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.
Bovine serum albumin (BSA) and poly(diallyldimethylammonium chloride) (PDADMAC) spontaneously form, over a range of ionic strength I and pH, dense fluids rich in both macroions. To study their nanostructure, these coacervates were prepared at low I and high pH (strong interaction) or at high I and lower pH (weaker interaction), with polymer MWs ranging from 90K to 700K, and then examined by dynamic light scattering (DLS) and rheology. DLS shows a dominant and surprisingly fast protein diffusional mode independent of polymer MW; accompanied by robust slow modes, slower by 1-2 orders of magnitude, which are also insensitive to MW and are present regardless of I, pH, and sample aging. High MW sensitivity was observed by rheology for the terminal time (order of milliseconds), which increased as well with the strength of polyelectrolyte-protein interaction. Viscoelastic behavior also indicated a tenuous network, solidlike at low strain but re-forming after breakage by shear. Two models, both of which have strengths and defects, are put forward: (I) macroion-rich domains dispersed in a continuum of macroion-poor domains near the percolation limit and (II) a semidilute solution of PDADMAC chains with interchain friction modulated by transient BSA-PDADMAC association.
Turbidity measurements performed at 450 nm were used to follow the process of simple coacervation when 1% (w/v) aqueous alkali processed gelatin (type-B) solutions were titrated with methanol, ethanol, propanol, and tert-butyl alcohol at various pHs of the solution ranging from pH = 5 to 8 and ionic strengths varying from I = 0.01 to 0.1 M NaCl. The titration profiles clearly established the transition points in terms of the percentage of volume of alcohol added relative to that of solvent corresponding to the first occurrence of turbidity (Vt) and a point of turbidity maximum (Vp). Addition of more alcohol drove the system toward precipitation. The values of Vt and Vp characterized the initiation of intramolecular folding and intermolecular aggregate formation of the charge neutralized gelatin molecules and the subsequent micro coacervate droplet formation. The state of intermolecular aggregates and that of folded gelatin molecules could be characterized by dynamic laser light scattering experiments, which implied spontaneous segregation of particle sizes preceding coacervation. The aggregates constitute the coacervate phase while the folded gelatin molecules mostly stay dispersed in the supernatant. The data taken together reveal the role played by solution entropy in addition to that of electrostatic and solute-solvent interactions, which had been overlooked hitherto.
The sol–gel transition in dilute gels of gelatin are studied by differential scanning calorimetry (DSC) and static light scattering techniques. The sol–gel phase diagram clearly shows the existence of an upper critical solution temperature in this system. In addition, the DSC data conclusively exhibits two more phase curves in the sol state—one pertaining to the coexistence of monomers and aggregates and the second one separating the random coil and helix domains in the solution phase. The Ferry–Eldridge equation has been used to determine the enthalpy of the melting of the gel structure (ΔHg) which is equal to (30±2.0) kcal mol−1. The gelation temperature Tg is correlated to Flory’s statistical model of gelation, which gives the enthalpy of melting of pure gelatin crystallites (ΔHv) as ΔHv=(35±2.0) kcal cm−3 and crystallite melting temperature (Tm) as Tm=(588±20.0) K. The Flory–Huggins interaction parameter (χ) has been determined as χ=0.49±0.05. Interesting scaling behavior has been observed through light scattering measurements. The intensity of scattered light, Is∼ε−γ and Is∼ε0−β; ε=(T/Tg−1); T≥Tg and ε0=(t/tg−1); t≥tg, where t is the time and tg is the gelation time for ε and ε0≤0.5 with γ≂β≂0.02±0.005. The initial phase of cooling of the sol has been analyzed within the framework of Smoluchowski aggregation kinetics. On the basis of the above results it is being proposed that the sol–gel transition path in this polymer has three distinct steps—monomer aggregation, coil–single-helix transition, and single-helix–triple-helix transition followed by gelation.
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