Chapter 8
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Complexation Between Poly (dimethyldiallylammonium chloride) and Globular Proteins Mark A. Strege, Paul L. Dubin, Jeffrey S. West, and C. Daniel Flinta Department of Chemistry, Indiana University—Purdue University at Indianapolis, Indianapolis, IN 46223
Studies of complexation between globular proteins and the synthetic strong polycation, poly(dimethyldiallylammonium chloride), were undertaken to gain insight into protein-polyelectrolyte complexation selectivity. Investigations of the protein-polyelectrolyte phase boundary, via turbidimetric titrations, suggest that phase separation may be a consequence of the saturation of protein binding sites on the polymer. Comparisons of the phase boundaries of various proteins reveal that the net protein surface charge density does not control phase separation, but rather suggests the importance of charge patches on the protein surface. Quasi-elastic light scattering measurements provide strong evidence for the existence of a stable soluble complex. Size exclusion chromatography, via the Hummel-Dreyer method, provides additional information on the binding equilibria for such soluble complexes.
Ο p p o s i t e l y charged polyelectrolytes interact to form complexes. Depending primarily on the molecular weights and the linear charge densities of the polyelectrolytes i n v o l v e d , 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 be a l i q u i d "complex coacervate" (8) or a solid precipitate. Examples of the former have been observed for gelatin and polyphosphate (9), and serum albumin and poly(dimethyldiallyl-ammonium chloride) (10). Systems that exhibit p r e c i p i t a t i o n i n c l u d e h e m o g l o b i n a n d d e x t r a n sulfate (11), carboxyhemoglobin and potassium poly(vinyl alcohol sulfate) i n the presence of poly(dimethyldiallylammonium chloride) (12), lysozyme a n d p o l y ( a c r y l i c acid) (13), and R N A polymerase and poly(ethyleneimine) (14). 0097-6156/90/0419-0158$06.00/0 © 1990 American Chemical Society
8. STREGE ET AL.
Complexation ofPoly(dimethyldiallylammonium chloride) 159
The ability of polyelectrolytes to remove oppositely charged proteins from solutions has been exploited through the incorporation of protein-polymer precipitation steps into a variety of protein purification procedures, wherein precipitated proteins are recovered f r o m the insoluble complex aggregate v i a redissolution by p H or ionic strength adjustment (14-16). Furthermore, preferential c o m p l e x a t i o n of polyelectrolytes w i t h specific proteins has been substantiated (13). A l t h o u g h there are reports of the optimization of bulk complexation yield through the adjustment of solution parameters such as p H and ionic strength (17), very little has been accomplished i n the optimization of the selectivity of complex formation. The recovery of proteins through the formation of insoluble complexes w i t h polyelectrolytes appears to be a very attractive nondenaturing separation process. After precipitating various enzymes with polyacrylic acids, Sternberg and Hershberger reported high recoveries of enzymatic activities, indicating that little or no denaturation h a d taken place during the separation (13). Other workers have reported the nondenaturing fractionation at slightly alkaline p H of intracellular proteins using synthetic polycations (14,16,17). Compared to other methods used for protein separation, such as gel filtration chromatography, the utilization of water-soluble, charged polymers as precipitating agents offers great economy w i t h regard to materials a n d process, a n d , furthermore, is virtually unlimited i n scale. Thus, an elucidation of the principles governing protein selectivity i n polyelectrolyte separation w o u l d be of considerable applied significance. In the present work, the mechanism of polyelectrolyte-protein interaction was studied using two approaches. To gain insight into the cooperativity of binding, and to determine whether complexes are intra- or interpolymer, complexes were characterized by size exclusion chromatography (SEC) and quasi-elastic light scattering (QELS). Size exclusion chromatography was also used to determine complex stoichiometry, i . e. the number of protein molecules bound per polyion. Such methods w i l l also lead to the determination of the number of b i n d i n g sites per polymer molecule, a n d the intrinsic association constant. O u r second approach involved the analysis of plots of the ionic strength dependence of the critical p H (the p H at which an abrupt increase i n turbidity is observed). The effects of proteimpolymer stoichiometry and protein type on such phase diagrams were studied.
EXPERIMENTAL Turbidimetric Titrations. Poly(dimethyldiallylammonium chloride) ( P D M D A A C ) , a commercial sample "Merquat 100" from Calgon Corp. (Pittsburgh, P A ) w i t h nominal molecular weight 2 χ 10 and reported polydispersity of M / M =10, was dialyzed and freeze-dried before use. A l l proteins were obtained from Sigma Chemical C o r p . Solutions were 5
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prepared as mixtures of P D M D A A C (0.05 - 1 g/1) and protein (0.25 - 25 g/1), corresponding to protein/polymer weight ratios (r) ranging from 0.25 to 200, at pH
