Structure, mobility, and enzyme activity in protein-polyelectrolyte complexes
Abstract
The critical conditions for complex formation in BSA-polyelectrolyte systems were examined. The interactions leading to complex formation were found to occur on a local level, between a sequence of charges on the protein surface and a complementary set of charges along a short segment of the polyelectrolyte. In contrast to simple models wherein binding is assumed to be controlled by the net protein charge, polyelectrolyte charge density, and ionic strength, critical conditions for complex formation and the degree of cooperativity in the binding mechanism were also found to be dependent upon protein size, polymer flexibility, and macromolecular concentration ratio. From a compositional analysis of BSA-polycation coacervates it was found that the wt % polymer in the coacervate was directly proportional to the protein charge, regardless of the ionic strength. The %H2O in the coacervates ranged from 70 to 86%. This range was invariant with ionic strength. The influence of coacervation on the structure/activity of trypsin, pepsin, and BSA was also examined. Coacervation of BSA with a polycation stabilized the protein against enzymatic digestion by trypsin in the dilute phase. Coacervation of trypsin with a polyanion led to a reduction in activity, in the absence of significant structural changes. This reduction was accompanied by an increase in the rate of auto-digestion, implying that the change in activity is correlated to a reduction in enzyme concentration via self-digestion. Polyelectrolyte coacervation of BSA under low salt conditions decreased the protein stability to pH induced structural changes, in contrast to the enhanced stability observed for BSA and pepsin in coacervates formed under moderate salt conditions. Rheological measurements of BSA-polycation coacervates indicate that the coacervates behave as Newtonian fluids, implying that coacervation is a bulk phenomenon, occurring in the absence of any gross structural changes in the primary complex that would lead to subsequent polyelectrolyte chain entanglement. Macromolecular probe (charged and uncharged) diffusion in the coacervates was indistinguishable from that in Dextran solutions of identical mass concentration, suggesting that the electrostatic environment within the coacervate could be modeled as a charge continuum, rather than a concentrated collection of discreet charges.
Degree
Ph.D.
Advisors
Dubin, Purdue University.
Subject Area
Biochemistry|Analytical chemistry
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