Bovine Erythrocyte Carbonic Anhydrase Research Articles

Synthesis of novel hybrid pharmacophore of N-((4-sulfamoylphenyl)carbamothioyl)alkanamides as potent carbonic anhydrase-II and 15-lipoxygenase inhibitors.

A series of N-((4-sulfamoylphenyl)carbamothioyl)alkanamides (5a-j) were synthesized by the reaction of sulphanilamide in dry acetone with freshly prepared alkyl and acyl isothiocyanates (5a-j). The structures of products were confirmed by IR, 1 H, and 13 C NMR. The synthesized compounds were screened as inhibitors of the bovine erythrocyte carbonic anhydrase isoform II (bCA II) and 15-lipoxygenase enzyme (15-LOX). Most of the derivatives showed significant activity against bCA-II while only few compounds were found active against 15-LOX. Molecular docking studies of most active compounds were carried out against bCA II as well as 15-LOX to rationalize the binding mode and interactions of compound in the active sites. Additionally, the pharmacokinetic properties of the compounds were predicted through computational tools, which reflect that these compounds possess acceptable pharmacokinetic profile and good drug-likeness.

Immobilization of Carbonic Anhydrase in Glass Micropipettes and Glass Fiber Filters for Flow-Through Reactor Applications.

There are various ways of immobilizing carbonic anhydrase (CA) on solid materials. One of the final aims is to apply immobilized CA for the catalytic hydration of carbon dioxide (CO2) as a first step in the conversion of gaseous CO2 into solid products. The immobilization method investigated allows a straightforward, stable, and quantifiable immobilization of bovine erythrocyte carbonic anhydrase (BCA) on silicate surfaces. The method is based on the use of a water-soluble, polycationic second-generation dendronized polymer with on average 1000 repeating units, abbreviated as de-PG21000. Several copies of BCA were first covalently linked to de-PG21000 through stable bisaryl hydrazone (BAH) bonds. Then, the de-PG21000-BAH-BCA conjugates obtained were adsorbed noncovalently either on microscopy glass coverslips, inside glass micropipettes, or in porous glass fiber filters. The apparent density of the immobilized BCA on the glass surfaces was about 8–10 pmol/cm2. In all three cases, the immobilized enzyme was highly active and stable when tested with p-nitrophenyl acetate as a model enzyme substrate at room temperature. The micropipettes and the glass fiber filters were applied as flow-through systems for continuous operation at room temperature. In the case of the glass fiber filters, the filters were placed inside a homemade flow-through filter holder which allows flow-through runs with more than one filter connected in series. This offers the opportunity of increasing the substrate conversion by increasing the number of BCA-containing filters.

A new affinity gel for the purification of α-carbonic anhdrases

The new affinity gel reported in this study was prepared using EUPERGIT C250L as a chromatographic bed material, to which etylenediamine spacer arms were attached to prevent steric hindrance between the matrix and ligand, and to facilitate effective binding of the CA-specific ligand, of the aromatic sulfonamide type for the purification of α-carbonic anhydrases (Cas; EC 4.2.1.1). Indeed, the aminoethyl moieties of the affinity gel were derivatized by reaction with 4-isothiocyanatobenzenesulfonamide, with the formation of a thiourea-based gel, having inhibitory effects against CAs. Both bovine erythrocyte carbonic anhydrase BCA and human (h) erythrocyte CA isoforms I, II (hCA I and II) have been purified from hemolysates, by using this affinity gel. The greatest purification fold and column yields for BCA and for cytosolic (hCA I + II) enzymes were of 181-fold (21.07%) and 184-fold (9.49%), respectively. Maximum binding was achieved at 15 °C and I = 0.3 ionic strength for α-carbonic anhydrases.

New aminobenzenesulfonamide–thiourea conjugates: Synthesis and carbonic anhydrase inhibition and docking studies

A variety of 1-substituted-3-(3-aminosulfonylphenyl)thioureas (3a–k) and two new 1-aroyl-3-(4-aminosulfonylphenyl)thiourea derivatives (5a and 5b) were synthesized by reaction of 3-aminobenzenesulfonamide and 4-aminobenzenesulfonamide respectively with freshly prepared aroyl/heteroaryl isothiocyanates in dry acetonitrile. FTIR, 1H NMR, 13C NMR, GC–MS and elemental analyses data confirmed the assigned structures to the synthesized compounds. Further structure of compound (3g) was also confirmed by single crystal XRD analysis. The compounds were investigated as inhibitors of the bovine erythrocyte carbonic anhydrase isoform II (bCA II). The inhibition constants of these compounds against bCA II were in the range 0.011–17.1 μM. Among the evaluated compounds, 1-substituted -3-(3-aminosulfonylphenyl)thiourea derivatives 3h and 5a were the most potent inhibitors with IC50 of 0.052 and 0.011 μM, respectively. In silico docking and molecular dynamics simulation studies were performed against bCA II and human CA II enzymes to rationalize the inhibitory properties of these compounds.

Opioid peptides derived from food proteins suppress aggregation and promote reactivation of partly unfolded stressed proteins

A new view of the opioid peptides is presented. The potential of small peptides derived from precursor food proteins, to bind to partly unfolded stressed proteins to prevent their irreversible aggregation and inactivation has been demonstrated in various in vitro test systems: dithiothreitol-induced aggregation of alpha-lactalbumin (LA), heat-induced aggregation of alcohol dehydrogenase (ADH), and aggregation and inactivation of bovine erythrocyte carbonic anhydrase (CA) in the process of its refolding after removal of stress conditions. Using dynamic light scattering (DLS), turbidimetry, fluorescence, and circular dichroism measurements protective effects of the synthetic opioid peptides: exorphin C from wheat gluten (Tyr-Pro-Ile-Ser-Leu), rubiscolin-5 from spinach ribulose-bisphosphate-carboxylase/oxygenase (Rubisco) (Tyr-Pro-Leu-Asp-Leu), and hemorphin-6 from bovine hemoglobin (Tyr-Pro-Trp-Thr-Gln-Arg) have been revealed. We have demonstrated the concentration-dependent suppression of light scattering intensity of aggregates of LA and ADH in the presence of the peptides, the population of nanoparticles with higher hydrodynamic radii being shifted to the lower ones, accompanied by an increase in the lag period of aggregation. The presence of the peptides in the refolding solution was shown to assist reactivation of CA and enhance the yield of the CA soluble protein. The results suggest that bioactive food protein fragments may be regarded as exogenous supplements to the endogenous defense mechanisms of the human organism under stress conditions.

Thermodynamic Analysis of the Low- to Physiological-Temperature Nondenaturational Conformational Change of Bovine Carbonic Anhydrase

The stability curve - a plot of the Gibbs free energy of unfolding versus temperature - is calculated for bovine erythrocyte carbonic anhydrase in 150 mM sodium phosphate (pH = 7.0) from a combination of reversible differential scanning calorimetry measurements and isothermal guanidine hydrochloride titrations. The enzyme possesses two stable folded conformers with the conformational transition occurring at ~30 degrees C. The methodology yields a stability curve for the complete unfolding of the enzyme below this temperature but only the partial unfolding, to the molten globule state, above it. The transition state thermodynamics for the low- to physiological-temperature conformational change are calculated from slow-scan-rate differential scanning calorimetry measurements where it is found that the free energy barrier for the conversion is 90 kJ/mole and the transition state possesses a substantial unfolding quality. The data therefore suggest that the x-ray structure may differ considerably from the physiological structure and that the two conformers are not readily interconverted.

Potent enzyme inhibitors derived from dromedary heavy-chain antibodies.

Evidence is provided that dromedary heavy-chain antibodies, in vivo-matured in the absence of light chains, are a unique source of inhibitory antibodies. After immunization of a dromedary with bovine erythrocyte carbonic anhydrase and porcine pancreatic alpha-amylase, it was demonstrated that a considerable amount of heavy-chain antibodies, acting as true competitive inhibitors, circulate in the bloodstream. In contrast, the conventional antibodies apparently do not interact with the enzyme's active site. Next we illustrated that peripheral blood lymphocytes are suitable for one-step cloning of the variable domain fragments in a phage-display vector. By bio-panning, several antigen-specific single-domain fragments are readily isolated for both enzymes. In addition we show that among those isolated fragments active site binders are well represented. When produced as recombinant protein in Escherichia coli, these active site binders appear to be potent enzyme inhibitors when tested in chromogenic assays. The low complexity of the antigen-binding site of these single-domain antibodies composed of only three loops could be valuable for designing smaller synthetic inhibitors.

Synthesis of zwitterionic acrylic acid buffers for isoelectric focusing in immobilized pH gradients.

A range of zwitterionic acrylic acid derivatives, buffering in the neutral and basic pH ranges, have been synthesized by the Mannich reaction of malonic acid, formaldehyde and a secondary amine. These compounds include 2-(4-morpholinomethyl)propenoic acid pK2 7.59 +/- 0.03 (23 degrees C), 2-[bis(2-hydroxyethyl)aminomethyl]propenoic acid pK2 approximately 8.6 (20 degrees C), 2-[bis(2-hydroxypropyl) aminomethyl]propenoic acid PK2 approximately 8.7 (20 degrees C), 2-[N-(2-hydroxyethyl)-N-methylaminomethyl]propenoic acid pK2 9.22+/-0.08 (22 degrees C), 2-[N-ethyl-N-(2hydroxyethyl)aminomethyl]-propenoic acid pK2 approximately 9.6 (20 degrees C), and 2-[4-(2-carboxyprop-2-enyl)piperazinylmethyl]propenoic acid, which has a sigmoidal buffering profile over the pH range 3-10. These zwitterionic acrylic acid buffers were successfully copolymerized with acrylamide to prepare immobilized pH gradients (IPGs) in the neutral to alkaline portion of the pH range. Bovine erythrocyte carbonic anhydrase isozymes were resolved on a pH 5-8 IPG prepared using 2-[4-(2-carboxyprop-2-enyl)piperazinylmethyl]propenoic acid as the immobilized buffer, and horse heart myoglobin was focused on pH 7.1-8.1 and pH 7.5-7.7 IPGs, using 2-(4-morpholinomethyl)propenoic acid as the immobilized buffer. In both cases the pK 9.3 Immobiline compound was used as the strongly basic titrant. These new compounds, besides possessing more hydrophilic residues than the corresponding commercial basic acrylamido buffers (Immobilines), resist hydrolysis at alkaline pH values.

High-resolution density gradient electrophoresis of subcellular organelles and proteins under nondenaturing conditions.

We have developed a density gradient electrophoresis device (DGE) and used it for the preparative separation of various endocytic organelles that are hard to separate by other means. Our separation by DGE of late endosomal vesicles, recycling vesicles, early endosomes and plasma membranes is unmatched. Using the same DGE device, we performed preparative high-resolution rate zonal separation of proteins using amphoteric buffers as originally described by Bier (Electrophoresis 1993, 14, 1011-1018). Isoforms of bovine beta-lactoglobulin, human apo-transferrin, and bovine erythrocyte carbonic anhydrase that have isoelectric points within 0.8 pH units were readily separated even in the absence of nonionic detergents. The DGE apparatus is inexpensive and has unique separation abilities for vesicles and proteins.

The Inhibition Kinetics of Bovine and Human Erythrocyte Carbonic Anhydrase Isozymes with Some Active Cations

In this study, the in vitro effects of some active cations on carbonic anhydrase (CA) isozymes were investigated. Bovine (BCA) and human (HCA-I and H CA-ll) isozymes were purified by affinity chromatography from bovine and human erythrocytes. In order to make the kinetic studies, esterase activities of CA isozymes were determined. By using AI{^3+}, Mn{^2+}, Sr{^2+}, Hg{^2+}, Ni{^2+}, Ca{^2+}, and Cd{^2+}ions, K _iconstants for CA isozymes were found by means of Linewear-Burk graphics. While A1{^3+} showed competitive inhibition, the others displayed noncompetitive inhibition. In addition, the inhibitor concentrations halving the enzyme activity (I{_50} values) were determined. The determined I{_50} values for BCA, HCA-I and CA-ll were fit to the obtained Ki values.

The effects of some pesticides on human and bovine erythrocyte carbonic anhydrase enzyme activitiesin vitro

Abstract The influence of Penncozeb [Mangan‐zinc ethylenediamine‐bisdithicarbamate], Dithane, [Manganese etylenebisdithiocarbamate], Cupravit [Cuper oksichloride], Bayleton [1‐(4‐ chlorophenoxy)‐3,3‐dimethyl‐1‐(1H‐1.2.4‐Thiazol‐l‐yl)‐2.2‐butanon], Baythroid [Cyano‐(4 fluoro‐3‐phenoxyphenyl)‐methyl‐3‐(2.2‐dichloroethyl ‐2. 2 metyl ‐ cyclo‐propancarboxylate], Mavrik [‐cyano‐3‐phenoxybenzyl N‐(2‐chloro—trifluoro ‐ p ‐tolyl], [alstar [2‐methyl biphenyl‐3‐ylmethyl(z)‐(IRS,3RS)‐3‐(2‐chloro‐3.3.3 ‐triflouroprop ‐1‐enyl)=Dimethylcyclo propenecarboxyate], Endosulfan [6.7.8.9.10.10‐hekzachloro‐1.5.5a.6.9.9a ‐ hekzahydro‐6. 9‐methano‐2.3.4 ‐ benzole dioxi atehiepin 3‐oxide] has been investigated on human erythrocyt carbonic anhydrase isozymes (HCA‐I, HCA‐II) and bovine erythrocyte carbonic anhydrase (BCA) in vitro. Among the chemicals; Baythroid, Talstar, Mavrik were determined to have inhibition effect, on bovine CA and human CA isoenzymes. Dithane had effect of inhibition only on BCA. The I50 values of chemicals c...

Rapid separation of carbonic anhydrase isozymes using cellulose acetate membrane electrophoresis

A method is described for the rapid separation of carbonic anhydrase (CA) isozymes by cellulose acetate membrane electrophoresis in which CA activity is detected using the pH-indicating dye, bromcresol purple. This method can detect bovine erythrocyte CA in a 0.3 mm 3 sample applied at a concentration of 100 ng cm -3 (total of 30 pg applied) while at higher concentrations three isozymes were observed. It was found, using a potentiometric technique, that intact cells of Anabaena flos-aquae (Cyanophyceae) and Chlorella ellipsoidea had no detectable activity while C. saccharophila and Chlamydomonas reinhardtii (Chlorophyceae) had external CA activity

PH-dependent association of carbonic anhydrase (CA) with gastric light microsomal membranes isolated from bovine abomasum. Partial characterization of membrane-associated activity

1. 1. The effect of pH on the association of carbonic anhydrase (CA) with bovine gastric light microsomal membranes (LMMs) was investigated (a) by washing LMMs containing CA activity with solutions of different pHs; (b) by studying the adsorption at various pHs of soluble bovine erythrocyte CA to washed gastric LMMs. In both cases, the association of CA with gastric LMMs was dependent on pH, being lower at neutral or alkaline pH. 2. 2. The amount of soluble CA associated with gastric LMMs at pHs 8.0 and 9.0 was reduced when 140 mM K +/10 mM Na + was added to the incubation medium. 3. 3. Two sources of CA activity in bovine gastric LMMs were assumed: a loosely- and a firmly-membrane-associated activity. Both CA activities were dose-dependently inhibited by acetazolamide (I 50: 3.6 × 10 −9 and 8.4 × 10 −9 M, respectively) and by chloride, acetate, iodide, bromide and nitrate at 100 mM. Firmly-membrane-associated activity appeared to be less sensitive to inhibition by acetazolamide, chloride and iodide. 4. 4. Both activities exhibited different behavior and stability following treatment with alkaline Triton X-100. 5. 5. The possible importance of a membrane-associated CA actyvity in gastric LMMs related to gastric acid secretion is discussed.

Metallotropic forms of zinc, copper and cadmium carbonic anhydrases and zinc carboxypeptidases reflected in differential pulse polarography

Bovine erythrocyte carbonic anhydrase II (ZnCA), its metal substituted analogs CuCA, CdCA and bovine pancreatic carboxypeptidase A (ZnCP) were studied by means of differential pulse polarography (DPP) in buffered media in the pH range of 7.0 to 9.0. Each of the metalloproteins studied yielded several DPP peaks which were attributed to the reduction of several types of metal centre. These centres arose through migration of the single Me(II) to other donor groups (forms B and D) than the groups known from crystals (forms A). The peak potentials of the known structures A of ZnCA and ZnCP allowed insight into the structures B and D. Surface denaturation and the interaction of metallotropic forms of the enzymes with inhibitors are discussed.

Metallotropic forms of zinc, copper and cadmium carbonic anhydrases and zinc carboxypeptidase reflected in differential pulse polarography

Bovine erythrocyte carbonic anhydrase II (ZnCA), its metal substituted analogs CuCA, CdCA and bovine pancreatic carboxypeptidase A (ZnCP) were studied by means of differential pulse polarography (DPP) in buffered media in the pH range of 7.0 to 9.0. Each of the metalloproteins studied yielded several DPP peaks which were attributed to the reduction of several types of metal centre. These centres arose through migration of the single Me(II) to other donor groups (forms B and D) than the groups known from crystals (forms A). The peak potentials of the known structures A of ZnCA and ZnCP allowed insight into the structures B and D. Surface denaturation and the interaction of metallotropic forms of the enzymes with inhibitors are discussed.

Use of Carbon Oxysulfide, a Structural Analog of CO2, to Study Active CO2 Transport in the Cyanobacterium Synechococcus UTEX 625

Carbon oxysulfide (carbonyl sulfide, COS) is a close structural analog of CO(2). Although hydrolysis of COS (to CO(2) and H(2)S) does occur at alkaline pH (>9), at pH 8.0 the rate of hydrolysis is slow enough to allow investigation of COS as a possible substrate and inhibitor of the active CO(2) transport system of Synechococcus UTEX 625. A light-dependent uptake of COS was observed that was inhibited by CO(2) and the ATPase inhibitor diethylstilbestrol. The COS taken up by the cells could not be recovered when the lights were turned off or when acid was added. It was concluded that most of the COS taken up was hydrolyzed by intracellular carbonic anhydrase. The production of H(2)S was observed and COS removal from the medium was inhibited by ethoxyzolamide. Bovine erythrocyte carbonic anhydrase catalysed the stoichiometric hydrolysis of COS to H(2)S. The active transport of CO(2) was inhibited by COS in an apparently competitive manner. When Na(+)-dependent HCO(3) (-) transport was allowed in the presence of COS, the extracellular [CO(2)] rose considerably above the equilibrium level. This CO(2) appearing in the medium was derived from the dehydration of transported HCO(3) (-) and was leaked from the cells. In the presence of COS the return to the cells of this leaked CO(2) was inhibited. These results showed that the Na(+)-dependent HCO(3) (-) transport was not inhibited by COS, whereas active CO(2) transport was inhibited. When COS was removed by gassing with N(2), a normal pattern of CO(2) uptake was observed. The silicone fluid centrifugation method showed that COS (100 micromolar) had little effect upon the initial rate of HCO(3) (-) transport or CO(2) fixation. The steady state rate of CO(2) fixation was, however, inhibited about 50% in the presence of COS. This inhibition can be at least partially explained by the significant leakage of CO(2) from the cells that occurred when CO(2) uptake was inhibited by COS. Neither CS(2) nor N(2)O acted like COS. It is concluded that COS is an effective and selective inhibitor of active CO(2) transport.

Carbonic anhydrase catalysis: An experiment on enzyme kinetics

This experiment utilizes bovine erythrocyte carbonic anhydrase; after preliminary specific activity determinations, each student develops his or her own laboratory project.

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.

Interaction of hydroflumethiazide and 2,4-disulfamyl-5-trifluoromethylaniline with cyclic AMP phosphodiesterase and carbonic anhydrase.

The inhibitory effect of hydroflumethiazide (HFT) and its metabolite, 2,4-disulfamyl-5-trifluoromethylaniline (DTA) on cyclic AMP phosphodiesterase and the binding of HFT and DTA to carbonic anhydrase was studied in vitro. Significant inhibition of rat kidney low-Km cyclic AMP phosphodiesterase was observed with DTA concentration above 2.5 X 10(-4) mol/l and with HFT concentration above 1 X 10(-4) mol/l. 50% inhibition was observed at a DTA concentration of 1 X 10(-3) mol/l. Binding of DTA and HFT to commercially obtained bovine erythrocyte carbonic anhydrase was demonstrated by equilibrium dialysis. Data were consistent with one class of binding sites. The product of n (number of binding sites) and Kass (association constant) was 5 X 10(5) M for DTA and 3.3 X 10(4) M for HFT at 2 degrees. In human blood in vitro at 37 degrees, the equilibrium erythrocyte/plasma concentration ratio was 18 for DTA and 1.6 for HFT. It is concluded that HFT and DTA have approximately the same potency as cyclic AMP phosphodiesterase inhibitors, whereas DTA is more extensively bound by erythrocyte carbonic anhydrase.

Renaturation of bovine erythrocyte carbonic anhydrase B denatured by acid, heat, and detergent.

Renaturation of bovine erythrocyte carbonic anhydrase B denatured by acid, heat, and detergent.