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).