Jiulin Xia

Protein-Polyelectrolyte Complexes

Proteins interact strongly with both synthetic and natural polyelectrolytes. Ample evidence exists for the binding of polyanions and polycations to proteins below and above their isoelectric points, respectively. These interactions may result in soluble complexes [1,2], complex coacervation [3–6], or the formation of amorphous precipitates [7–9]. The practical consequences of these phase changes may include the use of polyelectrolytes for protein separation [10–16] and immobilization or stabilization of enzymes in polyelectrolyte complexes [17–18]. In these two applications, the optimal physical states of the system are different. In the case of enzyme immobilization, highly deaggregated states may be less active. In purification or separation processes involving settling or filtration, aggregation is desirable. For efficient settling, close-packed aggregates are preferred, whereas in filtration processes more open-textured aggregates are needed to allow adequate solvent penetration. However, in both cases the aggregation should be essentially reversible.

The effect of cations on the interaction between dodecylsulfate micelles and poly(ethyleneoxide)

Abstract The effect of the micelle counterion on the interaction of poly (ethyleneoxide) with dodecylsulfate micelles has been studied, using dye solubilization to determine the critical micelle concentration and dynamic light scattering to measure the relative amount of uncomplexed micelles. In the presence of 0.10- M Na + , Li + , and NH 4 + , the CMC-lowering effect of the polymer is strongly dependent on the nature of the cation. A parallel influence of the cation is seen in the distribution of scattering intensities between well-defined modes corresponding to free micelle and complex. These results are taken as evidence for a direct role of the cation in the stabilization of the complex, in which the cation interacts simultaneously with the micelle (through electrostatic forces) and with the polymer (via coordination complexation). This type of association may occur simultaneously with other interaction forces.

Complexation of trypsin and alcohol dehydrogenase with poly(diallyldimethylammonium chloride)

Complexation of alcohol dehydrogenase (ADH) and trypsin with poly(diallyldimethyl-ammonium chloride) (PDADMAC) in dilute electrolyte solution was studied by turbidimetric titration, quasi-elastic light scattering (QELS), and electrophoretic light scattering (ELS). Both QELS and turbidimetric titration show that PDADMAC forms complexes with ADH and trypsin in 0.01M NaCl solution at pH ≥ 6.8 and pH ≥ 9.2, respectively. These complexes take the form of stable coacervates in 0.01M, pH 11.0, phosphate buffer solution. QELS shows sizes of 400 and 315 nm for the coacervates of ADH-PDMDAAC and trypsin-PDMDAAC, respectively, while ELS reveals that these coacervates carry a net positive charge. Activity measurements show that both ADH and trypsin are enzymatically active in their coacervated states. Complexation of trypsin and PDADMAC was also studied by fluorescence in 0.01M, pH 11.0, phosphate buffer, and the protein emission was found to be quenched by complexation. The fluorescence quenching data show that trypsin retains its three-dimensional structure in the complex. These and other results are consistent with the quenching of the two tryptophans on the protein surface, but not the interior ones.© 1997 John Wiley & Sons, Inc.

Light scattering, CD, and ligand binding studies of ferrihemoglobin-polyelectrolyte complexes.

Quasi-elastic light scattering (QELS), electrophoretic light scattering (ELS), CD spectroscopy, and azide binding titrations were used to study the complexation at pH 6.8 between ferrihemoglobin and three polyelectrolytes that varied in charge density and sign. Both QELS and ELS show that the structure of the soluble complex formed between ferrihemoglobin and poly(diallyldimethylammonium chloride) [PDADMAC] varies with protein concentration. At fixed 1.0 mg/mL polyelectrolyte concentration, protein addition increases complex size and decreases complex mobility in a tightly correlated manner. At 1.0 mg/mL of greater protein concentration, a stable complex is formed between one polyelectrolyte chain and many protein molecules (i.e., an intrapolymer complex) with apparent diameter approximately 2.5 times that of the protein-free polyelectrolyte. Under conditions of excess polyelectrolyte, each of the three ferrihemoglobin-polyelectrolyte solutions exhibits a single diffusion mode in QELS, which indicates that all protein molecules are complexed. CD spectra suggest little or no structural disruption of ferrihemoglobin upon complexation. Azide binding to the ferrihemoglobin-poly(2-acrylamide-2-methylpropanesulfonate) [PAMPS] complex is substantially altered relative to the polyelectrolyte-free protein, but minimal change in induced by complexation with an AMPS-based copolymer of reduced linear charge density. The change in azide binding induced by PDADMAC is intermediate between that of PAMPS and its copolymer.

Stoichiometry and the Mechanism of Complex Formation in Protein-Polyelectrolyte Coacervation

Abstract The complexation and coacervation of bovine serum albumin (BSA) with poly(dimethyldiallylammonium chloride) (PDMDAAC) in pH 7.86 buffer solutions, at various ionic strengths (I), was investigated by turbidimetric titration, quasi-elastic light scattering, and electrophoretic light scattering. 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. 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 to be ∼ 700 nm by optical microscopy.

Chromatographic evaluation of the binding of lysozyme to poly(dimethyldiallylammonium chloride)

Abstract Size-exclusion chromatography on Superose columns was used to examine the binding of lysozyme to a strong polycation, poly(dimethyldiallylammonium chloride). A modified Hummel-Dreyer method was employed to determine the number of protein molecules bound per polymer chain as a function of protein concentration, in 0.325 M buffer, at pH 9.0. Even though this pH is smaller than the isoelectric point, the protein binds to this polycation. The binding data could be fit to Hills equation, and the resulting fitting parameters indicate that the binding is cooperative.

Dynamic and Electrophoretic Light Scattering of Poly(dimethyldiallylammonium chloride) in Salt-Free Solutions

SYNOPSIS Dynamic and electrophoretic light scattering were used to study the diffusion and electrophoretic mobility of poly(dimethyldiallylammonium chloride) as a function of polymer molecular weight in salt-free solutions. Two relaxation modes characterized as fast diffusion (D,) and slow diffusion (D,) were obtained from dynamic light scattering. Although the slow diffusion coefficient D, strongly depends on molecular weight (M,), the fast diffusion coefficient D, was found to be independent of M, over the range in the study. The fast diffusion was considered as the diffusion of a part of the polymer chain; the slow diffusion was interpreted by multichain diffusion. Electrophoretic light scattering results in the saltfree solution show that the electrophoretic mobility of the polymer is independent of M,. 0 1996 John Wiley & Sons, Inc.