino acid residues in a protein to give what is called the Bigelow number.’ With the discovery by Lee and Richards3 that the surface of globular proteins also contained nonpolar amino acids, interest in the hydrophobic interactions between a protein and its environment increased which resulted in the emergence of methods that quantify the surface hydrophobicity rather than the total overall hydrophobicity. Of the methods that measure surface hydrophobicity, there are the hydrophobic probe meth-
0141-0229/96/$15.00 PII SU141-0229(96)00062-2
Papers
Materials and Methods
ods, the hydrophobic binding methods, reversed-phase chromatography (RPC), and hydrophobic interaction chromatography (HIC). With the advent of chromatographic systems such as HPLC and F’PLC which reduce the time involved in specific measurements, chromatography as a method for measuring the surface hydrophobicity of proteins became popular.
Partitioning
P-lactoglobulin A, a-lactalbumin, bovine serum albumin, oonalbumin, and hen egg lysozyme were purchased from Sigma (Poole, England). Lysozyme was a gift from STC Laboratories Inc., Winnipeg, Canada. Table I shows the isoelectric points and molecular weights of the proteins. Acetonitrile was HPLC grade and trifluoroacetic acid (TFA) was SpectrosoL grade. All solvents were filtered through a 0.45 pm filter and degassed prior to use. All other reagents used were the highest purity available.
in aqueous two-phase systems
The partition coefficient of a protein in an aqueous twophase system (ATPS), K, is defined as C& where C, and C, represent the equilibrium concentrations of the partitioned protein in the top and bottom phases, respectively. It is possible, therefore, to separate a mixture of proteins by choosing a system where the respective K values of the proteins are widely different. The partitioning of molecules between the two phases is a complex phenomenon because of the involvement of many factors in the interactions between the solute and the phase-forming components like hydrogen bonding, charge interactions, van der Waals forces, hydrophobic interactions, and steric effects. This makes the molecular weight and chemical properties of the polymer and the size and the chemical properties of the partitioned solute of extreme importance. Since the surface of a protein is made of different types of amino acids, two properties of the protein surface, namely its charge and hydrophobicity, play important roles in the partition of proteins in ATPS. If systems are chosen where the effect of the potential difference on the partitioning of proteins is nullified, factors other than charge dominate, especially the hydrophobic effect. To make such systems, neutral salts are usually added at very low concentration to a PEG/dextran system. Under such conditions, Johansson and Hartman found that most proteins maintain the same K value independently of pH. Due to the complexity of the partitioning phenomenon, it is difficult to predict protein behavior and select separation conditions for the rational planning of experiments. Models that can predict separation a priori are, therefore, very useful; however, most of the models available to date involve a large number of parameters to be calculated which hinders their utilization in bioseparations. The model of Eiteman and Gainer5 is promising since it relates the partition coefficient to two parameters, the hydrophobicity of the solute and the weight difference of one of the polymers between the phases. Both can be easily measured. The first of these parameters, namely the protein hydrophobicity, however, might pose a problem because there are many methods normally used for measuring it and in many cases the values obtained correlate poorly. In this paper, we compare two modes of chromatography widely used to measure the functional hydrophobicity of proteins, RPC and HIC, and also introduce a parameter for measuring this property (l/m*) that differs from the former ones in that it is a measure of functional hydrophobicity in solution without the aid of a matrix. These methods are then evaluated as a means of estimating the hydrophobic behavior of proteins in ATPS. 508
Materials
Enzyme Microb. Technol.,
Equipment
and methods
The HPLC system employed for RPC consisted of a Perkin Elmer Binary LC pump 250 (Norwalk, CT) and a Rheodyne Model 7 125 valve equipped with a 20 ~1 sample loop. The experiments were performed on a Nucleosil 300-7 Cl8 reversed phase column (length, 25 cm; i.d., 4.6 mm; Hichrom, Reading, UK). It had a pore size of 30 nm and a particle size of 5.7 + 1.5 pm. The effluent of the column was monitored at 280 nm using a UV-VIS Sepectroflow 757 spectrophotometer with a Kratos analytical detector. The chromatograms were obtained under gradient elution at a flow rate of 1 ml min-’ and at ambient temperature. After each run, the column was equilibrated for a period of approximately 20 min. with 10% eluant A. The profile of the gradient was as follows: 3 min at 10% A after which the sample was injected; 30 min from 10% to 100% A; and 3 min from 100% to 10% A where A was acetonitrile + 0.1% TFA (v/v) and B was water + 0.1% TFA (v/v). Proteins were dissolved in solvent B at a concentration of 1 mg ml-’ except for lysozyme which was dissolved at a concentration of 0.5 mg ml-‘. All samples were filtered through a 0.2 pm filter before application to the column. Reported retention times are the average of four replicates. Values obtained were within * 5%. Fast protein liquid chromatography (FPLC, Pharmacia) was used for HIC. The column HR 5/5 (1 ml) was packed with lowsubstituted, fast-flow Phenyl Sepharose (Pharmacia). Detection was carried out at 280 nm for all proteins and the gradient employed was: 3 min at 100% 1.5 M ammonium sulfate in 0.05 M sodium phosphate buffer pH 7.0; 30 min linear gradient from 1.5 M to 0.0 M ammonium sulfate in the same buffer; and 3 min at 100% sodium phosphate buffer. Proteins were dissolved in buffer and filtered (0.2 km filter) before elution at a flow rate of 1 ml mini. The column was considered equilibrated when a stable baseline was obtained. Retention time is reported as the average of 4-5 runs. For calculating the average capacity factors of the proteins, the method of Quarry et ~1.~ was followed which involved eluting the proteins using the same column and elution conditions under two different gradient run times. In the RPC experiments, the gradient run times employed were 30 and 17 min whereas in the HIC experiments, they were 30 and 15 min. The value of t,, (dead retention time) was found from the spike at which the change in
Table 1 Molecular the proteins
weights (MW) and isoelectric points (pl) of
Protein a-Lactalbumin P-Lactoglobulin A Bovine serum albumin Conalbumin Lysozyme
1996, vol. 19, November
15
MW (Da) 14,000 37,100 66,500 77,000 14,300
PI 4.2-4.5 5.13 4.9 5.88 10.3
Hydrophobic the mobile front occurred, and t, (delay time before the system starts the gradient) was found by running a gradient with a UVabsorbing solvent (acetone) as one of the two mobile phase solvents. For the HPLC-RP system, to and t,, were 3.40 and 3.35 min, respectively. For the FPLC-HIC system, the values were 1.2 and 0.6 min, respectively.
Precipitation
of proteins
Proteins were dissolved in 0.05 M sodium phosphate buffer pH 7.0 at a concentration of 2 mg ml-‘. To a measured volume of each solution, solid ammonium sulfate was added slowly and with continuous stirring to reach a certain saturation according to Scopes.’ After all of the salt had dissolved, the solutions were left to equilibrate for I5 min in a water bath at 24°C. After centrifugation (25,000 g for 20 min), an aliquot of the supematant was assayed for protein concentration. To the rest of the supematant, more ammonium sulfate was added, thus making the concentration higher. Again, the solution was left to equilibrate, was centrifuged, and the new supematant was assayed for protein after dilution. A blank was prepared for each level of saturation and diluted in the same way as the solution containing the protein.
Protein determination Protein was measured by a modified Bradford dye-binding assay.8 Standard curves were prepared for each protein. Blank systems were made to account for the interference of ammonium sulfate. For the PEG/dextran systems, absorbance of the diluted top and bottom phases were measured at 280 nm against a blank. Concentrations of the proteins were calculated from standard curves in which absorbance at 280 nm was plotted against increasing concentration of the same protein.
Phase systems PEG of molecular weight 8000 was purchased from Sigma while analytical grade K,HPO, - 3H,O and NaH,PO, * 2H,O were purchased from BDH Phase systems with compositions of 8% (w/w) PEG 8,000 and 12% (w/w) phosphate were prepared from stock solution of PEG (50% w/w) and phosphate solution (40% w/w). The phosphate solution was pH 7.0. Sodium chloride was added to attain the following final concentrations in the system: 0,0.48,4.8, 9.6, and 17.6% (w/w). In the 17.6% (w/w) system, the salt was added as a solid. The protein concentration in the initial stock solution was 2 mg/ml-‘. Low speed centrifugation was used (1,200 g, for 5 min) to accelerate the phase separation after gentle and thorough mixing of the system. Samples from the top and bottom phases were assayed for protein concentration. All partition experiments were done at room temperature. For the experiments on the effect of molecular weight on the separation of proteins, two PEG/phosphate systems were chosen. One contained 4.8% (w/w) sodium chloride and the other contained 9.6% (w/w) sodium chloride. Additional proteins used include immunoglobulin G (human, 150,000 Da), ol-amylase (Bacillus subrilis, 55,000 Da), ovalbumin (chicken egg, 45,000 Da), trypsin (bovine pancreas, 23,800 Da), transfenin (human, 77,000 Da), and glutathione reductase (Bakers’ yeast, 100,000 Da). All proteins were purchased from Sigma. PEG/dextran systems were prepared containing 4% PEG, 5% dextran. 0.15 M (0.73% w/w) NaCl, and 0.01 M sodium phosphate buffer.
Results and discussion Measuring
the hydrophobic@
of proteins
Hydrophobicity by chromatography. Five globular teins
of different
molecular
weights
and isoelectric
propoints
partitioning
of proteins:
F. Hachem
et al.
(Table 1) were eluted in RPC and HIC. In addition to being a powerful separating and purifying technique, RPC has been used as a method to determine the hydrophobicity of
peptides and proteins. g*lo More hydrophobic proteins will have higher retention times; the retention time is lower with higher concentrations of the modifier. In the case of HIC, the high concentration of the salt in the initial buffer may cause a small salting-out of the protein from solution but also an increase in the hydrophobic interactions with the hydrophobic tails of the matrix in the chromatography column causing adsorption; therefore. solutes that are hydrophobic bind strongly to the matrix and elute at lower concentrations of the salt when a decreasing gradient is used. In both RPC and HIC, the way to quantify the hydrophobic character of the solute is by reporting their retention times (or volumes) or their respective capacity factors. The capacity factor is the standardized retention quantity which normalizes the effects of the dimensions of the column usually used for isocratic elution. In the case of gradient elution which is often used for proteins, an average capacity factor, k”, can be used. This factor can be calculated from retention times obtained using two different gradients.h.” Table 2 shows the retention times (tR) and average capacity factors (K’) for RPC and HIC for the five proteins. From the elution profiles (Table 2), it is observed that the order of hydrophobicity of the proteins is different when comparing RPC values of retention times to those of HIC, e.g., BSA, which elutes second in RPC, elutes last in HIC. A similar behavior was observed for values of the capacity factors, K’. This type of behavior has been obtained by Fausnaugh et al. I2 who compared the retention of 12 proteins on a phenyl-acetyl column CHIC) and a Synchropack RP-8 column (RPC) and found that proteins which were more hydrophilic on the HIC column (e.g., cytochrome c and myoglobin) were retained by RPC (less hydrophilic). On the other hand, P-glucosidase. a protein that adsorbed to the phenyl-acetyl column, was only weakly retained by the RP-8 column. Disaccordance in the elution order has also been observed when milk proteins were chromatographed using a phenyl-Superose column.” a C-8 column,14 and a C-6 column. I5 The difference in elution behavior of a protein between RPC and HIC can be explained by the different conformational changes which proteins undergo when interacting with the matrices and the mobile phase. Although the two
Table 2 The retention times (tR) average capacity factors (I’) of the different proteins on RPC-Cl8 and I-W Phenyl Sepharose columns during a 30 min gradient RPC Protein a-Lactalbumin @-Lactoglobulin A Bovine serum albumin Conalbumin Lysozyme
Enzyme Microb. Technol.,
HIC
fR (min)
K
tR (min)
K
20.5 21.7 19.8 20.0 19.4
1.53 2.48 2.23 1.67 1.77
14.0 10.3 16.3 8.6 10.4
4.83 5.43 4.58 3.78 6.69
1996, vol. 19, November
15
509
Papers
,
-21 Concentration
A
3
2
1
I
4
of ammonium
Concentration
sulphate
084 7
2
3
of ammonium
4
sulphate
084 -
02 -
o,o-
h
c 29 ul
4
C
-0,2
-
-0,4
-
-0,6
-
-033
3
2‘4
Concentration
2.6
2‘0
of ammonium
Concentration
3,2
3,4
Concentration
4
of ammonium
sulphate
modes of chromatography exploit the hydrophobicity of the proteins, they may have different selectivity. In RPC, the harsh conditions employed (organic modifkrs and a very hydrophobic and dense stationary phase) lead to the partial denaturation of the protein structure. In contrast in the HIC mode, the mild hydrophobic surface and the high antichaotropic salt concentration that stabilizes the protein structure result in the separation of the proteins in their native forms. Hydrophobicity by precipitationkolubility. In the process of precipitating proteins by the addition of electrolytes, a small increase in the concentration of salt transfers the proteins from being relatively soluble to being largely precipitated; l6 therefore, it is essential to know the concentration of salt at which a protein starts to precipitate which is done by plotting the logarithm of solubility against the concentration of salt. 570
Enzyme Microb. Technol.,
2
3,6
sulphate
3
2 E
3,0
4
3
of ammonium
sulphate
Figure 1 Protein solubility curves (log S) as a function of concentration (MI of ammonium sulfate at 25°C. Conalbumin, A; lysozyme, B; BSA, C; p-lactoglobulin, D; and a-lactalbumin, E
It should be noted that the shape of the solubility curve is determined by the interaction between the surface of the protein and the salt used. The salubility curves of the five proteins used in this study consist of two segments (Figure I). The first corresponds to a region where protein solubility changes very little with the concentration of salt. This behavior is observed at low concentrations of protein (e.g., l-2 g 1-l) such as the work reported in this paper. At high concentrations of protein, a salting-in region is observed at low salt concentrations where protein solubility increases with salt concentration.9 The second segment corresponds to the salting-out region in a way consistent with theory. The concentration of salt at which the discontinuity occurs (point at which the protein starts precipitating given a fixed initial concentration, e.g., 1 g 1-l) serves as a good reference for measuring the solubility or hydrophilicity of a protein; therefore, the inverse of the concentration of salt at
1996, vol. 19, November
15
Hydrophobic
partitioning
of proteins:
F. Hachem
et al.
showed how the slope of the solubility curve changed sharply at a certain concentration. Values of dS/dM are obtained as follows: ‘I log S = p - K, M d(log S)/d(M)
(2)
= - K,
(3)
since d(ln S) = d(S)/S and d(ln S) = 2.303 d(log S) (4) then 0.2
I
I
I
I
16
17
18
19
time
(min)
0.2 _I-____/ 6
8
10
Retention
12
time
14
16
(min)
Figure 2 Correlation between the corrected retention times in RPC and HIC and the experimental value of l/m*for the proteins studied. The abbreviations are: Lac, a-lactalbumin; Lag, 8-lactoglobulin; BSA, bovine serum albumin; Conal, conalbumin; and Lys, lysozyme. RPC using a C-18 column, (A); and HIC using Phenyl Sepharose (B)
this discontinuity point could be used as a good indicator of the protein hydrophobicity in solution. We have called the concentration of the salt at the point of discontinuity, m*, after Przybycien and Bailey. I7 The value is used as a comparative measure of the solubility or hydrophilicity; its inverse is a measure of the hydrophobicity of the proteins in solution (l/m *). Precipitation of proteins occurs at relatively high salt concentrations (above 1.0 M). At such concentrations, charge interactions of proteins are neutralized. Przybycien and Bailey17 found the value of m* on the solubility curve by the following expression: m* = (p - In S,)/ K,
S)
(5)
and
! 15
Retention
d(log S) = d(S)/(2.303
(1)
where S, is the initial concentration of the protein and p and K, are parameters from the equation of solubility in the salting-out region. To find the value of m* experimentally, the solubility data on the five proteins was fitted into two equations; one for the region to the left of point m* which corresponds to the region reflecting a very small effect of the salt on the solubility of the protein and one for the saltingout region. The point of intersection of the two curves determines the salt concentration, m* (Figure I). By differentiating the solubility curve (&Y&4), Stanley I8
d(S)/d(M) = -2.303 S K, (6) when d(S)ld(M) is plotted as a function of the molarity (M), a good value of the discontinuity m* is thus obtained.’ ‘.I9 In Table 3, the parameters derived from the solubility curves for the five different proteins are shown. There is very good agreement between the values of m * as calculated from the salting-out curve by equating S to the initial concentration of the protein in solution (m*_,) and as derived when the two segments of the solubility curve are fitted to two separate curves (m*,,J. Also, the values of m* derived from the differential graphs using d(S)/d(M) (m*& are very close to the values determined experimentally and by calculation. In most cases, m*diff is closer to m*,_. The value of the parameter m* can be easily determined from experimental data and thus becomes a useful parameter for determining the solubility (and thus hydrophilicity) of a protein in salt solutions and, as already discussed, we will use l/m* as a measure of the protein hydrophobicity in solution. The parameter l/m* was compared with the hydrophobicity values for the same proteins obtained from RPC and HIC as shown in Figure 2. These Figures show that there was little or no correlation between the hydrophobicity values of the proteins determined by RPC and the values of the parameter l/m* for the five proteins under study. In the cases of HIC, the hydrophobicity values of the proteins also did not correlate with those of I/m*. The retention values from an HIC column reflect the interaction between the salt, the matrix, and the protein while the value of l/m* reflects the interaction between the salt and the protein only. Using the normalized retention data, i.e., the capacity factors, a small improvement in the correlation between K’ and I/m* was observed in the case of RPC but not in the case of HIC” (data not shown). The correlation of the hydrophobicity measured by RPC, Table 3 The salting-out constant (KS), the extrapolated solubility to zero molarity of ammonium sulfate (6), and the point of discontinuity between the two segments of the solubility curve determined experimentally (m*,,,,), calculated (IT-I’,,,), and derived from the differential graphs (m*&
KS
P
mxmp
m*cal
m*dirr
Protein
M-’
g I-’
M
M
M
a-Lactalbumin 8-Lactoglobulin A Bovine serum albumin Conalbumin Lysozyme
1.82 2.48 2.09 1.68 1.14
5.34 8.79 6.87 4.79 2.89
2.83 3.48 3.21 2.90 2.39
2.77 3.42 3.14 2.67 2.26
2.84 3.49 3.20 2.59 2.33
Enzyme Microb. Technol.,
1996, vol. 19, November
15
511
Papers HIC, and precipitation with the hydrophobic behavior proteins in ATPS is studied in the next section.
Partitioning in aqueous two-phase evaluation of hydrophobicity
centrations >4% (w/w) added salt (NaCl) and a-lactalbumin prefers the upper phase when the salt concentration is approximately 14% (w/w) while BSA, B-lactoglobulin A. and conalbumin prefer the lower salt-rich phase at all concentrations of NaCl. Johanssonz3 has noted that in PEG-salt systems, most proteins strongly favor the lower salt-rich phase. This is especially true when the molecular weight (MW) of the polymer is high because with increasing MW, the concentration of the protein and the salt will increase in the “free” solvent in the polymer-rich phase causing a strong partition of the protein to the lower phase.24 With the use of PEG 8,000 in the polymer phase, it is not surprising that all of the proteins resided in the lower salt-rich phase at 0% (w/w) NaCl. The same result was obtained by Cascone el &.*”
of
systems and
Previous publications have shown that the partition coefficients of certain proteins can be increased quite dramatically when high concentrations of NaCl are added to ATPS”)-‘* whereas others are not much affected by this. This effect has been attributed to possible hydrophobic interactions. Hence, ATPSs with different levels of NaCl in the range O-17.6% were chosen. Figure 3 shows that proteins behave differently when the concentration of NaCl is increased in the system. Lysozyme prefers the upper PEG-rich phase at con-
1
“II 2-
1
0
x
X
4
4
-1
-1 -
-2d
-3,
Concentration
A
0
20
of NaCl (Ohwh)
10
Concentration
B
X
X
4
3 -2 -
-22
-31
-31
of N&l
20
(OAw/w)
1
0
10
Concentration
C
20
of NaCl (% w/w)
0
D
10
Concentration
20
of NaCl (Oh w/w)
‘I
0
X
4 - .
lb
0 E
512
Concentration
Enzyme Microb.
2b
of NaCl (% wb)
Technol.,
Figure 3 The effect of NaCl concentration on the partition coefficient of proteins in a system containing 8% PEG 8,000 and 12% PO, of pH 7.0. Each point on the curve represents the average of 3-4 experiments cariied out in duplicate ‘(results were within f 5%). Lysozyme (A); BSA (B); 4actalbumin (C); P-lactoglobulin (D); and conalbumin (E)
1996, vol. 19, November
15
Hydrophobic when partitioning thaumatin in PEG 6,OOO/PO, systems. In the first instance with no NaCl added, thaumatin preferred the lower phase; however, increasing the NaCl concentration resulted in a drastic increase in its partition coefficient. To investigate the effect of the hydrophobicity of proteins, the logarithms of the partition coefficients for the five proteins in the different PEG/PO, systems used were plotted against the logarithms of the volumetric fractions of organic solvent at which these proteins eluted from a RP-C 18 column (log p). the logarithms of the inverse of the molar salt fractions at which they eluted from an HIC phenyl Sepharose column (as a measure of hydrophobicity), and the logarithms of the parameter proposed to measure hydrophobicity in solution (I/m*). The results are shown in Figures 4, 5, and 6. Volumetric fractions of organic solvent in RPC will be referred to as hydrophobicity since more hydrophobic proteins will elute at higher solvent concentrations in gradient elution. Similarly, the inverse of the molar fractions of salt in HIC will be used as a measurement of hydrophobicity since more hydrophobic proteins will elute at lower salt concentrations in a (decreasing) gradient elution. Figure 4 shows that a good correlation does not exist between log p and log K in the PEG/PO, systems used. This indicates that the hydrophobicity determined by RP-HPLC does not correlate with the protein partitioning behavior in the ATPS used for the five proteins under study. To verify whether or not the method used to determine hydrophobicity was the cause of the poor correlation, HIC using phenyl Sepharose as the hydrophobic matrix and a decreasing gradient of (NH&SO, was used. The results are shown in Figure 5. In these Figures, the hydrophobicity of the proteins is taken as the inverse of the molar fraction of salt needed for their elution with the initial salt concentration of 1.5 M representing 100%. This method for measuring hydrophobicity, which was successful for evaluating the hydrophobicity of a family of chemically modified proteins with different hydrophobicities’” but with the same charge and MW, did not improve the correlation between hydrophobicity of the proteins and the partition coefficient (Figure 5) for the five proteins studied. The poor correlation between the hydrophobicity measured by RP-HPLC and K is not totally surprising in view of the fact that the use of organic solvents in the former leads to at least partial denaturation of the native proteins which affects the opening up of the molecule and the exposure of the hydrophobic residues that were previously hidden in the core of the protein. In the case of HIC, the proteins are not denatured but hydrophobicity depends on the interaction between the matrix used and the hydrophobic patches on specific areas on the protein. Hence, distribution of localized hydrophobic patches on the surface may be playing a role. Figure 6 shows the correlation between log (l/m*), which corresponds to the evaluation of hydrophobicity in solution by precipitation, and log K. There exists a good linear relationship between the hydrophobicity of the proteins measured as a function of their solubility and their partitioning coefficient in systems of PEG/salt with a high concentration of NaCl for the five proteins studied. This strongly suggests that the ATPSs of PEG and salt with a high concentration of NaCl principally exploit hydrophobic
partitioning
of proteins:
F. Hachem
et al.
O-
x
Y
*Lys
he m
Q
-1 -
W
Cond El -2 -
q
%&
-3)
-0.24
-0.23
-0.22
-0.21
-0.20
-0.19
-0.18
1% p
A I
X
4
.Lys ImC
0
-1 -
0 BSA 0 0
-2 -
-3-m -0.24
-0.23
-0.22
2 1
4
Q
-0.21
-0.20
-0.19
-0.18
-0.20
-0.19
-0.18
log p
B
X
w
Cord
Ws 0 i
I
0-l -1
Cond
1
-0.24
0
-0.23
C
-0.22
La q
-0.21
1% p
Figure 4 The relationship between log K of the five proteins in a two-phase system (8% PEG and 12% PO,, at pH 7.0) and hydrophobicity (log p) in RPC-HPLC. Abbreyiations same as in Figure 2. 0% (w/w) NaCl (A); 0.48% (w/w) NaCl (B); and 9.8% (w/w) NaCl (C)
differences for partitioning and that the parameter m *, originally proposed as a measurement of protein solubility, is also a good estimator of protein hydrophilicity/hydrophobicity in solution. On the other hand, the results show that the correlation is very sensitive to ahanges in the system. An increase of as little as 0.48% (w/w) of NaCl in the PEG/phosphate system increased the correlation coefficient between log (l/m*) and log K from 0.457 to 0.837. The results show that the concentration of added NaCl need not be high in order to promote hydrophobic interactions
Enzyme Microb. Technol.,
1996, vol. 19, November
15
513
Papers Table 4 The calculated values of f? and the intrinsic hydrophobicity, log PO, of the ATPSs at 20°C Two-phase 8% 8% 8% 8% 8%
-31
0.3
0.2
0.1
0.4
log (wu
A
PEG, PEG, PEG, PEG, PEG,
system (wt%)
12% 12% 12% 12% 12%
PO, PO, PO, PO, PO,
+ + + +
0.48% NaCl 4.8% NaCl 9.6% NaCl 17.6% NaCl
R 5.4 8.3 14.9 22.4 22.8
log
po
-0.23 -0.29 -0.38 -0.45 -0.45
show that by adding NaCl to a PEG/phosphate system, the hydrophobic difference between the phases is increased and NaCl may also increase the hydrophobic interaction between the protein and the PEG.
A correlation
x 4
A correlation for predicting the partition coefficient of proteins in ATPS that takes into account the effect of hydrophobicity has been proposed”,25J6
Conal Q
-2
0
log K = R log(P/P,)
(7)
log K = R log P - R log PO
(8)
or -31 0.2
0.1 B
0.3 log
(
0.4
l/M)
2G l0
24 w 0
-1
-
-2
1
hC
Cond m
a
BSA ID
w 0
1 -3 C
! 0.1
I 02
I 0.3 log
I 0.4
(1/M)
Figure 5 The relationship between log K of the five proteins in a two-phase system (8% PEG and 12% P04, at pH 7.0) and hydrophobicity in HIC, log (l/molar fraction of ammonium sulfate) at which the proteins eluted from a Phenyl Sepharose column. Abbreviations same as in Figure 2. 0% (w/w) NaCl (A); 0.48% (w/w) NaCl (B); 9.6% (w/w) NaCl VIZ)
between PEG and the proteins which is confirmed by an almost doubling of the correlation coefficient between O-0.48% (w/w) NaCl for the five proteins studied in PEG/ phosphate systems. Since the values of the other correlation coefficients fluctuate around 0.813 at 4.8% salt, 0.915 at 9.6%, and 0.857 at 17.6%, it might well be that the small addition of 0.48% (w/w) NaCl brings the unstructured water zone to the concentration of salt where all the protein is salted-out. It can be seen in Table 4 that the resolution for hydrophobicity of the ATPSs, R, increases with increasing NaCl concentration between 0.48-9.6%. These results 514
Enzyme Microb. Technol.,
where P = (l/m*) is the protein hydrophobicity in solution measured by precipitation and log POrepresents the intrinsic hydrophobicity of the given ATPS. Log PO = log P when K = 1, thus with more negative values of log P,, the proteins tend to partition more favorably to the top phase. R represents the hydrophobic resolution which is the ability of the system to discriminate between solutes with different hydrophobicities. Table 4 shows the calculated values of R and the intrinsic hydrophobicity, log PO, of the ATPS used at 20°C. Clearly, the systems with higher concentrations of NaCl give a higher resolution to exploit the protein hydrophobicity in partitioning which is given by the value of the slope, R, in Table 4. Also, with increasing concentration of NaCl, log P,, has more negative values and hence the proteins will partition more favorably to the top phase.
PEG/dextran systems In order to compare the hydrophobic behavior of proteins in PEG/salt systems to that in PEG/dextran, the model proteins were partitioned in a PEG/dextran system at their respective isoelectric points and in another PEG/dextran system having the same concentrations of the polymers at pH 7.0 to which 0.15 M NaCl was added. In both systems, the effect of charge is minimized. In the first, because partitioning takes place at the p1 and in the second because the interfacial potential is made close to zero by the addition of a neutral salt at a low concentration. 27,28When log K was plotted as a function of hydrophobicity measured by chromatography (RP-HPLC or HIC), no trend was observed” (data not shown); however, a trend was observed when log K was plotted against log(l/m*) as shown in Figure 7. The values of R in these systems, however, are considerably smaller than in PEG/phosphate systems (Table 5).
1996, vol. 19, November
15
Hydrophobic
partitioning
of proteins:
F. Hachem
et al.
1
0
X
4
_2f
X
-1
-3L-----l -0.6
A
-0s
v
-0.4
-0.3
log l/m*
-0.6
-0.5
-1
-0.4
log l/m*
A
2I1
-I
I
J
x
X
4
I?
w
O-
Lyz
kc
-
p
CL.1
0 BcJ*
-1 -
-4 -I -0.6
I -0.5 log
B
I -0.4
J -0.3
I/m*
I
-2
-(
log l/m*
B
4
r
-0.4
-0.5
-0.6
1 Figure 7 The relationship between log K and hydrophobicity determined by precipitation and log (l/m*) in a two-phase system of 4% PEG and 5% dextran. Abbreviations same as in Figure 2. Points on the curve represent the average of five experiments carrier out in duplicate at a pH equal to the pl of the proteins (A) and at pH 7 with 0.15 M NaCl (B)
X
4 shown in Figure 8. It can be seen that the proteins do not exhibit a clear correlation between their MW and partition coefficients in the PEG/PO, system as has been demonstrated for PEG/dextran systems2g.30 where the value of K -0.6
C
-0.5
-0.4
-0.3 3 ,
log l/m*
2Figure 6 The relationship between log K and hydrophobicity determined by precipitation, log (7/m*) in a two-phase system (8% PEG and 12% PO,, at pH 7.0). Abbreviations same as in Figure 2. 0% (w/w) NaCl (A); 0.48% (w/w) NaCl (8); 9.6% (w/w) NaCl (C)
y
Amy1 o
l-
* x
O-
Y
-1-m
. hc
Oval ~
-2 Molecular
weight of proteins
-3 -
In order to evaluate whether the MW of the protein has an important effect on partitioning in the PEG/salt systems used in this work, proteins with MWs varying between 14,000-150,000 Da were partitioned in two systems, one that showed the highest correlation coefficient between hydrophobicity and partition coefficient, i.e., a system to which 4.8% (w/w) NaCl was added and another which contained 9.6% (w/w) NaCl (where hydrophobic resolution was the highest). The results for the system with 9.6% NaCl are
Glut o
G
Conal P
W 0
m~htut BSA
Izg
-I
-4:
0
50000
100000
150000
200000
MW (Da) Figure 8 The relationship between the MW of proteins and log Kin 8% PEG/12% PO., systems at pH 7 with 9.6% NaCI. Abbreviations same as in Figure 2, plus IgG, lmmunoglobulin G; Amyl, cY-amylase; Oval, ovalbumin; Try, trypsin; Tran, transferrin; Glut, glutathione reductase.
Enzyme Microb. Technol.,
1996, vol. 19, November
15
515
Papers Table 5 The calculated values of Rand the intrinsic bicity, log P,,, of the PEG/dextran systems
hydropho-
References I.
Two-phase
systems
(wt%)
R
log p0 7.
4% PEG, 5% dextran, 4% PEG, 5% dextran,
pH = pl 0.15 M NaCI, pH 7.0
2.2 2.4
-0.38 -0.40 3. 4.
tends to decrease with increasing protein MW. Clearly, the results reported in this paper regarding the effect of hydrophobicity are not affected by protein MW.
5.
Conclusions
6.
A parameter for measuring the hydrophobicity of proteins (Z/m*) has been derived from precipitation curves by addition of a salt such as (NH,),SO,. The correlation between the proposed parameter l/m* based on the solubility of proteins and retention times in RPC and HIC for the five proteins was rather poor. Hydrophobicity, measured by the volumetric fraction of organic solvents from RPC or the inverse of the molar fraction of salts from HIC columns needed to elute proteins or their capacity factors, does not correlate with partition coefficients in the PEG/PO, systems with or without added NaCl. Measuring the hydrophobicity of proteins in solution using precipitation curves from which the parameter log(I/m*) was derived shows a linear correlation with partition coefficient (log K) in the PEG/PO, systems, particularly in systems with NaCl. The parameter log (l/m*) also shows a good correlation with log K in PEG/dextran systems that exploit the hydrophobicity of the solutes, i.e., systems at the p1 of the native proteins and a system with neutralized electric potential difference of approximately zero between the phases. Hydrophobicity of proteins is better exploited in PEG/ PO, systems than in PEG/dextran systems since in the former systems, a much higher resolution R, can be obtained. The addition of NaCl to PEG/PO, systems increases the hydrophobic difference between the phases and promotes hydrophobic interaction between the proteins and PEG. The MW of proteins do not contribute to their partitioning in PEG/PO, systems confirming that partitioning is due to attractive interactions of a hydrophobic nature between the proteins and the polymer. A simple correlation for the prediction of partitioning in a specific ATPS based on the parameter (I/m*) has been used. It describes the hydrophobic resolution of the system, R, which is a measure of the separation power. R was higher in PEG/PO, systems with higher concentrations of NaCl.
Acknowledgments
Enzyme Microb. Technol.,
8.
9.
10.
11.
12.
13. 14. 15. 16. 17.
18.
19. 20.
21.
22.
23.
24.
Financial assistance from the European Community, the Hariri Foundation, and the Agricultural and Food Research Council (AFRC) are gratefully acknowledged.
516
I.
25.
1996, vol. 19, November
Tanford. C. Contribution of hydrophobic interactions to the stability of the globular conformation of proteins. J. Am. Chem. Sac,. 1962, 84,42404247 Bigelow. C. On the average hydrophobicity of proteins and the relation between it and protein structure. J. Theor. Bio/. 1967, 16, 187-211 Lee. B. and Richards, F. M. The interpretation of protein structures: Estimation of static accessibility. J. Mol. Biol. 1971, 55, 379-400 Johansson, G. and Hartman, A. Partitioning of proteins in two-phase systems containing charged poly(ethylene glycol). In: Proceedings of the International Solvent Extmction Conference Vol. 1 (Thornton, J. D., Naylor. A.. McKay. H. A. C., and Jeffreys, G. V. Eds.). Society of Chemistry Industry, London, 1974 Eiteman, M. A. and Gainer, J. L. A model for the prediction of partition coefficients in aqueous two-phase systems. Bioseparation 1989, 2, 3141 Quarry, M. A., Grab, R. L., and Snyder, L. R. Prediction of precise isocratic retention data from two or more gradient elution runs. Analysis of some associated errors. Anal. Chem. 1986, 58,907-9 I7 Scopes, R. K. Protein Purification: Principles and Practice. Springer-Verlag. New York, 1982 Sedmak, J. J. and Grossberg, S. E. A rapid, sensitive, and versatile assay for protein using Coomassie Brilliant Blue G2.50. Anal. Biothem. 1977, 79, 544-552 Melander, W. and Horvath, C. Salt effects on hydrophobic interactions in precipitation and chromatography of proteins: An interpretation of the lyotropic series. Arch. Biochem. Biophys. 1977, 183, 200-2 15 El Rassi, Z., Lee, A. L. and Horvath, C. Reversed-phase and hydrophobic interaction chromatography of peptides and proteins. In: Separation Processes in Biotechnology (Asenjo. J. A.. Ed.). Marcel Dekker, New York, 1990, 447494 Hachem, F. Hydrophobic behaviour of five model proteins in chromatography, precipitation, and aqueous two-phase systems. Ph.D. Thesis, University of Reading, UK, 1992 Fausnaugh, J. L., Kennedy, L. A. and Regnier, F. E. Comparison of hydrophobic-interaction and reversed-phase chromatography of proteins. J. Chromatogr. 1984, 317, 141-155 Chaplin, L. C. Hydrophobic interaction fast protein liquid chromatography of milk proteins. J. Chromatogr. 1986, 363, 329-335 Diosady, L. L. and Bergen, I. High performance liquid chromatography of whey proteins. Milchwissenschaft 1980, 35(1 l), 67 l-674 Pearce, R. R. Analysis of whey proteins by high performance liquid chromatography. Aust. J. Dairy Technol. 1983, 38, 114-l 17 Green, A. A. Studies in the physical chemistry of the proteins. J. Biol. Chem. 1931, XC111 (2), 495-516 Przybycien, T. M. and Bailey, J. E. Solubility-activity relationships in the inorganic salt-induced precipitation of a-chymotrypsin. Enzytne Microb. Technol. 1989, 11, 264-276 Stanley, P. G. Salting-out of multi-component systems with and without the use of constant final volumes. Biochim. Biophys. Acta 1963. 75,442444 Dixon, M. and Webb, E. C. Enzyme fractionation by salting-out: A theoretical note. Adv. Prot. Chem. 1961, 16, 197-219 Cascone, 0.. Andrews, B.A. and Asenjo, J. A. Partitioning and purification of thaumatin in aqueous two-phase systems. Enzyme Microb. Technol. 1991, 13, 629-635 Schmidt, A. S., Ventom, A. M. and Asenjo, J. A. Partitioning and purification of cY-amylase in aqueous two-phase systems. Enzyme Microb. Technoi. 1994, 16, 131-142 Hodgson, C. Two-phase aqueous systems for the separation and purification of tPA (tissue plasminogen activator). Ph.D. thesis. University of Reading, UK, 1992 Johansson, G. Partitioning of proteins. In: Partitioning in Aqueous Two-Phase Systems: Theory. Methods, Uses and Applications in Biotechnology (Walter, H.. Brooks, D. E., and Fisher, D.) Academic Press, London, 1985, 161-226 Baskir, J. N., Hatton, T. A., and Suter, U. W. Protein partitioning in two-phase aqueous polymer systems. Biotechnol. Bioeng. 1989,34, 541-558 France, T. T., Andrews, A. T., and Asenjo, J. A. Use of chemically modified proteins to study the effect of a single protein property on
15
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partitioning in aqueous two-phase systems: Effect of surface hydrophobicity. BiotechnoL Bioeng. 1996, 49, 3OC308 Asenjo, J. A.. Schmidt, A. S., Hachem, F., and Andrew% B. A. A model for predicting the partition behaviour of proteins in aqueous two-phase systems. J. Chromat. 1994, 668, 47-54 Reitherman, R., Flanagan, S. D., and Barondes, S. H. Electromotive phenomena in partition of erythrocytes in aqueous polymer twophase systems. Biochem. Biophys. Acta 1973, 297, 193-202
28.
29. 30.
partitioning
of proteins:
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et al.
Gaiscone, P. S. and Fisher, D. The dependence of cell partition in two-polymer aqueous phase systems on the electrostatic potential between the phases. Biochem. Sot. Truns. 1984. 12(6), 1085-1086 Albertsson, P. A. Partition of proteins in liquid polymer-polymer two-phase systems. Nature 1958. 182, 709-71 I Sasakawa. S. and Walter. H. Partition behaviour of native proteins in aqueous dextran-poly(ethylene glycol) phase systems. Biochrmistry 1972, 11, 2760-2765
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