pH-Induced Coacervation in Complexes of Bovine Serum Albumin and Cationic Polyelectrolytes K. Kaibara, † T. Okazaki, † H. B. Bohidar, ‡ and P. L. Dubin* ,‡ Department of Chemistry, Faculty of Sciences, Kyushu University, Higashi-ku, Fukuoka 812, Japan, and Department of Chemistry, Indiana-Purdue University, Indianapolis, Indiana 46202 Turbidity and light scattering measurements, along with phase contrast microscopy, were used to follow the processes leading to coacervation when aqueous solutions of bovine serum albumin (BSA) and poly- (dimethyldiallylammonium chloride) (PDADMAC) were brought from pH ) 4 to 10. The state of macromolecular assembly of complexes formed between BSA and PDADMAC prior to and during the pH-induced coacervation could be characterized by specific pH values at which recognizable transitions took place. In addition to the two characteristic pH values (pH crit and pH φ ) previously identified through turbidimetry, other transitions were explicitly established. On the basis of the pH-induced evolution of scattering intensity measurements, we concluded that the formation of soluble primary protein-polymer complexes is initiated at pH crit and proceeds until “pH′ crit ”. A subsequent increase in scattering intensity at “pH pre ” may arise from the assembly of quasi-neutralized primary complexes as their net positive charge decreases with increase in pH. Subsequently, a maximum in scattering intensity at pH φ is observed coincident with the appearance of turbidity and also corresponding to the first microscopic observation of coacervate droplets. The temperature independence of pH crit and pH φ suggests that hydrophobic contributions are negligible for the initial BSA-PDADMAC interactions and the subsequent coacervation process. The pH dependence of scattering intensity profiles allowed the identification of two other transitions beyond pH φ . Spherical microcoacervate droplets first observed around pH φ subsequently displayed morphological changes at “pH morph ”, followed by the transformation to solid or flocculant substances at pH precip. Introduction Protein-polyelectrolyte interactions, primarily arising from electrostatic forces, often lead to coacervation as described in the pioneering work of Bungenberg de Jong. 1 During this process, a homogeneous aqueous solution undergoes liquid- liquid-phase separation giving rise to a dense protein-rich phase. This phenomenon has been of interest from a basic physicochemical point of view, as well as from the perspec- tive of the development of a large variety of possible applications. The unique characteristics of the coacervate phase suggest it as a model for proteins in cytoplasm-like environments. The properties of biological macromolecules in self-organized systems can be examined by studies of the coacervate state. Investigation of protein-polyelectrolyte complexes can prove useful in development of medical devices and artificial organs including cell attachment and scaffolding in biological tissues. Protein-polyelectrolyte coacervation may be applied to a protein separation pro- cess, 2,3 in which purification and recovery of a target protein depend on control of coacervation via pH or ionic strength. 4-6 Protein-polyelectrolyte coacervation may be used to im- mobilize enzymes, an attractive alternative to microcapsu- lation, if these enzymes can be more active, selective, or stable in polyelectrolyte complexes or coacervates. 7-9 Thus, investigations of basic aspects of coacervation of protein- polyelectrolyte complexes provide a foundation not only for the basic understanding of these supramolecular structures but also for their practical applications to protein-related industrial processes. In the past, several systematic investigations of micro- and macroscopic phase behavior have been carried out in aqueous solutions of bovine serum albumin (BSA) and poly(dimeth- yldiallylammonium chloride) (PDADMAC). These revealed that BSA-PDADMAC interactions initially lead to soluble “primary complexes”. The subsequent coacervation process may be described either as stoichiometric or nonstoichio- metric. 10,11 Two specific pH values, pH crit and pH φ , were used to parametrize the phenomenological results. Primary complex formation, initiated at pH crit , was viewed as a microscopic transition on the molecular scale, whereas coacervate droplet formation at pH φ was viewed as a global phase transition associated with a characteristic length scale of 10-100 nm. 12,13 Factors affecting pH crit and pH φ values were examined by a variety of experimental methods including static, dynamic, and electrophoretic light scattering measurements. It was found that pH φ but not pH crit was a function of BSA/PDADMAC ratio. 14a pH crit is seen as the point of incipient polycation binding which occurs when some sufficient local negative charge develops on the protein and is related but not equivalent to the isoelectric point of * Corresponding author: dubin@chem.iupui.edu. † Kyushu University. ‡ Indiana-Purdue University. 100 Biomacromolecules 2000, 1, 100-107 10.1021/bm990006k CCC: $19.00 © 2000 American Chemical Society Published on Web 02/05/2000