Electrophoretic and quasi-elastic light scattering of soluble protein-polyelectrolyte complexes

Xia, Jiliang; Dubin, Paul L.; Kim, Yesook; Muhoberac, Barry B.; Klimkowski, V. J.

doi:10.1021/j100119a046

Cited by 130 publications

(137 citation statements)

References 11 publications

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“…50 The rather linear increase in turbidity as pH descended below pI corresponds to progressively stronger heparin-protein interaction, possibly leading to association of primary intrapolyanion complexes as they approach charge neutrality. 51 Designating the initial formation of soluble complexes as "pH c " and eventual phase separation condition as "pH " [52][53][54] we see pH c > pI+1, and pH < pI-2. For heparin and bovine serum albumin in 30 mM NaCl, we observed pH c ) pI + 2 and pH ≈ pI.…”

Section: Figure 1 Shows the Results Of Turbidimetric Titrations In Whichmentioning

confidence: 99%

Suppression of Insulin Aggregation by Heparin

et al. 2008

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The aggregation of insulin near its isoelectric point and at low ionic strength was suppressed in the presence of heparin. To understand this effect, we used turbidimetry and stopped-flow to study the pH-and ionic strength (I)-dependence of the aggregation of heparin-free insulin. The results supported the role of interprotein electrostatic interactions, contrary to the commonly held view that such forces are minimized at pH ) pI. Electrostatic modeling of insulin (DelPhi) revealed that attractive interactions arise from the marked charge anisotropy of insulin near pI. We show how screening of the interprotein attractions by added salt lead to maximum aggregation near I ) 0.01 M, corresponding to a Debye length nearly equal to the diameter of the insulin dimer, consistent with a dipole-like protein charge distribution. This analysis is also consistent with suppression of aggregation by heparin, a strong polyanion that by binding to the positive domain of one protein, inhibits its interaction with the negative domain of another.

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Section: Figure 1 Shows the Results Of Turbidimetric Titrations In Whichmentioning

confidence: 99%

Suppression of Insulin Aggregation by Heparin

et al. 2008

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“…To some extent small ions can compensate this charge imbalance but evidently not to the extent to produce zero mobility at the point of incipient coacervation. 72,74 The electrostatic energy of a protein transiently at the periphery of a soluble complex is larger than one less distal because it is removed from the center of mass of polyelectrolyte segments; this form of surface energy promotes coalescence, providing an enthalpic driving force for aggregation which is accompanied by the favorable entropy of the release of counterions. This process is kinetically driven by a form of polarization, in which the protein-rich (net negative) region of one complex can induce and interact with the polyelectrolyte-rich (positive) domain of another.…”

Section: Molecular Modelsmentioning

confidence: 99%

Mesophase separation and probe dynamics in protein–polyelectrolyte coacervates

et al. 2007

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Protein-polyelectrolyte coacervates are self-assembling macroscopically monophasic biomacromolecular fluids whose unique properties arise from transient heterogeneities. The structures of coacervates formed at different conditions of pH and ionic strength from poly(dimethyldiallylammonium chloride) and bovine serum albumin (BSA), were probed using fluorescence recovery after photobleaching. Measurements of self-diffusion in coacervates were carried out using fluorescein-tagged BSA, and similarly tagged Ficoll, a non-interacting branched polysaccharide with the same size as BSA. The results are best explained by temporal and spatial heterogeneities, also inferred from static light scattering and cryo-TEM, which indicate heterogeneous scattering centers of several hundred nm. Taken together with previous dynamic light scattering and rheology studies, the results are consistent with the presence of extensive dilute domains in which are embedded partially interconnected 50-700 nm dense domains. At short length scales, protein mobility is unobstructed by these clusters. At intermediate length scales, proteins are slowed down due to tortuosity effects within the blind alleys of the dense domains, and to adsorption at dense/dilute domain interfaces. Finally, at long length scales, obstructed diffusion is alleviated by the break-up of dense domains. These findings are discussed in terms of previously suggested models for protein-polyelectrolyte coacervates. Possible explanations for the origin of mesophase separation are offered.

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“…6,7 The formation of intrapolymer complexes, while not a true phase transition, appears as a discontinuity in such properties as scattering intensity, diffusivity, and electrophoretic mobility and is designated as pH c . 5,6,8,9 While pH φ depends on polyelectrolyte molecular weight, MW, the protein and polymer concentrations (stoichiometry), pH c , are influenced solely by ionic strength. This is because pH c , corresponding to the formation of soluble complexes, is governed only by the interactions between a charged domain on the protein and its associating sequence of polyelectrolyte charges.…”

Section: Introductionmentioning

confidence: 99%

Entering and Exiting the Protein−Polyelectrolyte Coacervate Phase via Nonmonotonic Salt Dependence of Critical Conditions

2009

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Critical conditions for coacervation of poly(dimethyldiallylammonium chloride) (PDADMAC) with bovine serum albumin were determined as a function of ionic strength, pH, and protein/polyelectrolyte stoichiometry. The resultant phase boundaries, clearly defined with this narrow molecular weight distribution PDADMAC sample, showed nonmonotonic ionic strength dependence, with the pH-induced onset of coacervation (at pH φ ) occurring most readily at 20 mM NaCl. The corresponding onset of soluble complex formation, pH c , determined using highprecision turbidimetry sensitive to changes of less than 0.1% transmittance units, mirrored the ionic strength dependence of pH φ . This nonmonotonic binding behavior is attributable to simultaneous screening of short-range attraction and long-range repulsion. The similarity of pH c and pH φ was explained by the effect of salt on protein binding, and consequently on the number of bound proteins relative to that required for charge neutralization of the complex, a requirement for phase separation. Expansion of the coacervation regime with chitosan, a polycation with charge spacing similar to that of PDADMAC, could be due to either the charge mobility or chain stiffness of the former. The pH φ versus I phase boundary for PDADMAC correctly predicted entrance into and egress from the coacervation region by addition of either salt or water. The ability to induce or suppress coacervation via protein/polyelectrolyte stoichiometry r was found to be consistent with the proposed model. The results indicate that the conjoint effects of I, r, and pH on coacervation could be represented by a three-dimensional phase boundary.

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Electrophoretic and quasi-elastic light scattering of soluble protein-polyelectrolyte complexes

Cited by 130 publications

References 11 publications

Suppression of Insulin Aggregation by Heparin

Suppression of Insulin Aggregation by Heparin

Mesophase separation and probe dynamics in protein–polyelectrolyte coacervates

Entering and Exiting the Protein−Polyelectrolyte Coacervate Phase via Nonmonotonic Salt Dependence of Critical Conditions

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