Refolding Of Carbonic Anhydrase Research Articles

Role of Arginine in the Stabilization of Proteins against Aggregation

The amino acid arginine is frequently used as a solution additive to stabilize proteins against aggregation, especially in the process of protein refolding. Despite arginine's prevalence, the mechanism by which it stabilizes proteins is not presently understood. We propose that arginine deters aggregation by slowing protein-protein association reactions, with only a small concomitant effect on protein folding. The associated rate effect was observed experimentally in association of globular proteins (insulin and a monoclonal anti-insulin) and in refolding of carbonic anhydrase. We suggest that this effect arises because arginine is preferentially excluded from protein-protein encounter complexes but not from dissociated protein molecules. Such an effect is predicted by our gap effect theory [Baynes and Trout (2004) Biophys. J. 87, 1631] for "neutral crowder" additives such as arginine which are significantly larger than water but have only a small effect on the free energies of isolated protein molecules. The effect of arginine on refolding of carbonic anhydrase was also shown to be consistent with this hypothesis.

Control of Aggregate Formation in Refolding of Carbonic Anhydrase at High Urea Concentrations and Effects of Urea Removal.

Refolding of CAB was studied at high concentrations of protein and urea. Aggregate formation was decreased by using higher concentrations of urea in refolding buffer. Refolding yields of 60% were obtained at high concentrations of CAB by using urea concentrations of 3.0-4.0 M in renaturation mixture. Comparison of the refolding yield and the amount of soluble CAB in renaturation mixture indicated that the apparent specific activity of refolded CAB was lower than that of native one at higher concentrations of urea. Difference in the steric structures of native and refolded CAB was observed in CD spectra of these samples. However, removal of urea from loosely folded CAB solution by dialysis recovered the native structure of CAB, as well as its native specific activity. Hence the refolding efficiency after dialysis was improved to about 70-80% at CAB concentrations of 3-5 kg/m3. Effect of incubation time of renaturation mixture before dialysis was studied, and it was shown that incubation time less than 3 hours increased aggregate formation and decreased the refolding yield. Therefore, formation of more stable intermediates in the refolding pathway decreases aggregate formation during urea removal. These results lead to a consecutive dilution-dialysis method for refolding at high concentrations of proteins.

Protein refolding using stimuli-responsive polymer-modified aqueous two-phase systems

The function of a stimuli-responsive polymer was studied for the utilization of protein unfolding and refolding in protein separation using aqueous two-phase systems (ATPS). Poly(ethylene glycol) (PEG) bound to a thermo-reactive hydrophobic head (poly(propylene oxide)-phenyl group (PPO-Ph group)) was used as the functional ligand to modify the PEG phase of the aqueous two-phase systems. Firstly, refolding of carbonic anhydrase from bovine (CAB) was examined in the presence of PPO-Ph-PEG at various temperatures. The refolding yield of CAB was strongly enhanced and aggregate formation was suppressed by addition of PPO-Ph-PEG at a specific temperature (50–55°C). The change in the local hydrophobicity of CAB and PPO-Ph-PEG was characterized using the aqueous two-phase partitioning method and a hydrophobic fluorescent probe. The local hydrophobicity of CAB was maximized at 60°C. The local hydrophobicity of PPO-Ph-PEO was also found to be increased above 45°C. A simple model for CAB refolding, which includes (i) PPO-Ph-PEG complex formation and CAB in the intermediate state and (ii) refolding and release of native CAB from the PPO-Ph-PEG surface, is suggested based on the evaluated surface hydrophobicity.

3F1115 Mechanical unfolding and refolding of carbonic anhydrase B in buffer studied by atomic force microscope

3F1115 Mechanical unfolding and refolding of carbonic anhydrase B in buffer studied by atomic force microscope

Molecular chaperone-like activity of hydrogel nanoparticles of hydrophobized pullulan: thermal stabilization with refolding of carbonic anhydrase B.

We have been studying the formation of hydrogel nanoparticles by the self-aggregation of hydrophobized polysaccharide and the effective complexation between these nanoparticles as a host and various globular soluble proteins as a guest. This paper describes a new finding that refolding of the heat-denatured enzyme effectively occurs with the nanoparticles and beta-cyclodextrin according to a mechanism similar to that of a molecular chaperone. In particular, the irreversible aggregation of carbonic anhydrase B (CAB) upon heating was completely prevented by complexation between the heat-denatured enzyme and hydrogel nanoparticles formed by the self-aggregation of cholesteryl group-bearing pullulan (CHP). The complexed CAB was released by dissociation of the self-aggregate upon the addition of beta-cyclodextrin. The released CAB refolded to the native form, and almost 100% recovery of the activity was achieved. The thermal stability of CAB was drastically improved by capture of the unfolded form which was then released to undergo refolding.

Cyclodextrins as Protein Folding Aids

Aggregation of proteins is a frequent occurence during their transition from random coil to native structure. The influence of cyclodextrins in the refolding of carbonic anhydrase under aggregating conditions was studied. Cyclodextrin prevented formation of protein aggregates during renaturation of carbonic anhydrase. In addition, over 90 % of active enzyme was recovered even at protein concentrations as high as 67 μM. The enhanced protein reactivation by cyclodextrins may be due to their ability to bind to hydrophobic sites in protein folding intermediate(s) followed by their subsequent removal as the protein refolds.

Reassessment of the putative chaperone function of prolyl- cis/trans-isomerases

The folding of proteins can be assisted by two unrelated groups of helper molecules. Chaperones suppress non-productive side reactions by stoichiometric binding to folding intermediates, and folding enzymes catalyze slow rate-limiting steps of folding. We reinvestigated, whether peptidyl-propyl- cis/trans-isomerases of the cyclophilin type act simultaneously as chaperones and as folding catalysts in the reactivation of human carbonic anhydrase II, as reported recently [Freskgård, P.-O et al. (1992) Science 258, 466–468; Rinfret, A. et al. (1994) Biochemistry 33, 1668–1673]. No increase in the yield of carbonic anhydrase-II could be detected in the presence of three different propyl isomerases, when reactivation was followed by a sensitive assay for an extended time of 4 h. We conclude that the role of propyl isomerases in the refolding of carbonic anhydrase can be explained solely by their isomerase activity. There is no need to invoke simultaneous functions as chaperones for these folding catalysts.