Refolding Of Bovine Carbonic Anhydrase Research Articles

L-Argininamide improves the refolding more effectively than l-arginine

L-arginine (Arg) is a widely used additive for suppressing protein aggregation during refolding. Systematic screening of Arg analogs provides superior additives that enhance the refolding yield more effectively than Arg. The refolding yield of hen egg lysozyme in the presence of 500 mM L-argininamide (ArgAd) increases 1.7-fold higher than Arg. Thermal unfolding experiments indicate that ArgAd has a greater denaturing effect than Arg. The refolding yield positively relates to the net charge of Arg analogs. Moreover ArgAd was also effective for the refolding of bovine carbonic anhydrase. High potency to increase the refolding yield of ArgAd compared to Arg results from high positive net charge and the denaturing property.

Polyvinylpyrrolidone 40 Assists the Refolding of Bovine Carbonic Anhydrase B by Accelerating the Refolding of the First Molten Globule Intermediate

Protecting proteins from aggregation is one of the most important issues in both protein science and protein engineering. In this research, the mechanism of enhancing the refolding of guanidine hydrochloride-denatured carbonic anhydrase B by polyvinylpyrrolidone 40 (PVP40) was studied by both kinetic and equilibrium refolding experiments. The reactivation and refolding kinetics indicated that the rate constant of refolding the first refolding intermediate (I(1)) to the second one (I(2)) is promoted by the addition of PVP. Fluorescence quenching studies further indicated that PVP could bind to the aggregation-prone species I(1), resulting in the protection of the exposed hydrophobic surface, a minimization of the protein surface, and more importantly, an increase of the refolding rate of I(1). These properties were quite different from those of poly(ethylene glycol) (PEG), which has been shown to have a strong and stoichiometric binding to I(1) and does not interfere with the refolding pathway. Unlike PEG, the binding of PVP to I(1) does not block the aggregation pathway directly but decreases the energy barrier for I(1) to refold to I(2) and thus reduces the accumulation of I(1). These results suggested that PVP works by a quite different mechanism from those well established ones in chaperones and chemical promoters. PVP is more like a folding catalyst rather than a chemical chaperone. The distinct mechanism of enhancing protein aggregation by PVP is expected to facilitate the attempt to develop new chemical compounds as well as new strategies to protect proteins from aggregation.

Influence of the Zn(II) Cofactor on the Refolding of Bovine Carbonic Anhydrase after Denaturation with Sodium Dodecyl Sulfate

This paper uses capillary electrophoresis to follow a globular metalloprotein--bovine carbonic anhydrase II (BCA, EC 4.2.1.1)--on unfolding upon treatment with sodium dodecyl sulfate (SDS) and refolding upon removal of SDS, both in the presence and the absence of its Zn(II) cofactor. This research demonstrates that the Zn(II) cofactor is not required for refolding into a nativelike conformation, does not remain associated with the unfolded protein, and does not significantly change the rate of refolding. The presence of the Zn(II) cofactor, however, does increase the total amount of recovered protein by a factor of 2. Capillary electrophoresis could distinguish between native and denatured protein, based on the difference in electrophoretic mobility between the native protein and the aggregate of denatured protein and SDS. In addition, the active site was probed by observing binding of BCA to a charged arylsulfonamide using affinity capillary electrophoresis. These studies provide a foundation for future physical-organic studies using BCA as a model to examine interactions between proteins and SDS.

Artificial Chaperone-Assisted Refolding of Bovine Carbonic Anhydrase Using Molecular Assemblies of Stimuli-Responsive Polymers

An artificial chaperone, which can decrease the protein aggregation and increase the reactivation yield of denatured protein in a fashion similar to natural chaperone, was newly developed using stimuli-responsive polymers. It has previously been reported that the addition of poly(propylene oxide)-phenyl-poly(ethylene glycol) (PPOn-Ph-PEG) with the unit number of PPO (n) 33 could enhance the refolding of bovine carbonic anhydrase (Kuboi et al. J. Chromatogr. B 2000, 243, 213). PPO-Ph-PEG with a large PPO chain (n = 50) was synthesized and the surface properties were characterized by both the relative fluorescence intensity of 1-anilino-8-naphthalene sulfonate (ANS) and the fluidity determined by diphenylhexatriene (DPH). The variation of ANS intensity and DPH fluidity is shown in a diagram as functions of temperature and polymer concentration. The high values of ANS intensity and fluidity of PPO50-Ph-PEG were obtained in a relatively wide conditional range (more than 0.08 mM and more than 15 degrees C) although the conditions showing the high values of PPO33-Ph-PEG were restricted (more than 0.1 mM and more than 40 degrees C). It was also found that molecular assemblies of PPOn-Ph-PEG with diameters of 7-18 nm were formed in the above conditions. On the basis of the surface properties of their polymer self-assemblies, the possibility of using them as an artificial chaperone was investigated. The effect of the addition of PPOn-Ph-PEG on the reactivation yield of a model protein, carbonic anhydrase from bovine (CAB), and the optical density of the solution was examined at various temperatures and concentrations. The reactivation yield of CAB was strongly enhanced and the aggregate formation (the optical density) was suppressed by adding PPOn-Ph-PEG in the above conditions, which show high ANS intensity and DPH fluidity. Especially in the presence of 0.1 mM PPO50-Ph-PEG, the reactivation yield of CAB reached approximately 100% at 40-55 degrees C. It was thus found that self-assemblies of the present polymer could be utilized as an artificial chaperone by selecting suitable stimuli conditions.

Inhibition of aggregation by media selection, sample loading and elution in size exclusion chromatographic refolding of denatured bovine carbonic anhydrase B

The effects of gel media, sample application and elution flowrate on the activity recovery and aggregation in refolding of bovine carbonic anhydrase B (CAB) by size exclusion chromatography (SEC) were investigated. Variation in aggregation was demonstrated visibly by the comparison of refolding profiles under different operating conditions. Some principles with regard to practical application were proposed. Meanwhile, the analysis of relationship between peak resolution and activity recovery provided evidences for the mechanism of size exclusion chromatography protein refolding.

Chaperone-like activity and surface hydrophobicity of 70S ribosome

Ribosomes have been shown to mediate refolding of proteins in vitro. In order to understand the mechanism of action, we have explored the 70S ribosome surface for hydrophobicity, one of the important aspects in chaperone–target protein interaction. We find that the 70S ribosome displays significant hydrophobicity on its surface when probed with the hydrophobic fluorophore 8-anilino-1-naphthalene sulfonate. To understand the functional significance of this hydrophobicity we investigated the ability of the ribosome to prevent aggregation of insulin B chain and α-lactalbumin induced by reducing the interchain and intrachain disulfide bond respectively with dithiothreitol (DTT) and photo aggregation of γ-crystallin at 37°C. The 70S ribosome offers complete protection towards light-induced aggregation of γ-crystallin (at 1:2 (w/w) ratio of crystallin:ribosome) and DTT-induced aggregation of α-lactalbumin (at 1:3) and there is appreciable protection (at 1:3) against the aggregation of insulin B chain. We also investigated the role of 70S ribosome in refolding of bovine carbonic anhydrase. Ribosomes improved the folding yield in a concentration-dependent manner. These results clearly demonstrate a general chaperone-like activity of 70S ribosome and implicate its surface hydrophobicity.

Immobilized liposome chromatography for refolding and purification of protein

Small unilamellar liposomes were utilized as a kind of aqueous two-phase system and artificial chaperone which specifically recognize protein conformation with fluctuated structure. Liposomes showed highly selective binding ability to conformationally changed proteins treated with various concentrations of guanidinium hydrochloride, as evaluated by immobilized liposome chromatography (ILC). In refolding of proteins, liposomes bound to refolding intermediate of proteins and prevented them from forming intermolecular aggregates. Refolding of bovine carbonic anhydrase, lysozyme and ribonuclease A was significantly improved in the presence of liposomes. Furthermore, by utilizing ILC, refolding of proteins was also successfully and simply carried out with considerable high reactivation yield.

The role of proline in the prevention of aggregation during protein folding in vitro.

Proline effectively inhibits protein aggregation during the refolding of bovine carbonic anhydrase. Other osmolytes used such as glycine and ethylene glycol fail to exhibit the 'aggregation-blockade' role shown by proline. Results of viscosity and ANS fluorescence (1-anilino-8-naphthalene sulphonic acid) experiments suggest that proline at high concentrations forms an ordered supramolecular assembly. Based on these results, it is proposed that proline behaves as a protein folding chaperone due to the formation of an ordered, amphipathic supramolecular assembly. To our knowledge, this is the first report wherein proline is proposed as a protein folding aid.

Polyethylene glycol enhanced refolding of bovine carbonic anhydrase B. Reaction stoichiometry and refolding model.

Polyethylene glycol (PEG) inhibited aggregation during refolding of bovine carbonic anhydrase B (CAB) through the formation of a nonassociating PEG-intermediate complex. Stoichiometric concentrations of PEG were required for complete recovery of active protein during refolding at aggregating conditions. For example, a PEG (Mr = 3350) to CAB molar ratio ([PEG]/[CAB]) of 2 was sufficient to inhibit aggregation during refolding at 1.0 mg/ml (33.3 microM) protein and 0.5 M guanidine hydrochloride. In addition, the PEG concentration required for enhancement was dependent upon the molecular weight and only molecular weights between 1000 and 8000 were effective in inhibiting aggregation. In the presence of PEG, the rate of refolding was the same as that observed for refolding without the formation of associated species. Refolding in the presence of PEG resulted in the rapid formation of a PEG complex with the molten globule first intermediate, and this PEG-intermediate complex did not aggregate. The CAB refolding kinetics in the presence of PEG were determined and used to develop a model of the PEG enhanced refolding pathway. The mathematical model was validated by independent activity measurements of CAB refolding. This model predicted that PEG enhanced refolding of CAB occurred by a specific interaction of PEG with the molten globule first intermediate to form a nonassociating complex which continued to fold at the same rate as the first intermediate. The predicted pathway and binding properties of PEG indicate that PEG enhanced refolding may be analogous to chaperonin mediated protein folding.

Transient association of the first intermediate during the refolding of bovine carbonic anhydrase B.

Many proteins which aggregate during refolding may form transiently populated aggregated states which do not reduce the final recovery of active species. However, the transient association of a folding intermediate will result in reduced refolding rates if the dissociation process occurs slowly. Previous studies on the refolding and aggregation of bovine carbonic anhydrase B (CAB) have shown that the molten globule first intermediate on the CAB folding pathway will form dimers and trimers prior to the formation of large aggregates (Cleland, J. L.; Wang, D. I. C. Biochemistry 1990, 29, 11072-11078; Cleland, J. L.; Wang, D. I. C. In Protein Refolding; Georgiou, G., De-Bernardez-Clark, E., Eds.; ACS Symposium Series 470; American Chemical Society: Washington, DC, 1991; pp 169-179). Refolding of CAB from 5 M guanidine hydrochloride (GuHCl) was achieved at conditions ([CAB]f = 10-33 microM, [GuHCl]f = 1.0 M) which allowed complete recovery of active protein as well as the formation of a transiently populated dimer of the molten globule intermediate on the refolding pathway. A kinetic analysis of CAB refolding provided insight into the mechanism of the association phenomenon. Using the kinetic results, a model of the refolding with transient association was constructed. By adjusting a single variable, the dimer dissociation rate constant, the model prediction fit both the experimentally determined active protein and dimer concentrations. The model developed in this analysis should also be applicable to the refolding of proteins which have been observed to form aggregates during refolding. In particular, the transient association of hydrophobic folding intermediates may also occur during the refolding of other proteins.(ABSTRACT TRUNCATED AT 250 WORDS)

Two slow stages in refolding of bovine carbonic anhydrase B are due to proline isomerization

Kinetics of refolding of bovine carbonic anhydrase B have been studied by the "double-jump" technique (i.e. the dependence of protein refolding on delay time in the unfolded state after fast unfolding). It is shown that two stages (the slow with a relaxation time of t1/2 approximately equal to 120 s and the superslow with t1/2 approximately equal to 600 s) observed during refolding of bovine carbonic anhydrase B are due to trans-cis isomerization of proline residues. The dependences of rate constants of these processes on temperature and on the final denaturant concentration were measured. Activation energies of both processes are the same, Ea = 18(+/- 2) kcal/mol. The rate constants of protein refolding do not depend on the final concentration of urea under native conditions. In addition, the rate of isomerization of essential proline residues in the "molten globule" intermediate state of bovine carbonic anhydrase was measured and found to be equal to that for unstructural polypeptides. The effect of several proline residues on carbonic anhydrase refolding is discussed.

Sequential mechanism of refolding of carbonic anhydrase B

The kinetics of refolding of bovine carbonic anhydrase B was studied by a variety of methods over a wide range of times (from milliseconds to hours). It has been shown that protein refolding proceeds through three stages. At the first stage ( t 1 2 ≈0.03 s) hydrophobic clusters and a compact state of the chain are formed. At the second stage ( t 1 2 ≈140 s) hydrophobic clusters are desolvated and the rigid native-like hydrophobic core is formed. At the third stage ( t 1 2 ≈600 s) the native active protein is formed.

Multiparameter kinetic study on the unfolding and refolding of bovine carbonic anhydrase B.

The kinetics of unfolding and refolding of bovine carbonic anhydrase B by guanidinium chloride have been studied by simultaneously monitoring several spectroscopic parameters, each of which reflects certain unique conformational features of the protein molecule. In the present report, far-UV circular dichroism (CD) was used to follow the secondary structural change, UV difference absorption was used to follow the exposure or burying of aromatic amino acid residues, and near-UV CD was used to follow tertiary structural changes during unfolding and refolding. The unfolding is described by two unimolecular rate processes, and refolding is described by three unimolecular rate processes. The minimum number of conformational species involved in the mechanism is five. The refolding of the protein followed by the above three parameters indicates that the process consists of an initial rapid phase in which the random-coiled protein is converted to an intermediate state(s) having secondary structure comparable to that of the native protein. This is followed by the burying of the aromatic amino acid residues to form the interior of the protein molecule. Subsequently, the protein molecule acquires its tertiary structure and folds into a unique conformation with the formation of aromatic clusters.

Kinetics and mechanism of refolding of bovine carbonic anhydrase. A probe study of the formation of the active site

In kinetic studies of the folding of bovine carbonic anhydrase from disorganized to native structure, an azosulfonamide, 2-(4-sulfomylphenylazo)-7-acetamido-1-hydroxynaphthalene-3,6-disulfonate (I), has been used as a probe to follow the dynamics of formation of the active site region. The probe is a specific inhibitor of the native enzyme that binds in the active site crevice. The experiments, with previous data (Yazgan, A., and Henkens, R. W. (1972), Biochemistry 11, 1314), show that a tight binding site for I forms at an intermediate stage in the folding process. A subsequent conformational change perturbs the visible absorption and circular dichroism of bound I and could result in even tighter binding. The subsequent change completes formation of the active site. This is shown by results from separate experiments on the kinetics of recovery of activity (p-nitrophenyl acetate as substrate). Similar probe methods could be used with other proteins and enzymes to study the kinetics and mechanism of regeneration of specific sites--for example, the active site.