Protein Separation via Polyelectrolyte Complexation - American

Chapter 5

Protein Separation via Polyelectrolyte Complexation Mark A. Strege, Paul L. Dubin, Jeffrey S. West, and C. Daniel Flinta

Downloaded by CORNELL UNIV on June 3, 2017 | http://pubs.acs.org Publication Date: June 12, 1990 | doi: 10.1021/bk-1990-0427.ch005

Department of Chemistry, Indiana University-Purdue University, Indianapolis, IN 46205-2810

The complexation of proteins with synthetic strong polycations may lead to selective coacervation. For two proteins, A and B, selectivity may be defined as S = ([A]c/[B]c)([A]s/[B]s)-1, where the subscripts c and s refer to coacervate and solution phases. We have measured S for mixtures of two proteins and the polycation poly(dimethyldiallylammonium chloride), and found it to be a function of solution pH and protein isoelectric point. Protein-polyelectrolyte coacervation occurs abruptly at a critical pH which depends strongly on the ionic strength I. Plots of pHcritvs.I may thus be viewed as phase boundaries. Analysis of the effects of protein:polyion stoichiometry on the phase boundary suggests that phase separation may be a consequence of the saturation of binding sites on the polymer with protein. Assuming that electroneutrality of the complex occurs at the point of coacervation, it is possible to estimate the number of proteins bound per polycation at the point of phase separation, n . Comparison of the phase boundaries for several proteins reveals that the net protein surface charge density does not control phase separation, but instead suggests the importance of charge patches on the protein surface. Size-exclusion chromatography, via the Hummel-Dreyer method, reveals the existence of a primary complex in which the degree of intrapolymer protein binding depends on pH. The average number of proteins bound per polymer as obtained by this technique, n, is seen to depend upon thefreeprotein concentration. The value of n corresponding to polymer saturation is in fair agreement with n from phase separation studies. crit

crit

Oppositely charged polyelectrolytes interact to form complexes. Depending primarily on the molecular weights and linear charge densities, these complexes may be amorphous solids (1), liquid coacervates (2,3), gels (4), fibers (4), or soluble aggregates (5-7). One particular case of inter-macroion complex formation involves synthetic polyelectrolytes and globular proteins. The formation of these complexes is generally evidenced by phase separation, where the denser, polymer-rich phase may 0097-6156/90/0427-0066$06.00/0 © 1990 American Chemical Society

Ladisch et al.; Protein Purification ACS Symposium Series; American Chemical Society: Washington, DC, 1990.


5. STREGEETAL.

Separation via Polyelectrolyte Complexation

67

be a liquid "complex coacervate" (8) or a solid precipitate. Examples of the former have teen observed for gelatin and polyphosphate (9), and serum albumin and poly(dimethyldiallylammonium chloride) (10). Systems that exhibit precipitation include hemoglobin and dextran sulfate (11), carboxyhemoglobin and potassium polyvinyl alcohol sulfate) in the presence of poly(dimethyldiallylammonium chloride) (PDMDAAC) (12), lysozyme and poly(acrylic acid) (13), and R N A polymerase and poly(ethyleneimine) (14). The ability of polyelectrolytes to remove proteins from solutions represents tremendous potential in the area of protein fractionation. Protein separation may occur at two steps in the protein complexation/recovery process: at the point of complex formation, or at the point of complex redissolution. The second approach has recently been exploited through the incorporation of protein-polymer precipitation stages into a variety of protein purification procedures, wherein the precipitated proteins are selectively recovered from the insoluble complex aggregate via step-wise selective redissolution by pH or ionic strength adjustment (14-16). However, selectivity in the first step in the complexation/recovery process, complex formation, has not been thoroughly studied. Earlier demonstrations of preferential complexation of polyelectrolytes with specific proteins (13,17), suggest the value of further investigations along this line. The use of polyelectrolyte-precipitation to separate proteins offers several advantages over other protein fractionation techniques. The recovery of proteins through the formation of insoluble complexes with polyelectrolytes appears to be a non-denaturing process, inasmuch as Sternberg and Hershberger reported high recoveries of activities for enzymes precipitated with polyacrylic acid (13), while other workers have reported the non-denaturing fractionation at slightly alkaline pH of intracellular proteins using synthetic polycations (14,16,18). Furthermore, compared to other methods for protein separation, e.g. chromatography, selective precipitation offers great economy with regard to materials and process, and, furthermore, is virtually unlimited in scale. Thus, an elucidation of the principles governing protein selectivity in polyelectrolyte separation would be of considerable applied significance. Evidence for the existence of a stable, soluble intrapolymer B S A P D M D A A C complex has been obtained (20). The model for protein-polyelectrolyte coacervation may thus prove to be similar to that proposed by Tsuchida, Abe, and Honma for the coacervation of linear oppositely charged polyelectrolytes (21). The initial (possibly cooperative) binding of proteins via electrostatic interaction leads to soluble complex formation. A subsequent increase in the energy of binding occurs in response to an increase in protein surface charge, and results in the exclusion of water molecules. At the same time the net charge of the complex approaches zero. The complex thus acquires hydrophobic character, leading to aggregation and phase separation . This process is schematically depicted in Figure 1. Studies of complexation between globular proteins and synthetic strong polycations were undertaken to gain insight into protein-polyelectrolyte coacervation selectivity. In the present work, two approaches were applied toward the study of the mechanism of protein-polyelectrolyte interaction. The first approach involved the investigation of phase separation, and the analysis of the phase boundary plots that show the ionic strength dependence of the critical pH. Since selective binding in soluble complexes is believed to be prerequisite to coacervation selectivity, a second approach involved the characterization of soluble complexes at conditions prior to phase separation. The goal of this second approach is the determination of fundamental parameters, such as protein-polymer binding constants, from which coacervation selectivity can be predicted. Soluble complexes were characterized by size-exclusion chromatography (SEC) to gain insight into the cooperativity of



PROTEIN PURIFICATION


5. STREGE ET Al»


69

binding, the structure of the complex, and the complex stoichiometry, i.e.. the number of protein molecules bound per polyion. In principle, such methods may also yield the number of binding sites per polymer molecule, and the intrinsic association constant. EXPERIMENTAL Materials: Poly(dimethyldiallylammonium chloride) (PDMDAAC), a commercial sample "Merquat 100" from Calgon Corp. (Pittsburgh, PA) possessing a nominal molecular weight of 2x10^ and a reported polydispersity of M / M « 10, was dialyzed and freeze-dried before use. A l l proteins were obtained from Sigma Chemical Corp.


w

n

Turbidimetric Titrations: Solutions were prepared as mixtures of P D M D A A C (0.05 -1 gA) and protein (0.25-25 g/1), corresponding to protein/polymer weight ratios (r) ranging from 0.25 to 200, at pH 4 - 5, in dilute (0.05-0.3 M) NaCl. The optical probe (2 cm path length) of a Brinkmann PC600 fiber optics probe colorimeter, and a pH electrode connected to an expanded scale pH meter (Orion 811, or Radiometer model 26), were both placed in the solution. Changes in turbidity were monitored as %T, relative to a blank (polymer-free) solution, as the pH was adjusted by the addition of dilute (0.01-0.10 M) NaOH. Critical pH, the pH at which phase separation takes place, was determined using the method described elsewhere (10). Coacervation Selectivity Measurements: In order to determine the selectivity of synthetic polycations toward complexation with specific proteins, the following experimental procedure was devised. A mixture of P D M D A A C and two proteins, in 0.01 M NaCl, was titrated with NaOH until a desired pH or percent transmittance was reached. The titrated solution was centrifuged (2000 rpm, 10-15 minutes), the supernatant was removed, and the coacervate centrifugate was resuspended in a small volume (2.0-10.0 ml) of acidified (pH 4-5) 0.4 M NaCl buffer. The protein contents of both supernatant and redissolved coacervate were then analyzed via SEC (all protein peaks were baseline resolved). Size-Exclusion Chromatography: SEC was carried out on an apparatus comprised of a Minipump (Milton Roy), a model 7012 injector (Rheodyne) equipped with a 100 μΐ sample loop, an R401 differential refractometer (Waters), and a Model 120 U V detector (λ = 280 nm) (Gilson). A Superose-6 column (30cm χ 1cm OD) (Pharmacia) was eluted at 0.53 ml/min. Column efficiency, determined with acetone, was at least 12,000 plates/meter. Injections were performed in mobile phases at pH 8.0, which is below critical pH at this ionic strength. To determine complex stoichiometry, we have used the Hummel-Dreyer (HD) method (22, 23), as recently applied to dextran-hemoglobin complexes (19). H D experiments were carried out employing a 0.25 M NaOAc buffer as mobile phase, and the protein concentration in the mobile phase varied from 0.10 to 8.00 mg/ml. Polymer samples (2.0 mg/ml) were filtered (0.2 μπι ) before injection. To decrease the degree of polymer M W polydispersity, P D M D A A C was fractionated via SEC, prior to use in H D experiments. The fractionation of P D M D A A C was carried out using a mobile phase of 0.4 M NaOAc buffer, pH = 7.0, which has been found to sufficiently repress adsorption effects, especially in regard to the polycation (24). 40.0 mg of polymer were applied to a Superose-6 prep, gel column (100 ml gel bed) via a 1.0 ml sample loop. The mobile phase was eluted through the column at a velocity of 2.0 ml/min, and the eluent was monitored


70

PROTEIN PURIFICATION

using a R401 differential refractometer (Waters). The injected sample was separated into nine fractions, collected at 6.7 ml intervals following the beginning of sample elution, possessing average polydispersities (calculated through the use of a pullulan calibration curve) of 1.5. RESULTS A N D DISCUSSION


Coacervation Selectivity Experiments: Although separative coacervation of proteins by synthetic polyelectrolytes has been reported (13-18), no parameter describing the selective binding has been formally described. Thus, we define proteinpolyelectrolyte coacervation selectivity as the comparative tendency of a given protein to be removed, in the presence of a second protein, by an oppositely charged polyelectrolyte. Since the concentrations of proteins present in both the coacervate and soluble phases, after centrifugation, may readily be determined, it is convenient to define the selectivity as =

[Ay[B] [A]^B]

c

S

where [ A ] and [ B ] are the weight concentrations of protein A and protein Β in the pelleted coacervate, respectively, and [ A ] and [B] are the weight concentrations of the two proteins in the supernatant. For S>1, protein A must have an isoelectric point (pi) (the pH at which a protein has zero electrophoretic mobility) equal to or lower than that of protein B . Complex formation and coacervation occur in response to electrostatic interactions. Since the charge of P D M D A A C is independent of pH, the surface charge density of the protein molecule or some related variable must - along with the ionic strength - govern phase separation. The pi of a protein should therefore reflect its tendency toward complex formation. For example, in a mixture of B S A (pi = 4.8) and lysozyme (pi = 11.0), the former, with a greater net negative surface charge density at any solution pH, is expected to complex more strongly at intermediate pH. At pH = 6.4, analysis of the coacervate of BSA and P D M D A A C in the presence of lysozyme revealed the phase separation of 90% of the B S A and only 4% of the lysozyme initially present in solution , corresponding to S = 190±10. On the other hand, coacervation of a mixture of B S A and ribonuclease (pi = 9.0), at pH = 5.7, revealed infinite selectivity, with no ribonuclease in the coacervate, showing that coacervation selectivity is not simply a function of ΔρΙ. We propose that the strength of the binding depends not only on the global or net charge, but is also sensitive to the protein surface charge distribution. Further evidence from turbidimetric titrations in support of this suggestion will be discussed later. In a solution of two proteins possessing different pi, S would be expected to be largest at a pH where the potential created by the surface charges on the more basic protein is less than some critical value. For B S A and ribonuclease at I = 0.01, S is plotted vs. pH in the pH region intermediate to the pi's of the two proteins in Fig. 2. S is infinite at pH 5.7, and rapidly drops as pH approaches 9.0. Thus, at pH < 5.7, it appears that the negative charges distributed on the surface of ribonuclease are not capable of generating a negative potential of sufficient magnitude to bind the polycation. c

c

s

s

Turbidimetric Titrations: Since selectivity depends upon coacervation, it is important to study the dependence of phase separation upon solution variables. Turbidimetric titrations were utilized to determine the critical pH of solutions of BSA and Merquat


5. STREGEETAL.

71


100 over a range of I and bulk solution protein:polymer ratio (r). The results are shown in Figure 3 as plots of critical pH vs. I (phase boundaries) over a range of r. In addition, the effect of total solute concentration on critical p H was obtained from Type I titrations at r = 0.25 at B S A concentrations from 0.05 g/1 to 1.0 g/1, for 0.01

Protein Separation via Polyelectrolyte Complexation - American

Recommend Documents