Hydrophobic core of molten-globule state of bovine carbonic anhydrase B

The interaction which stabilizes the intermediate state of the protein folding and/or unfolding is important for understanding the structure formation mechanism of proteins. The partitioning of a hydrophobic fluorescence probe, pyrene, into the core of a ‘molten globule’ structure of bovine carbonic anhydrase B was measured, revealing a partition coefficient of about 10⁴. The result leads to the conclusion that the compact structure of the molten-globule state is formed by the hydrophobic interaction, as detergent micelles are formed by the same interaction.     

Influence of cyclodextrin ring substituents on folding-related aggregation of bovine carbonic anhydrase.

A common obstacle to proper renaturation of an unfolded protein is aggregation, an intermolecular side reaction of immense importance in biotechnology and in the pathogenesis of several neurodegenerative diseases. Cyclic sugars known as cyclodextrins have been used as protein-folding aids. The effect of cyclodextrin chemistry on aggregation and refolding of carbonic anhydrase was evaluated in this study. Size-exclusion HPLC showed that cyclodextrins inhibit aggregate formation without interfering with the correct renaturation of carbonic anhydrase. PAGE of refolded enzyme provides further evidence of inhibition of folding-related aggregation by natural and chemically modified cyclodextrins. Although the amount of aggregate formed and recovery of active enzyme was dependent on cavity size, the nature of the chemical substituents found on the rims of the sugar molecule seems to play a more important role in cyclodextrin-assisted refolding of carbonic anhydrase. In general, neutral or cationic cyclodextrins with small cavities were found to be better folding aids than anionic cyclodextrins with larger holes. Although the exact prediction of the effect of a cyclodextrin substitution on protein refolding is not possible at present, these results clearly show that modified cyclodextrins can be designed that effectively inhibit protein aggregation.     

Application of constrained regularization analysis to low-angle quasi-elastic light scattering

A constrained regularization procedure has been applied to a low-angle quasi-elastic light scattering system in order to determine particle size distributions. The conditions under which this procedure may be successfully applied to low-angle photon correlation spectroscopy have been characterized. Acquisition of photon count data over a short time period, relative to the long exponential decay constants of correlation functions obtained at low forward angles, resulted in particle size distributions which were stable with regard to peak width and weighted mean particle radius. Irrespective of the number of photon counts obtained, peak resolution and position on the particle size scale were not optimized unless anomalies in the correlation function due to transient increases in the mean photon counting rate were removed from the photon count data prior to autocorrelation. When such measures were taken, reasonable size distributions were obtained for well characterized protein standards and for liposomal suspensions.     

Reactivation of kinetics of guanidine denatured bovine carbonic anhydrase B.

Denaturation and reactivation of bovine carbonic anhydrase B was studied with particular attention to the anomalous behavior in the transition region (about 2 m guanidine hydrochloride) that had been reported by previous workers. The denaturation curve based on the partition coefficient of gel chromatography was markedly different from the one based on the ultraviolet difference spectroscopy. Intrinsic viscosity and fluorescence intensity were also measured in guanidine solution. Reactivation of esterase activity of the enzyme was over 90% complete with an average half time of 9 ± 1 min when the protein was fully denatured in 5 m guanidine hydrochloride. Similar reactivation from 2 m guanidine solution showed the dependence of the extent of final activity regain on the time of incubation in 2 m guanidine solution. It decreased to a plateau value of 40–50% of the native activity after 24 h in 2 m guanidine. The irreversibly inactivated fraction could be reactivated if it was transferred to 5 m guanidine before reactivation experiment. Renaturation kinetics followed by ultraviolet difference spectroscopy showed a fast phase ($\text{t}\text{1}\text{2}\text{< 30}\text{sec}$) and a slow phase ($\text{t}\text{1}\text{2}\text{= 7 ± 1}\text{min}$).     

A quasi-elastic laser light scattering study of tubulin and microtubule protein from bovine brain

Quasi-elastic light scattering has been used to characterize the oligomeric properties of solutions of glycerol-cycled bovine microtubule protein, and the properties of the 30 S oligomeric species and 6 S tubulin heterodimer prepared by gel filtration on Sepharose 6B. It is shown that in dimer preparations, as little as 0.04% by number of 30 S rings would account for the difference between an observed mean diffusion coefficient D_{20, W} = 3.1 × 10^?7 cm² s^?1 and the value of D_{20, W} = 5.1 × 10^?7 cm² s^?1 calculated for tubulin dimer of M_rel 100,000. The 30 S ring has an observed diffusion coefficient of D_{20, W} = 0.49 × 10^?7 cm² s^?1. These values are not changed significantly by the presence of 4 m-glycerol, indicating the persistence of 6 S and 30 S forms for dimer and ring, respectively.Mixtures of ring and dimer components of this preparation behave as a non-interacting two-component system, indicating the absence of substantial re-equilibration between the species at 5 °C and pH 6.5.The effect of salt on ring and microtubule protein samples indicates partial dissociation, consistent with the formation of additional intermediate oligomeric forms.In quasi-elastic light scattering measurements adapted to kinetic studies, changes in the oligomeric composition of microtubule protein are detected in the early stages of the reversible assembly process at pH 6.5. A 25% decrease in scattered light intensity, without significant change in mean diffusion coefficient, indicates the lability of the ring oligomeric structures, which undergo partial transformation to alternative oligomeric species under these assembly conditions.     

Molecular weight determination of bovine carbonic anhydrase

The molecular weight of bovine carbonic anhydrase was determined by osmometric and sedimentation equilibrium methods. The solvents used were 0.15 M KCl and 6.0 M guanidinium chloride. The value found was 28300 ± 300 which is lower than the values found by other investigators.As a part of the studies the intrinsic viscosities of the enzyme in 4.5 M guanidinium thiocyanate and 6.0 M guanidinium chloride were also ascertained. The values found, 25.4 ml/g and 24.7 ml/g, respectively, are smaller than expected on the basis of the molecular weight. This finding, however, is in agreement with the low value. 0.72 × 10^?3 cm³ mol/g² of the second virial coefficient in 6.0 M guanidinium chloride.     

Membrane-associated carbonic anhydrase purified from bovine lung

We found carbonic anhydrase activity associated with particulate fractions of homogenates of rat, rabbit, human, and bovine lungs. These membrane-associated carbonic anhydrases were remarkably stable in solutions containing sodium dodecyl sulfate (SDS). The bovine enzyme was dissolved with SDS and purified by affinity chromatography and gel filtration. The purified enzyme contains glucosamine, galactose, and sialic acid; it is at least 20% carbohydrate. The apparent molecular weight by SDS-polyacrylamide gel electrophoresis (52,000) may be higher than the actual molecular weight due to the presence of carbohydrate. The enzyme contains cystine, an amino acid that is absent in bovine erythrocyte carbonic anhydrase. Dithiothreitol greatly accelerated the rate of inactivation of the membrane-associated enzyme in SDS, so disulfide bonds appear to stabilize this enzyme. The specific CO2-hydrating activity was about half that of the erythrocyte enzyme. Acetazolamide inhibits the membrane-associated enzyme (Ki = 10 nM) nearly as well as the erythrocyte enzyme (Ki = 3 nM). Antibody to bovine erythrocyte carbonic anhydrase did not inhibit the membrane-associated enzyme. Other investigators have accumulated a good deal of evidence for carbonic anhydrase on the luminal surface of pulmonary capillaries. The enzyme described here appears to be a new isozyme whose properties are consistent with such a localization.     

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.     

Trigger factor-assisted folding of bovine carbonic anhydrase II

Spontaneous refolding of GdnHCl denatured bovine carbonic anhydrase II (BCA II) shows at least three phases: a burst phase, a fast phase, and a slow phase. The fast and slow phases are both controlled by proline isomerization. However, we find that in trigger factor (TF)-assisted BCA II folding, only the fast phase is catalyzed by wild-type TF, suggesting that certain proline residues are accessible in folding intermediates. The refolding yields of BCA II assisted by wild-type TF and TF mutants which lack PPIase activity are about the same, which provides further experimental evidence that the PPIase and chaperone activities of TF are independent. The binding of TF to folding intermediates during BCA II refolding was characterized by chemical crosslinking and Western blotting. A scheme for TF-assisted BCA II folding is proposed and the possible role of the TF dimer as a "binding" chaperone in vivo is discussed.     

Study of bovine carbonic anhydrase by 13C-NMR-spectroscopy

The study of internal mobility in enzymes is of considerable importance for the understanding of their catalytic function, which cannot be adequately described as a property of a rigid protein. [13C]NMR spectroscopy permits simultaneous and selective observation of spectral lines from carbon atoms in many different residues in the enzyme with the chemical shift and relaxation parameters sensitive to structure, conformation and local motion. The changes in internal mobility in bovine carbonic anhydrase B (carbonate hydrolase, EC 4.2.1.1) in the native form and at various stages of denaturation are studied. Measurements of the relaxation parameters (T1, T1 rho) and of the NOE of 13C nuclei in the native protein showed that the extensive beta-sheet together with groups in the active center has a considerable internal librational mobility with tau G about 10(-11) s. This librational mobility is fairly uniform for all the alpha-carbons in the native enzyme. The use of a semiempirical modification of the motional theory proposed by Woessner allows to use simultaneously all the relaxation parameters measured in order to determine reliable values of the various correlation times.     

Immunohistochemical detection of bovine ruminal carbonic anhydrase isoenzyme

Summary Rabbits immunized with low-activity ruminal carbonic anhydrase (RCA) isoenzyme, extracted from ruminal epithelial cells isolated by digestion with trupsin, yielded anti-RCA sera which reacted specifically with bovine RCA in double agar gel diffusion and immunoelectrophoretic tests, but failed to cross-react with bovine erythrocyte CA. The localization of RCA was identified in histological sections and isolated ruminal epithelial cell preparations by indirect immunofluorescence and immunoperoxidase tests as the basal, spinosum and granulosum layers of ruminal mucous epithelium.     

Thermal inactivation and conformational lock of bovine carbonic anhydrase

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.     

Formation of amyloid fibrils by bovine carbonic anhydrase

Amyloids are typically characterized by extensive aggregation of proteins where the participating polypeptides are involved in formation of intermolecular cross beta-sheet structures. Alternate structure attainment and amyloid formation has been hypothesized to be a generic property of a polypeptide, the propensities of which vary widely depending on the polypeptide involved and the physicochemical conditions it encounters. Many proteins that exist in the normal form in-vivo have been shown to form amyloid when incubated in partially denaturing conditions. The protein bovine carbonic anhydrase II (BCA II) when incubated in mildly denaturing conditions showed that the partially unfolded conformers assemble together and form ordered amyloid aggregates. The properties of these aggregates were tested using the traditional Congo-Red (CR) and Thioflavin-T (ThT) assays along with fluorescence microscopy, transmission electron microscopy (TEM), and circular dichroism (CD) spectroscopy. The aggregates were found to possess most of the characteristics ascribed to amyloid fibers. Thus, we report here that the single-domain globular protein, BCA II, is capable of forming amyloid fibrils. The primary sequence of BCA II was also analyzed using recurrence quantification analysis in order to suggest the probable residues responsible for amyloid formation.     

Motility analysis of circularly swimming bull spermatozoa by quasi-elastic light scattering and cinematography.

The Rayleigh-Gans-Debye approximation is used to predict the electric field autocorrelation functions of light scattered from circularly swimming bull spermatozoa. Using parameters determined from cinematography and modeling the cells as coated ellipsoids of semiaxes a = 0.5 micrometers, b = 2.3 micrometers, and c = 9.0 micrometers, we were able to obtain model spectra that mimic the data exactly. A coat is found to be a necessary attribute of the particle. It is also clear that these model functions at 15 degrees may be represented by the relatively simple function used before by Hallett et al. (1978) to fit data from circularly swimming cells, thus giving some physical meaning to these functional shapes. Because of this agreement the half-widths of experimental functions can now be interpreted in terms of an oscillatory frequency for the movement of the circularly swimming cell. The cinematographic results show a trend to chaotic behavior as the temperature of the sample is increased, with concomitant decrease in overall efficiency. This is manifested by a decrease in oscillatory frequency and translational speed.     

Letter: Crystallization and preliminary x-ray investigation of bovine erythrocyte carbonic anhydrase

The major form of bovine erythrocyte carbonic anhydrase has been prepared by a new method in which the conventional chloroform-ethanol treatment is replaced by chromatography on DEAE-Sephadex. Single crystals, suitable for high resolution X-ray diffraction studies, have been obtained from enzyme prepared by this method. The space group is P6₁22. The unit cell contains 12 enzyme molecules and has the dimensions $\text{a = b = 68}\text{A}\text{?}$, $\text{c = 244}\text{A}\text{?}$.     

Interaction of lead ions with bovine carbonic anhydrase: further studies

Lead-substituted bovine carbonic anhydrase is investigated and the return to the holoenzyme form with exchange of Pb²⁺ by Zn²⁺ is followed by uv difference spectroscopy and by esterase activity methods. Equimolar amounts of Pb²⁺ added to apocarbonic anhydrase release one hydronium ion per molecule below pH 6. Above this pH there is a net gain of hydronium ions by the enzyme, due to Pb(OH)⁺ → Pb(OH₂)^{2 +}, when the metal is bound within the active site of the enzyme molecule. The reduced hydrolysis by lead when it is bound to the enzyme is relevant to the theory of Zn²⁺ hydrolysis as a mechanism for carbon dioxide hydration by the holoenzyme and to the idea of an altered pK_hydrolysis when Zn²⁺ is bound in the enzyme active site cavity. Lead appears to be bound to a His residue in the active site and to interact with a Tyr residue nearby. The Tyr interaction is disrupted by a high concentration of chloride ions, (also by lower concentrations of cyanide ions), but such anions do not displace lead from the enzyme. At pH 8.0 the buffer-free exchange of Pb²⁺ by Zn²⁺ is found to be consistent with a second-order process with an effective β = (95 ± 7) M^?1 sec^?1. Thus lead is more rapidly replaced by zinc than is Mn²⁺ or VO²⁺ whose replacement kinetics have been reported by others. Comparison of esterase-activation and spectral curves with second-order models shows that the effective β is both large and buffer dependent, indicating that a proton transfer process or buffer anion effects may be rate limiting in the buffer-free case.