Photo-induced unfolding and inactivation of bovine carbonic anhydrase in the presence of a photoresponsive surfactant

Mirarefi, Panteha; Lee, C. Ted

doi:10.1016/j.bbapap.2009.09.007

Cited by 18 publications

(26 citation statements)

References 66 publications

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“…Lee et al reported that hydrophobic association of azo-surfactants with proteins can be modulated by exposure to light. 44,45 Cationic azobenzene-containing surfactants under their apolar trans form were shown to favor an activity loss of CAB, 44 or the unfolding of bovine serum albumin, 45 whereas the cis form did not affect the conformation of albumin. Using azobenzene-containing PAA, we showed that the cis−trans isomerization and hydophobic assembly are effectively controlled in situ by exposure to UV light 46 and can modulate association with native bovine serum albumin 33 or cytochrome c. 34 All of these effects were ascribed to higher hydrophobic interactions between the blue-adapted (predominantly trans) azobenzene isomer as compared to the UV-adapted (predominantly cis) one.…”

Section: ■ Results and Discussionmentioning

confidence: 98%

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

2014

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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).

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Section: ■ Results and Discussionmentioning

confidence: 98%

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

2014

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“…To study the particle size distribution in the solutions, dynamic light scattering (DLS) technique is widely used due to its relative simplicity and reasonable accuracy [9,28]. Hydrodynamic radius of the macromolecules such as proteins and aggregates in heated samples was measured by dynamic light scattering Technique using a Zetasizer Nano ZS (Brookhaven, USA) equipped with a helium/neon Laser.…”

Section: Measuring Of Mean Hydrodynamic Diametermentioning

confidence: 99%

“…All of these isozymes have the same tertiary structure folding, containing a central 10-stranded beta-sheet as the dominant secondary structure segment [3]. The CA's active site geometry is a composition of a zinc ion which located in a cone-shaped cavity surrounded by three histidyl residues and a solvent molecule [3,8,9]. The catalytic mechanism of BCA has been investigated in full detail.…”

Section: Introductionmentioning

confidence: 99%

Thermal Inactivation and Conformational Lock of Bovine Carbonic Anhydrase

Alaei¹,

Moosavi-Movahedi

Hadi

et al. 2012

PPL

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The kinetics of thermal inactivation of bovine carbonic anhydrase (BCA) was studied in a 50 mM Tris-HCl buffer, pH 7.8 using p-nitrophenyl acetate as substrate in absorbance of 400 nm by UV-VIS spectrophotometry. The number of conformational locks and inter-subunit amino acid residues of BCA were obtained by thermal inactivation analysis. The cleavage bonds between dimers of BCA during thermal dissociation and type of interactions between specific amino acid residues were also detected. The thermal inactivation curves were plotted in temperatures ranging between 40-70°C. It was shown several phases for inactivation of BCA at 65°C. Analyses of the curves were done by the conformational lock theory. The subunits are dissociated and several intermediates appear during inactivation through increasing the temperature in comparison with native state. Dynamic light scattering measurements was done to study the changes in hydrodynamic radius during thermal inactivation. Three distinct zones were shown in DLS data. Biochemical computation using ligplot is performed to find the inter-subunit amino acid residues for BCA.

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“…The "model" of the a-CA family is the monomeric human carbonic anhydrase II (hCAII), illustrated in Figure 2(a). 224 The protein is composed of a single polypeptide chain containing 260 amino acids and has a molecular weight of 29 kDa. 224 The protein is composed of a single polypeptide chain containing 260 amino acids and has a molecular weight of 29 kDa.…”

Section: Alpha-casmentioning

confidence: 99%