Ind. Eng. Chem. Res. 2008, 47, 5203–5213
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Salting-Out of Lysozyme and Ovalbumin from Mixtures: Predicting Precipitation Performance from Protein-Protein Interactions Yu-Chia Cheng, Carolina L. Bianco, Stanley I. Sandler, and Abraham M. Lenhoff* Department of Chemical Engineering, UniVersity of Delaware, Newark, Delaware 19716
Protein-protein interactions, whether desirable or not, can have direct effects on protein separations. The most obvious of these are interactions between similar molecules, which determine the thermodynamic properties and phase behavior of protein solutions. Interactions between dissimilar molecules also affect the properties and phase behavior of protein mixtures and, therefore, protein separations. Here, we seek to quantify these effects by comparing precipitation behavior from binary solutions with direct measurements of protein-protein interactions using cross-interaction chromatography, which is a variant of affinity chromatography that provides data that can be interpreted in terms of the virial cross-coefficient. First, the effects of pH, ionic strength, different precipitants and the initial protein concentrations and their ratio on the binary precipitation of lysozyme and ovalbumin were investigated. Next, self- and cross-interaction measurements were used to suggest the optimal precipitation conditions for separating lysozyme from ovalbumin. The results show that protein interactions can explain anomalies and inconsistencies that frequently confound the extraction of meaningful general trends in separations analyses. Introduction Selective precipitation from a protein mixture is usually performed under the assumption that each protein can be precipitated at a different precipitant concentration at fixed pH and temperature. Dixon and Webb1 proposed that, if the solubility curves of a pair of proteins are well-separated, there can be two distinct precipitation steps in which precipitation of the less soluble protein is virtually complete before precipitation of the more soluble protein begins. If a clear separation of the two proteins cannot be obtained, adjustment of the initial protein concentrations or temperature and pH is required to shift the precipitation zone of the desired protein, although it may still be impossible to separate the two proteins completely at high precipitant concentration. Richardson et al.2 proposed an alternative systematic approach for fractionation utilizing the desired protein and total protein solubility profiles with increasing precipitant concentration. By comparing the purification factor at a given yield under various precipitation conditions (pH, temperature, and initial protein concentrations), the optimum process parameters for fractionation could be obtained. Most fractionation techniques, including those previously discussed, assume that each protein precipitates independently of others under the given solution conditions. This implies that the precipitation curve is the superposition of the precipitation solubilities of the constituent proteins, provided that the precipitant concentrations at which the respective proteins first precipitate do not coincide.1 However, there is little literature on the theoretical basis for this method, although the experimental precipitation curves of several proteins3–7 are in agreement with such predictions. If a precipitating protein coprecipitates other proteins, the precipitation curves of different proteins will be interdependent, and a sharp separation will not occur. Coen et al.8 found that the partitioning of ovalbumin and chymotrypsin in ammonium sulfate solutions could be enhanced by the presence of lysozyme, whereas the partitioning of lysozyme decreased in the presence of large proteins. This agrees * To whom correspondence should be addressed. Tel: +1-302-8318989. Fax: +1-302-831-4466. E-mail address:
[email protected].
with the observation that the larger protein partitions more strongly into the precipitate phase from a binary protein mixture.9 The interdependence of precipitation of proteins in a mixture is presumably a manifestation of protein cross-interactions, which have been measured by other methods. Lysozyme-bovine serum albumin (BSA) interactions were found to be pHdependent in measurements of reversible association, because of electrostatic attraction at low ionic strength.10 Association of ovalbumin and lysozyme has also been observed at neutral or slightly acidic pH and low ionic strength by sedimentation equilibrium,11,12 membrane osmometry,13 and fluorescence anisotropy.14 At pH 4 and 0.1 M acetate, however, ovalbumin has no measurable effect on lysozyme crystal growth.15 Attractive interactions between different proteins have also been reported in several systems via measurements of the osmotic second virial cross-coefficients (B23). Membrane osmometry measurements showed slightly attractive cross-protein interactions (slightly negative B23) between lysozyme and BSA in ammonium sulfate solutions, with a clear pH dependence.16 Patterns of protein cross-interactions have also been reported using chromatographic techniques. Ovalbumin-lysozyme and BSA-lysozyme interactions in aqueous salt solutions with ionic strengths between 0.1 m and 1.0 m are mostly attractive and strongly dependent on pH, ionic strength, and salt type.17 The cross-interactions of lysozyme-BSA and lysozyme-R-chymotrypsinogen have been measured in ammonium sulfate and sodium chloride solutions, and some counterintuitive trends for lysozyme-R-chymotrypsinogen interactions were observed.18 Protein-protein interactions can not only alter the solubility of a protein in a mixture, but also have a direct effect on performance measures of protein separations. The interactions between similar molecules determine the thermodynamic properties and phase behavior of protein solutions. George and Wilson correlated solution conditions that promote crystallization of various proteins with values of the osmotic second virial coefficient (B22).19 They proposed B22 as a predictor for protein crystallization, based on their observation that crystallization occurs in a fairly narrow range of slightly negative B22 values
10.1021/ie071462p CCC: $40.75 2008 American Chemical Society Published on Web 07/08/2008
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(-1 × 10-4 to -8 × 10-4 mol mL/g2), which they called the crystallization slot. Solutions in which the protein has more negative values of B22 were observed to lead to protein precipitation, whereas solutions with positive values of B22 did not display any phase separation. Values of B22 have been measured as a function of salt concentration and with different salts, in attempts to explain the Hofmeister series for saltingout precipitation.20–24 Interactions between dissimilar molecules can also affect other types of protein separations. For example, the ultrafiltration selectivity of protein mixtures, which involves heterogeneous association, can be adjusted by changing the repulsive protein-protein cross-interactions through changes in solution pH and ionic strength.25–33 These observations were quantified in a correlation between the second virial cross-coefficient and the lysozyme sieving coefficient in ultrafiltration from mixtures with BSA.34,35 In this work, we examine the binary precipitation of lysozyme and ovalbumin as a function of initial protein concentration, ionic strength, pH, and salt type, and we also study the correlation between protein-protein interactions and the selective precipitation of lysozyme and ovalbumin from their mixture. The goal is to be able to use information on protein-protein interactions as a predictor of the optimum conditions for the selective precipitation of proteins, to avoid a painstaking trialand-error process that would require numerous solubility and separation measurements under a large number of solution conditions. Materials and Methods Materials and Solutions. Lysozyme (L6878), ovalbumin (A2512), ammonium sulfate (A2939, ACS grade), bis-tris (B7535), citric acid (C1909), MES (2-(N-morpholino)ethanesulfonic acid, M8250), NHS (N-hydroxysuccinimide) (H7377), EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, E6383), ethanolamine (E9508), and sodium cyanoborohydride (S8628) were purchased from Sigma. Sodium chloride (S271-500, ACS grade), anhydrous sulfuric acid (A300), ammonium hydroxide (A669), anhydrous monobasic sodium phosphate (BP329), potassium phosphate (P288, ACS grade), and sodium hydroxide (S318) were purchased from Fisher Scientific. Anhydrous dibasic sodium phosphate (191440) was purchased from ICN Biomedicals, and 1 N hydrochloric acid (12421-0010) was purchased from Acros. Micro-BCA assay reagents (23231BP, 23232 BP, and 23234BP) were purchased from Pierce. Toyopearl AF-Formyl-650 M (08004) and AF-Amino-650 M (08002) chromatography particles were obtained from Tosoh Biosep. Ovalbumin was also obtained by repeated crystallization36 from the whites of two dozen fresh single-comb white Leghorn (SCWL) eggs, collected within 24 h of being laid. The crystalline ovalbumin was used for the binary precipitation measurements, because of the sensitivity of phase equilibrium measurements to small amounts of impurities, whereas the commercial ovalbumin was used for cross-interaction measurements to be consistent with the material used in previous such studies;17,23 the latter measurements are only slightly affected by the presence of impurities. Concentrated protein and salt stock solutions were prepared using distilled deionized (DI) water that was purified through a Millipore Milli-Q system. The protein and salt solutions for the precipitation measurements were prepared in 50 mM of pH 7 phosphate or pH 4 acetate buffers. Based on the chosen precipitant, protein and salt stock solutions were adjusted to the desired pH values with 1 N sodium hydroxide, 1 N
hydrochloric acid, 1 N sulfuric acid or 1 N ammonium hydroxide, and the pH measured with a Chemcadet 5984 digital pH meter. After pH adjustment, the solutions were filtered through 0.22 µm Millipore Millex-GV syringe filters and degassed for 5 s in a Branson 2510 ultrasonic cleaner before use. Protein-free buffer and salt solutions for chromatography were filtered through 0.22 µm Gelman bottle top filters (4632) into 2-L Corning sterile roller bottles (431133) and stored at room temperature. Binary Precipitation of Lysozyme and Ovalbumin. The effects of initial protein concentration, pH, and salt type on protein precipitation were examined. Protein and salt stock solutions and DI water were combined to produce the desired feed point for each precipitation experiment. The concentrations of the lysozyme, ovalbumin, sodium chloride, and ammonium sulfate stock solutions were 0.1, 0.1, 0.25, and 0.4 by mass fraction, respectively. The protein and salt solutions and any required DI water were gently and continuously mixed in a 1.5 mL Eppendorf tube on a Barnstead/Thermolyne Labquake shaker (8 rpm) at room temperature. After 2 h, mixing was stopped and the two phases were separated using a Beckman Coulter Optima L-100XP ultracentrifuge with a SW60Ti rotor at 47500 rpm for 30 min. After centrifugation, the supernatant phase was removed carefully by pipet into a vial and diluted with DI water. The precipitate in the ultracentrifuge tube was recovered by redissolving it gravimetrically with DI water for subsequent analysis. Assays. Protein concentrations in both the supernatant and dense phases in single-protein precipitation were determined by measuring the absorbance at 280 nm using a Perkin-Elmer Lambda 4B UV/Vis spectrophotometer. The extinction coefficients used for lysozyme and ovalbumin were 2.635 L/(g cm) (ref 37) and 0.705 L/(g cm) (ref 38), respectively. Solutions were diluted before measurement to keep the absorbance between 0.1 and 0.9. The dense phases all redissolved readily following dilution. Protein concentrations in binary mixtures were determined by cation-exchange chromatography using a 6.0 mm inner diameter (ID) × 3.6 cm long SP Sepharose FF column in a Waters 2695 separation module. Twenty microliters of each solution was eluted at a flow rate of 0.2 mL/min, using a linear salt concentration gradient over 20 min at pH 7 over a range of 0.1-0.9 M for (NH4)2SO4 or 0.2-0.8 M for NaCl solutions. Detection was by absorbance at 280 nm on a Waters 2996 photodiode array detector. The concentrations of lysozyme and ovalbumin in binary mixtures were calculated by converting the integrated peak areas to protein concentrations based on calibration curves constructed using pure protein samples of given concentrations in isocratic elution at 1.0 M (NH4)2SO4. Salt concentrations are expressed in terms of the ionic stength in units of molality (m). The concentrations of ammonium sulfate and sodium chloride were determined by comparing the conductivity of the solution measured using a Cole-Parmer 19820 conductivity meter with a previously prepared calibration curve of conductivities as a function of salt concentration. The difference between concentrations determined from the measured conductivity and gravimetrically determined concentrations was 80%) or shows a dependence on injection concentration,39 raising concerns about the contributions of multibody interactions on the measured second osmotic virial coefficients.17,18,39 Therefore, most CIC measurements were performed on the ovalbumin-immobilized columns at a surface coverage of 31%. Figure 6 shows the similar trends of B23 in both NaCl and ammonium sulfate solutions at pH 4 and pH 7, although the values are quantitatively different, which reflects the difference in the effectiveness of salts in inducing cross-jnteractions between the unlike proteins. At low NaCl ionic strength, the overall ovalbumin-lysozyme interactions are, based on the values of B23, slightly repulsive at pH 4 and attractive at pH 7, which is expected, based on the net charge of the proteins as a function of pH (see Figure 6a). At pH 4, both lysozyme and ovalbumin are positively charged, and the cross-interactions are correspondingly repulsive. At pH 7, the two proteins are oppositely charged and the measured interaction is attractive, but as the ionic strength increases, the electrostatic interactions are screened, making the interactions less attractive. At low (NH4)2SO4 ionic strength, the behavior of B23 is consistent with what is expected based on electrostatic interactions (see Figure 6b), although the B23 value measured in the ovalbumin column is slightly negative, indicating significant nonelectrostatic effects. This may be due to anisotropic local attractive electrostatic interactions that contribute disproportionately to the value of B23.53 The significant difference in Figure 6 of the pH trends of B23 at high ionic strength indicates that forces other than electrostatic interactions are involved, which may include van der Waals interactions or the effects of protein hydration.17,18,53 Protein hydration is a strong function of the protonation state of the amino acids, and increasing the pH from ∼4 to ∼5 leads to stronger hydration.54 The lesser extent of hydration at lower pH may lead to the stronger attraction observed at pH 4 than at
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Figure 7. Effects of pH and ionic strength on the ratio of lysozyme to ovalbumin partitioning in (a) NaCl ((O) pH 7, (]) pH 4) and (b) (NH4)2SO4 ((0) pH 7 and (4) pH 4) solutions. Initial concentration of each protein was 30 mg/g H2O.
pH 7, because the acidic residues are likely to be at least partially protonated at pH 4.23 Discussion Phase Behavior in Binary Precipitation. The lysozyme supernatant concentrations in binary precipitation in ammonium sulfate solutions at pH 7 and pH 4 (Figures 1 and 3, respectively) are qualitatively and quantitatively similar to those in lysozymeonly precipitation and to those reported previously for binary precipitation.55 However, the ovalbumin supernatant concentrations in binary precipitation show quantitative differences when compared with ovalbumin-only precipitation results. The observed independence on initial concentration of the lysozyme and ovalbumin supernatant concentrations in binary precipitation at a fixed lysozyme:ovalbumin ratio suggests that, in binary precipitation, each protein behaves as in single-protein precipitation. When the temperature and the salt concentration are fixed, the protein concentration in the supernatant at equilibrium is not affected by the amount of precipitate. The decrease in ovalbumin supernatant concentration with ionic strength can be correlated with the initial lysozyme:ovalbumin ratio and may result from lysozyme-ovalbumin association. Lysozyme and ovalbumin have been observed to associate at low ionic strength at various pH values and buffer concentrations.11–14 The measured second virial cross-coefficients (Figure 6b) suggest that the interactions between lysozyme and ovalbumin become increasingly attractive with increasing ionic strength at pH 4 and pH 7, which is consistent with increasing association at high ammonium sulfate concentrations. The precipitation results in NaCl solutions (see Figures 4 and 5) show two differences from those in ammonium sulfate solutions. At pH 7, only lysozyme is precipitated and ovalbumin remains soluble up to 4 m ionic strength at an initial concentration of 30 mg/g H2O. The precipitation of lysozyme in the binary mixture is independent of its initial concentration and is unchanged in the presence of ovalbumin. However, at pH 4, only ovalbumin precipitates, whereas lysozyme gels. The condition for lysozyme gelation and the supernatant concentration at which gelation occurs is dependent on the initial total protein concentration, the initial lysozyme:ovalbumin concentration ratio, and ionic strength. The different behavior in NaCl
solutions may reflect the greater effectiveness of NaCl than of (NH4)2SO4 (reverse Hofmeister series behavior)56 in precipitating lysozyme, and the greater effectiveness of (NH4)2SO4 over NaCl in precipitating ovalbumin.57,58 Measures of Separation Performance. Both recovery and selectivity must be taken into consideration when optimizing the conditions for separation. One possible design criterion for the relative recovery of the components in a mixture is the ratio of partition coefficients,8 which is shown for the various conditions studied here in Figure 7. However, this ratio can be misleading, because it fails to give a clear picture of how well a target species is concentrated or purified. For example, although the highest lysozyme partition ratio (Klys/Kova) in NaCl solutions is found at pH 7, the recovery of lysozyme at this pH is lower than that at pH 4. Moreover, the small (
