Stoichiometric and Nonstoichiometric Complex ... - ACS Publications

Chapter 17

Stoichiometric and Nonstoichiometric Complex Formation of Bovine Serum Albumin—Poly(dimethyldiallyl ammonium chloride) 1

1,3

1

2

Jiulin Xia , Paul L. Dubin , Lasker S. Ahmed, and Etsuo Kokufuta

Downloaded by CORNELL UNIV on June 16, 2017 | http://pubs.acs.org Publication Date: December 13, 1993 | doi: 10.1021/bk-1994-0548.ch017

1

Department of Chemistry, Indiana University—Purdue University at Indianapolis, Indianapolis, IN 46202 Institute of Applied Biochemistry, University of Tsukuba, Tsukuba, Ibaraki 305, Japan

2

Complexation between bovine serum albumin (BSA) and Poly(dimethyldiallyl-ammonium chloride) (PDMDAAC) was studied by static and quasi-elastic light scattering (QELS), electrophoretic light scattering and turbidimetric titration in dilute electrolyte solution. Both QELS and turbidimetric titration show that complexation occurs at pH ≥4.6 for the ionic strength of 0.01 M. The results obtained support the following mechanism. Upon addition of PDMDAAC to BSA at I = 0.01 M, a stoichiometric complex is initially formed. Subsequent addition of polymer causes this complex to form a coacervate with concomitant charge neutralization. The stoichiometric complex was found by static light scattering to have 120 BSA bound per polymer chain. At higher ionic strengths (0.05 and 0.10 M), the initial complex is nonstoichiometric, and coacervates are formed by the aggregation of the complex. These coacervates are observed as ca 700 nm droplets by optical microscopy. Proteins interact strongly with natural and synthetic polyelectrolytes mainly through electrostatic forces. These forces may lead to die formation of soluble complexes/1'2) complex coacervates(3"6) and amorphous piecipitates(7'9). The practical consequences of these phase changes may include (a) the use of polyelectrolytes for protein separation (10-12), (b) immobilization or stabilization of enzymes in polyelectrolyte complexes (13), and (c) the modification of proteinsubstrate affinity (14). Related phenomena are also undoubtedly significant in the cell, as in, for example, the Coulombic association of DNA with basic histones which leads to the collapse of the nucleic acid; and the effects of basic polypeptides such as polylysine on DNA behavior. Electrostatic interactions between proteins and nucleic acid are also likely to play a role in the transcription process (15). 3

Corresponding author

0097-6156/94/0548-O225$06.00/0 © 1994 American Chemical Society

Schmitz; Macro-ion Characterization ACS Symposium Series; American Chemical Society: Washington, DC, 1993.


226

MACRO-ION CHARACTERIZATION

We have been investigating the interaction between synthetic polyelectrolytes and proteins (16-19). One motive is the possible use of protein phase separation by polyelectrolytes for protein purification and recovery. In this process the target proteins are obtainedfirstthrough selective protein-polyelectrolyte phase separation, thenrecoveredby adjusting pH or ionic strength (10,20). The efficiency of final protein purification andrecoverydepends largely on the phase separation step. The phase separation of proteins by polyelectrolytes, commonly called precipitation, may in fact be more frequently coacervation. In coacervation, the concentrated phase is a second liquid phase, while precipitation corresponds to formation of a solid (21). The distinction between coacervation and precipitation becomes apparent upon centrifugation. It appears likely that precipitation is characteristic of oppositely charged polyelectrolytes with symmetric charge spacing at low ionic strength (22), while coacervation is typical of weakly charged and/or asymmetric polyelectrolyte systems. Macromolecular complex coacervation, of which protein-polyectrolyte coacervation is a special case, usually occurs when mixing two oppositely charged polyelectrolytes. This phenomenon wasfirststudied by Bungenberg de Jong (21). It was found that the coacervaterepresentsa kind of aggregate colloidal particle in aqueous solution. These particles appear as floating microscopic droplets, which may fuse with each other but do not mix with the solvent An important question is therelationshipbetween complex formation and phase separation. Even though protein-polyelectrolyte complexes have been examined for more than two decades, few studies have dealt with the mechanism of complex formation. In the purification of lysozyme from egg white by precipitation with polyelectrolytes, Glatz et al (11) first suggested that the protein-polyelectrolyte floes or coacervates are formed by aggregation of so called "primary particles'*. Recently, Berg et al (23)reporteda mechanism for floe formation of lysozyme-polyacrylic acid. It was concluded that coacervation is due to polymer bridging at high polymer MW (over one million), and to charge neutralization at low polymer MW (several thousand). In studies of both Glatz and Berg, the initial formation of the "primary particle" was not fully discussed; in particular, they did not elucidate mechanism of formation of the primary particle or its structure. Other studies (7, 13, 24) dealing with protein-polyelectrolyte complex composition and properties have provided more focus on the molecular level. For the complexation of bovine serum albumin (BSA), ribonuclease and lysozyme with both polycations and polyanions, Dubin et al (16,19) proposed formation of soluble complexes prior to phase separation. Kokufuta (25) employed colloidaltitrationto study complexation between human serum albumin (HSA) and poly(dimethyldiallylammonium chloride) (PDMDAAC) and potassium polyvinyl alcohol sulfate) (KPVS) in pure water. Titrating the protein with the polyelectrolytes, he found turbidity maxima (referred to as "end points") corresponding to conditions under which the mole numbers of quaternary ammonium groups in PDMDAAC and sulfate groups in KPVS were approximately identical to the contents of the acidic and basic groups in HSA. Therefore, it was concluded that the complexation between HSA and PDMDAAC and KPVS involves "stoichiometric" binding. Stoichiometric polyelectrolyte complexes have been thoroughly studied since the 1970s. The mechanism of complex formation is believed to be aresultof charge neutralization, i.e. theratiobetween the oppositely charged groups in the complexes is 1:1 (22, 26). One of the most important properties of the complex is insolubility in both water and organic solvents (22,26).



17. XIA ET AK

Stoichiometric and Nonstoichiometric Complex Formation 2

There are also reports of non-stoichiometric polyelectrolyte complexes. Kabanov (22) first reported that non-stoichiometric water-soluble polyelectrolyte complexes aie formed by poly(N^-dimethylaminoethylmethacrylate hydrochloride) and sodium polyphosphate. It was found that the complexes in aqueous salt media undergo considerable conformational change prior to phase separation. Conformation change was also found for some other non-stoichiometric proteinpolyelectrolyte complexes in salt solution (18,27-28). Consideration of the foregoing results suggests that stoichiometry may be related to salt concentration, but further understanding is needed. In particular, it is not clear how stoichiometric and non-stoichiometric binding are controlled, and how the two different binding processes lead to phase separation. In the present paper, we investigate complexation between BSA and PDMDAAC, focusing in particular on complex structure, and on the effect of ionic strength on binding stoichiometry and coacervate formation.

Experimental Materials: Poly(dimethyldiallylammonium chloride) with the following chemical structure,

PDMDAAC a commercial sample "Merquat 100" from Calgon Corporation (Pittsburgh, PA) possessing a nominal molecular weight of 2x10^ and M /M =10, was dialyzed and freeze-dried before use. Bovine serum albumin (pi = 4.9) was obtained from Sigma as 95-99% pure. Monobasic and dibasic sodium phosphate salts and sodium chloride with AR grades were obtained from Mallinckrodt Inc. Distilled and deionized water was used in all experiments. w

n

Sample Preparation Solutions for "type Γ titration were made in 0.01 M NaCl to maintain constant ionic strength (I). For type ΠΙ" titration, solutions were prepared in ionic strength I = 0.01 M pH 7.88 phosphate buffer. All solutions were made dust-free by filtering through Gelman 0.2 μιη syringe filters. N

Methods Turbidimetric Titration. Turbidimetric titrations were carried out at 22°C in solutions of the desired ionic strength. A 2 ml microburet was used to deliver titrant and the turbidity was followed with a Brinkmann PC600 probe colorimeter (420 nm, 2 cm pathlength). Solutions were always stirred, and turbidity values were obtained after several minutes of stabilization in all cases. Two types of titration were


228



involved in the study. In "type Γ titrations NaOH was added to an initial solution of PDMDAAC, BSA and NaCl at a pH around 4. A pH electrode connected to Beckmann Φ 34 pH meter was used to monitor pH change during the titration. The turbidity was monitored as a function of pH. In the other type of titration, referred as "type ΠΓ, BSA in phosphate buffer solution was titrated with PDMDAAC. The turbidity was recorded as a function of the concentration ratio of [PDMDAAC]/[BSA] = W (= 1/r). Quasi-Elastic Light Scattering (QELS). QELS measurements were made at scattering angles from 30° to 150° with a Brookhaven (Holtsville, NY) 72 channel BI-2030 AT digital Correlator and using a Jodon 15 mW He-Ne laser (Ann Arbor, MI). A 200 μηι pinhole aperture was used for the EMI photomutiplier tube, and decahydronaphthalene (decalin) was used as the refractive index matching fluid to reduce stray light We obtain the homodyne intensity-intensity correlation function G(q,t), with q, the amplitude of the scattering vector, given by q=(4jcn/X)sin(9/2), where η is the refractive index of the medium, λ is the wavelength of the excitation light in a vacuum, and θ is the scattering angle. For a Gaussian distribution of intensity profile of the scattered light, G(q,t) is related to the electric field correlation function g(q,t) by: 2

G(q,t) = A(l + bg(q,t) )

(1)

where A is the experimental baseline and b is a constant, which depends on the number of coherence areas that generates the signal (0 < b < 1). The quality of the measurements were verified by determining that the difference between the measured value of A and the calculated one was less than 1%. The electric field correlation function depends on the Fourier transform of the fluctuating number density of particles or molecules. For the center of mass diffusion of identical particles the following simple relation holds: g(q,t) = e -

t / x

(2)

where τ is decay time and D is diffusion coefficient More detailed discussions of QELS data analysis may be found in refs. (29) and (30). For polydisperse systems the correlation function g(q,t) can be expressed as an integral of die exponential decays weighted over the distribution of relaxation times ρ(τ): t/x

g(t) = Jee- p(x)dx

(3)

In principle, it is possible to obtain the distribution ρ(τ) by integral transformation of the experimental [G(t)/A-l] , but in practice this presents a formidable problem for numerical analysis, since taking the inverse Laplace transform is numerically an illposed problem. Several numerical methods developed so far are devoted to calculating ρ(τ). In the present work, we analyze the autocorrelation functions by 1/2


17. ΧΊΑ ET AL

Stoichiometric and

Nonstoichiometric Complex Formation 22

using the CONTIN program, which employs the constrained regularization method (31). From equation (3), the mean relaxation time, , defined as the area of g(t), is given by = fg(t)dt


0

(4) oo oo = Jxp(x)dx/Jp(x)dx 0 0 This value can be resolved from each of the distribution modes of p(x), as the first moment of the normalized relaxation spectrum. Therefore, the diffusion coefficient, which corresponds to each value of , can be calculated using λ D =

2

(5)

j—^f 2

2

167C sin (9/2) From each D value we obtain Stokes radius, Rs, by the Einstein equation 1

6κΐ\Ό

() 6

where k is Boltzmann's constant, Τ is the absolute temperature and η is the viscosity of the solvent In the present study the experimental uncertainty in Rs is less than 10%.

S t a t i c L i g h t S c a t t e r i n g (SLS). SLS measurements were made with the same Brookhaven system described above. Intensity measurements were calibrated by pure (>99.S%) toluene and optical alignment was ensured by less than 3% deviation from linearity in the I· sinO vs θ plot. Each measurement was carried out for 5 seconds. The average of 10 such measurements was reported as I . These values were used to calculate Rayleigh ratio, Re. In this Re measurement we use as the reference solvent pure BSA solution with the same ionic strength and pH as complex-containing solution. For PDMDAAC, we used the refractive index increment, dn/dc = 0.186, determined by Burkhardt (32) with a differential refractometer. Attempts to measure dn/dc of the complex failed because of scattering effects. Since the complex is comprised of polymer and protein, we calculated (dn/dc) of the complex by using s

x

1 ίάηλ

ΛΛ

dcj ~ l+pldcjp x

|

β ίάηΛ l+pldcj,,

( 7 )

where the subscripts x, p, and pr represent complex, polymer and protein, respectively, β represents the mass ratio of bound protein to polymer, and (dn/dc)pr = 0.185 for BSA (33).



230

Static light scattering results are usually plotted as Zimm-diagrams corresponding to the equation: + 2A c 2

(8) where c is the mass concentration of polymer, Κ is a constant which contains the optical parameters of the system; M and Rg are the weight average molecular weight and the root mean square radius of gyration of the macromolecule, respectively; and A2 is the second virial coefficient


w

Electrophoretic Light Scattering (ELS). ELS measurements were made at four scattering angles (8.6°, 17.1°, 25.6° and 34.2°), using a Coulter (Hialeah, Florida) DELSA 440 apparatus. The electricfieldwas applied at a constant current of 5 mA. The temperature of the thermostated chamber was maintained at 25°C. In ELS, the photon-counting heterodyne correlation function for a solution with an electrophoretically monodisperse solute can be written as (34): 2

2

C(x) = β δ(τ)+α + Oj exp(-2Dq τ) + α exp(-Dq t)cos(Aoyt) 0

0

2

(9)

where βο, Oo> 5.80. This constant diameter of the complex supports the hypothesis of a stoichiometric intrapolymer complex. BSA-PDMDAAC Complexation at Various Ionic Strength, Mass Ratio and Constant pH. Figures 3-5 show Type ΙΠ turbidimetric titrations of various concentrations of BSA with PDMDAAC in pH 7.88 sodium phosphate buffer, at ionic strengths of 0.01, 0.05 and 0.10 M, respectively. The value of pH 7.88 was chosen to ensure coacervate formation on the basis of the type I titrations in Figure 1. All solutions probably exhibit turbidity maxima. In the case of the high BSA concentration (0.6g/L), the plateau is not meaningful, because the sensitivity of the photodetector is not sufficient to record accurate transmittances for such turbid solutions. For the lowest ionic strength, the position of this turbidity maximum is relatively independent of protein concentrations, i.e. the turbidity maximum reveals a certain stoichiometric character. For higher ionic strengths, the position of the maximum shifts to larger relative polymer concentrations with decreasing protein concentration.


231


232


Figure 1. Type Ititrationsof various PDMDAAC concentration in 0.60 g/L BSA and 0.01 M NaCl. r: 50 (Δ); 100 (•); 300 (x) and 500 (+). 400

?

300

3 S

200

ο

ο

δ îoo η. η

ο L—

ρΗ

Figure 2. The complex size obtained by QELS as a function of pH for PDMDAAC in 0.60 g/L BSA and 0.01 M NaCl with r = 300. 100

Ρ

0.1

0.2

0.3

[PDMDAAC]/[BSA]

Figure 3. Type ΙΠ turbidimetrictitrationsof various concentrations of BSA with PDMDAAC at ionic strength 0.01 M and pH 7.88 phosphate buffer solutions. BSA concentrations (g/L): 0.6 (+); 0.12 (O); 0.06 (•); and 0.03 (Δ).


17. XIA ET AJL

Stoichiometric and Nonstoichiometric Complex Formation


100

U.O

0.1

0.2

0.3

[PDMDAAC]/[BSA]

Figure 4. Type ΙΠ turbidimetric titrations of various concentrations of BSA with PDMDAAC at ionic strength 0.05 M and pH 7.88 phosphate buffer solutions. BSA concentrations (g/L): 0.36 (+); 0.12 (O); and 0.06 (•). 80!

U.0

•

•

0.1

•

'

0.2

1

0.3

[PDMDAAC]/[BSA]

Figure 5. Type ΙΠ turbidimetric titrations of various concentrations of BSA with PDMDAAC at ionic strength 0.10 M and pH 7.88 phosphate buffer solutions. BSA concentrations (g/L): 0.36 (+); 0.12 (O); and 0.06 (•).


23

234


In order to understand the distinction between the behavior at high and low ionic strength, measurements of the apparent size of the scattering species were conducted by QELS, at a protein concentration of 0.60 g/L, and at both low (I=0.01M) and high (1=0.10M) salt concentrations. Figure 6a and Figure 7 are the QELS results obtained at low and high ionic strengths, respectively. A dramatic difference between the two ionic strength is obvious. Under the former condition, a remarkably stable species with R «100 nm is observed to form at very low added polymer concentration, and persists as the sole scattering species until a polymer concentration close to the turbidity maximum is attained. The same concentration of polymer corresponds to the onset of a plateau in the electrophoretic mobility, as shown in Figure 6b. Below this concentration, the mobility exhibits a sign change after displaying a constant value of u = -1.3 μηι-cm/V-s. In contrast, QELS measurements at I = 0.10 show that the size of the scattering particle increases rapidly with added polymer even at very low polymer concentration.


s

Static Light Scattering of BSA-PDMDAAC Complex. Prior to static light scattering, the degree of protein binding was first estimated by the following procedure. An equilibrium solution of BSA and PDMDAAC at pH 7.88 and ionic strength 0.01, with r = 300, was centrifuged andfilteredthrough a 20 nm filter (Alltech, Deerfield, IL). Thefiltratewas found to be complex-free by QELS. The concentration of free BSA in this solution was then determined by optical density measurement at 280 nm using the reported extinction coefficient of 4.36x1ο (36). From the free BSA concentration, we obtained β = 39, which corresponds to 120 BSA bound per PDMDAAC chain. Static light scattering was carried out for the complex in 0.01 M phosphate buffer at pH 7.88 and varying C and r. The refractive index increment of the complex was calculated from equation (7) with β = 40. To analyze the SLS results, we ignore the particle interaction term, i.e. the second virial coefficient in equation (8). The error caused by this approximation is less than 5% (37,38) because of the very low polymer concentration (37). Therefore, equation (8) can be simplified to 4

p

where c and M are the concentration and molecular weight of the complex, respectively. c and M are related to the polymer concentration and polymer molecular weight by x

x

x

x

^=€ (1 + β) Μ,=αΜ (1+β) ρ

ρ

(13) (14)

where α is the degree of aggregation, i.e. number of polymer chains within one complex. Substitution of equations (13) and (14) into (12) yields

(15)


17. XIA ET A L


0.0 100

0.1

0.2

0.3

.4 3000 12000.


looo i

0.1 0.2 0.3 [PDMDAAC]/[BSA]

Figure 6. (a) Diameter of BSA-PDMDAAC complex as a function of polymer concentration in 0.60 g/L BSA and 0.01 M, pH 7.88 phosphate buffer. (Δ) is QELS results, and (O) represents the corresponding turbidity, (b). Mobility of BSA-PDMDAAC complex at the same conditions as those in (a).

Figure 7. Diameter of BSA-PDMDAAC complex as a function of polymer concentration in 0.60 g/L BSA and 0.1 M, pH 7.88 phosphate buffer. (•) is QELS results, and (O) represents the corresponding turbidity.


235


236

Therefore, we may use the known polymer concentration C to evaluate the molecular weight of the complex, by plotting the angular dependence of the scattered light as KCp/Re vs sin^G^). Assuming α = 1 in the limit of low Cp, the intercept yields β, and hence M , and the slope leads to a value for R . The linearity of the plots, as shown for example in Figure 8, indicates the validity of the low concentration approximation. Values of M for different C are calculated from the intercepts of KCp/Re vs sin (0/2) plots using α = 1. Such results are plotted in Figure 9 as M vs Cp. The constant molecular weight at low polymer concentration of M = 8.U10 corresponds to a β value of 40±2, which is consistent with the value obtained byfiltration.This initial constant molecular weight is consistent with the cc= 1 assumption, which corresponds to an intrapolymer complex structure. The radius of gyration of the complex may be obtained from the slope of Figure 8.; For different amounts of added polymer, values of R and corresponding hydrodynamic radii, obtained from QELS, as well as ρ = Rg/RH» are given in Table L Hydrodynamic theory (39) shows that ρ changes from infinity to 0.77S when the polymer structure changes from a long rod to a sphere. The random coil structure is believed to occupy an intermediate position between long rod and sphere. For random coils, ρ assumes values from 1.3 to 1.5 (39). In a previous study (40), we found through a similar analysis that the complex formed from PEO and SDS micelles behaves like a random coil polymer; and similar results were obtained for the complex of ribonuclease A and sodium polystyrene sulfonate (41). However, the values for ρ in Table I are intermediate between those of random coil and long rod. These values suggest a extended structure of BSA-PDMDAAC complexes. The extended structure could be the result of the inter-protein repulsive forces which prevail at low ionic strength and pH well above pL p

x

g

x

p

2

x

6

x


g

Table L Hydrodynamic Radius and Radius of Gyration of the BSAPDMDAAC Complex^ C„(g/L) R (nm) Rh (nm) r(=R°/Rh) 250 2.3 1.2x10-3 no 2.0 115 230 2.1x10-3 2.5 95 240 2.4x10-3 2.7 90 245 3.7x10-3 2.2 110 235 5.1x10-3 2.4 120 290 6.6x10-3 8.3x10-3 3.8 110 420 (a) Protein concentration 0.60 g/L, pH=7.88, ionic strength 0.01M. e

Electrophoretic Light Scattering of BSA-PDMDAAC Complex. Figure 10 shows the electrophoretic mobility u of the complex formed at r = 300, CBSA = 0.60 g/L, in 0.01 M NaCl as function of pH. The decrease in mobility is due to the simultaneous increase in BSA negative net charge and the number of BSA bound per polymer chain. One may note that the mobility changes sign between pH 4.8 and 5.9. The positive value for u at pH 5 suggests that the complex initially formed in pH £ 5 is not saturated by BSA. Upon further increase in pH above 5.9, the mobility changes linearly with pH. As discussed below, this may be explained on the basis of an intrapolymer complex saturated with BSA, the presence of which in this pH range is supported by


17.

XIA ET A L

Stoichiometric and Nonstokhiometric Complex Formation 237

3i

•

r

0.2

U.O

0.4

0.6

0.8

1.0

2

sin (θ 11) Downloaded by CORNELL UNIV on June 16, 2017 | http://pubs.acs.org Publication Date: December 13, 1993 | doi: 10.1021/bk-1994-0548.ch017

2

Figure 8. KCp/Re vs sin (0/2) plot obtained for BSA-PDMDAAC complex at C = 0.002 g/L in CBSA=060 g/L (r=300) and 0.01 M phosphate buffer solution of pH 7.88. p

3.0

-2

2.0

1.0

δ 33

o.o

•

' • 0.004 C (g/L)

1

1

0.008

p

Figure 9. M vs C for B S A - P D M D A A C complex in 0.01 phosphate buffer with CBSA=0.60 g/L. X

P

M

pH 7.88

pH

Figure 10. Mobility of BSA-PDMDAAC complex as a function of pH: r = 300, CBSA = 0.60 g/L and I =0.01 M NaCl.


238


the observation from QELS of constant diameter above pH 5.8. The mobility of the intrapolymer complex, u, can be written as u

__q f

p

P

p

+pqpro _ q + q p i o P

+

nf

f

pio

x

(16)

where q and q are the charges of polymer and protein, respectively; η is the number of proteins bound per polymer chain; and f , f and f are friction coefficients of polymer, protein and complex, respectively. Published pH titration curves (42) show that the net charge of BSA (q ) varies approximately linearly with pH in the range of 6 to 10: p

pro

p

pro

x


pf0

q =26.4-6.0pH pro

( 1 7 )

Since q is independent of pH, eqs. (16) and (17) suggest that the mobility will be linear with pH at 6 < pH < 10, if the friction coefficient of complex f is independent of pH. This result which we observe is consistent with a saturated state of the complex (i.e. η is constant). Substituting eq.(17) and n=120 obtained by SLS into eq.(16), we obtain f =3.2xl0~ Poise-cm, which is much larger than that obtained by the Einstein equation: f=6phR«2.4xlO" Poise-cm. The Einstein relationship is not expected to hold in the present case because of hydrodynamic and electrostatic interactions. Even though some errors exist in the estimation of fx due to ignoring counterion condensation, the linear dependence of u vs pH is qualitatively satisfied by both calculation and experimental results. In Figure 10 we observe a negative mobility at pH 6.4, which is corresponding to the turbidity maximum in Type I titration. This suggests that the turbidity maximum is not the point of charge neutralization. For neutrality of charge we have p

x

5

x

6

Z =Z +ηZ T

p

p r

=0

(18)

where Zp is the formal charge of the polyion (i.e., for homopolymers, the degree of polymerization), η is the average number of protein molecules bound per polyion chain, and Zpr is the net charge of a bound protein molecule. At pH 6.4, BSA has a negative net charge of Z = -12 (42), and therefore η = 110 is obtained from equation (18). This η value is close to but smaller than the binding capacity i.e., 120 BSA per PDMDAAC chain obtained by SLS. Substitution of η =120 into eq. (18) we obtain the net charge Ζχ = -228 for the complex. This net charge is consistent with the observed negative mobility. Therefore, the turbidity maximum in "type Γ titration may correspond to the position of BSA saturation. p r

Mechanism of BSA-PDMDAAC Complex Formation and Complex Coacervation. Results of the present study are consistent with the following model for complexation and phase separation. At low ionic strength and at pH 7.88, the binding of BSA to PDMDAAC is quite strong, and in the limit of r—« each polymer chain binds a full complement of protein molecules, i.e. on the order of 120 proteins per polymer chain. Because the proteins are highly charged at this pH, Ζ = -21 (42), strong repulsion among neighboring bound proteins causes the chain



17. XIA ET A L


to adopt an extended configuration, with a hydrodynamic radius approximately four times larger than the value of the protein-free polymer (Rh = 24 nm). The net negative charge of this complex precludes its further association. Therefore, the turbidity increases only because of an increase in the number of such species. Because of the large binding constant, there is essentially no free polymer in these systems, only the primary complex and free protein. When a sufficient amount of polymer has been added to bind all the free protein, further addition of polycation leads to the coacervation of the primary complexes. The instability of the coacervate and the very large related particle sizes produce the ultimate decrease in turbidity. The maxima observed at low ionic strength therefore display a stoichiometric character, in the sense that a doubling of the protein concentration produces an approximate doubling of the amount of polymer added at the point where the turt>idity no longer increases. In contrast to stoichiometric protein-polyelectrolyte complexes formed in pure water (24,25), and stoichiometric polycation-polyanion complexes (22) which are neutral particles, the BSA-PDMDAAC stoichiometric complex has a negative charge. This negative charge prevents the complex from forming coacervates through simple aggregation. Addition of polymer neutralizes this charge and overcomes the Coulombic resistance to coacervation. This model is consistent with the experimental results in Figure 5b, which show a constant negative mobility at low polymer concentration, and then an increase from negative to positive values. The initial constant mobility corresponds to the stable stoichiometric complex; the mobility change with polymer concentration corresponds to the coacervation of the complex upon additional polymer. As seen in Figure 7, the size of the complex formed at higher ionic strength always increases with addition of polymer. Thus, the complexation is not stoichiometric because there is no well-defined product Furthermore, the lowest value for R seen, ca 250 nm, is very much larger than the dimensions of the protein-free polymer at this ionic strength. This result indicates that "primary" complexes are not particularly stable, but instead strongly tend to associate to form higher-order aggregates. Phase separation occurs when these aggregates become very large. We suggest that the size of the aggregates is not a function of the bulk solution stoichiometry alone, but instead increases strongly with total solute concentration. Thus, in two solutions that have the same polymerrprotein stoichiometry, the one with the higher protein concentration will form larger aggregates. The turbidity is thus observed to increase more rapidly with the concentration ratio of the polymer to the protein (W) when the protein concentration is higher, as seen in Figures 4 and 5. Also the size required for bulk phase separation is attained at lower W in this case. There are two reasons why complexes may tend to aggregate more readily at higher ionic strength. First, the intrinsic polymer-protein binding constant may be expected to be smaller at larger I. Therefore, the number of proteins bound per polymer chain may be less and the charge of the "primary complex" closer to electrical neutrality. Second, the Debye length decreases from 30 A to 10 À as the ionic strength increases from 0.01 to 0.10 M. In the low I condition, two primary complexes can interact with each other in their entireties, and the net charge of the entire complex determines association or coacervation. In the high I case, two primary complexes may interact with specific regions within each other's domain. Thus, a protein-rich portion of one complex (negatively charged) could interact with a protein-poor region (positively charged) in another complex. The "polarizability" s


239


240


Figure 11. Optical photomicrograph of BSA-PDMDAAC coacervate.

Charge Neutralization

Stoichiometric Complex

PDMDAAC

BSA

7

·^Α>22?

>0

Nonstoichiometric Complex

Coaccrvation

. Aggregation

Figure 12. Schematic diagram of complex formation in protein separation by polyelectrolytes.


17. ΧΊΑ

ET AL.

Stoichiometric and

Nonstoichiometric Complex Formation

241


of the primary complex may therefore promote higher-order aggregation. In all the cases die higher aggregates or coacervates have a size about 700 nm (Figure 11) as observed by microscope. In summary, the mechanism we propose for BSA-PDMDAAC complex formation and complex coacervation is shown schematically in Figure 12. At low ionic strength, e.g. 1=0.01 M, BSA and PDMDAACfirstform a negatively charged stoichiometric complex with a diameter of ecu 200 nm. The 200 nm particles then coacervate, through charge neutralization, upon further addition of the polycation. In the case of higher ionic strength, the initial formed complexes are nonstoichiometric with a size of ca. 500 nm in diameter. The nonstoichiometric complexes bear less charge and may exhibit charge polarization. The polarized particles then aggregate to form coacervates.

Conclusions The structure of the BSA-PDMDAAC complex in solutions of Cp = 0.6,1 = 0.01 M and pH 7.88 depends on the polymer concentration. At low polymer concentration, a stoichiometric intrapolymer complex is saturated with BSA. The mass ratio of the bound BSA to polymer in the saturation limit is about 40. This intrapolymer complex aggregates upon increase in the polymer concentration. In the case of higher ionic strength I = 0.1 M, the initial formed complexes have nonstoichiometric interpolymer structure with a size of ca. 500 nm in diameter. Acknowledgments. This research was supported by grants from the National Science Foundation (DMR 9014945); American Chemical Society (ACSPRF#25532-AC7B); Eli Lilly Company, Reilly Industries, and the Exxon Education Fund.

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