Prevention of Aggregation and Renaturation of Carbonic Anhydrase via Weak Association with Octadecyl- or Azobenzene-Modified Poly(acrylate) Derivatives

Abstract

The prevention of aggregation during renaturation of urea-denatured carbonic anhydrase B (CAB) via hydrophobic and Coulomb association with anionic polymers was studied in mixed solutions of CAB and amphiphilic poly(acrylate) copolymers. The polymers were derivatives of a parent poly(acrylic acid) randomly grafted with hydrophobic side groups (either 3 mol % octadecyl group, or 1-5 mol % alkylamidoazobenzene photoresponsive groups). CAB:polymer complexes were characterized by light scattering and fluorescence correlation spectroscopy in aqueous buffers (pH 7.75 or 5.9). Circular dichroism and enzyme activity assays enabled us to study the kinetics of renaturation. All copolymers, including the hydrophilic PAA parent chain, provided a remarkable protective effect against CAB aggregation during renaturation, and most of them (but not the octadecyl-modified one) markedly enhanced the regain of activity as compared to CAB alone. The significant role of Coulomb binding in renaturation and comparatively the lack of efficacy of hydrophobic association was highlighted by measurements of activity regain before and after in situ dissociation of hydrophobic complexes (achieved by phototriggering the polarity of azobenzene-modified polymers under exposure to UV light). In the presence of polymers (CAB:polymer of 1:1 w/w ratio) at concentration ∼0.6 g L(-1), the radii of the largest complexes were similar to the radii of the copolymers alone, suggesting that the binding of CAB involves one or a few polymer chain(s). These complexes dissociated by dilution (0.01 g L(-1)). It is concluded that prevention of irreversible aggregation and activity recovery were achieved when marginally stable complexes are formed. Reaching a balanced stability of the complex plays the main role in CAB renaturation, irrespective of the nature of the binding (by Coulomb association, with or without contribution of hydrophobic association).

Refolding of Aggregation-Prone ScFv Antibody Fragments Assisted by Hydrophobically Modified Poly(sodium acrylate) Derivatives

“…Noncovalent protein/polymer interactions are known to protect various proteins, mainly small globular single‐domain enzymes, against stress‐induced (e.g., pH, temperature) denaturation or against aggregation during refolding. The protection and/or association was attributed primarily to hydrophobic binding of the polymers with unfolded proteins or folding intermediates and, less frequently, to Coulomb interactions . The protection of partly folded proteins was ascribed to the increased solubility of the protein:polymer complexes, for instance in the cases of carbonic anhydrase, lysozyme alpha‐chymotrypsin, and alpha‐amylase), to the hydrophobically mediated interfacial immobilization of proteins on polymer nanoparticles or on polymer micelles, or to confinement in nanogels (of pullulan or in PNIPAM).…”

“…Release of native proteins was achieved by changing the environmental conditions, such as the ionic strength, the pH, or by addition of a competitive molecule that weakened the interaction of the protein with its artificial chaperon. Our recent studies indicate that hydrophobically modified linear poly(sodium acrylate) derivatives can form dynamic complexes with bovine carbonic anhydrase that reach significant protein renaturation yields without additional weakening step …”

“…We recently reported the use of low concentrations of poly(acrylic acid) (PAA) and its amphiphilic derivatives to slow down the thermal aggregation of full antibodies (immunoglobulins G, IgG) and to renature soluble enzymes . In both cases, the protein colloidal stability was markedly enhanced by addition of polymer in a 1:1 w/w protein/polymer ratio, a value much lower than the usual concentrations of “stabilizing” excipients, such as osmolytes or detergents.…”

Reversible control of enzyme‐inhibitor interactions with light illumination using a photoresponsive surfactant

“…17 Azobenzene is a commonly used moiety for the photoregulation or photocontrol of biological macromolecules. 24,25 Various azobenzenemodified ligands and polymers have been utilized to initiate changes in protein unfolding, [26][27][28] often through conjugation to the protein backbone [29][30][31] or with replacement of an amino acid in the protein with a phenylazophenylalanine. 32 Notably, a nearly threefold increase in enzyme activity was observed with azobenzene-conjugated papain in the trans vs cis conformation.…”

Alumina nanoparticle-assisted enzyme refolding: A versatile methodology for proteins renaturation

“…In water, colloidal or interfacial assemblies with surfactants or nanoparticles provide conditions to control protein stability and folding/unfolding rearrangements and renaturation 3 . Several methods based on interface interactions for renaturation of proteins are available, which in general can be divided into three main groups: (I) methods to remove the denaturants from the protein solution 4–6 ; (II) methods involving the control of the physical conditions used during the refolding process 7, 8 ; and (III) methods in which refolding agents are added during the renaturing processes 9–11 .…”