Characterization Of Carbonic Anhydrase Research Articles

Isolation, purification, and characterization of carbonic anhydrase from Azadirachta indica (neem) leaves and its CO2 sequestration efficiency

Carbonic anhydrase (CA) has been purified, immobilized, and characterized from a novel plant source Azadirachta indica (neem) leaves. The CA enzyme was purified to homogeneity to 19-fold. By SDS-PAGE the molecular mass of the purified CA was obtained to be 42.49 kDa. The pI value of the purified CA was found to be 4.95. Immobilization study of the purified CA was carried out with chitosan beads. The pH and temperature optima for free and immobilized enzyme were found to be 8.0, 30 °C and 9.0, 40 °C, respectively. From Lineweaver burk plot the km and Vmax values of the free and immobilized CA were found to be 0.89 mM, 0.118 μmol/min and 1.04 mM, 0.083 μmol/min, respectively. The storage stability of the free and immobilized CA was studied for 30 days where the immobilized CA was found to show better stability. The immobilized CA also showed satisfactory reusable capacity. Both free and immobilized neem leaves CA exhibited good CO2 sequestration capacity, forming CaCO3 formation in presence of CaCl2·2H2O under optimized conditions. The formed CaCO3 were further characterized by SEM, XRD, and FTIR.

Purification, Characterization of Carbonic Anhydrase from Turkey Muscle Tissue and Investigation of Inhibition Effects of Some Metal Ions and Pesticides

Purification, Characterization of Carbonic Anhydrase from Turkey Muscle Tissue and Investigation of Inhibition Effects of Some Metal Ions and Pesticides

Characterization of Carbonic Anhydrase 9 in the Alimentary Canal of Aedes aegypti and Its Relationship to Homologous Mosquito Carbonic Anhydrases.

In the mosquito midgut, luminal pH regulation and cellular ion transport processes are important for the digestion of food and maintenance of cellular homeostasis. pH regulation in the mosquito gut is affected by the vectorial movement of the principal ions including bicarbonate/carbonate and protons. As in all metazoans, mosquitoes employ the product of aerobic metabolism carbon dioxide in its bicarbonate/carbonate form as one of the major buffers of cellular and extracellular pH. The conversion of metabolic carbon dioxide to bicarbonate/carbonate is accomplished by a family of enzymes encoded by the carbonic anhydrase gene family. This study characterizes Aedes aegypti carbonic anhydrases using bioinformatic, molecular, and immunohistochemical methods. Our analyses show that there are fourteen Aedes aegypti carbonic anhydrase genes, two of which are expressed as splice variants. The carbonic anhydrases were classified as either integral membrane, peripheral membrane, mitochondrial, secreted, or soluble cytoplasmic proteins. Using polymerase chain reaction and Western blotting, one of the carbonic anhydrases, Aedes aegypti carbonic anhydrase 9, was analyzed and found in each life stage, male/female pupae, male/female adults, and in the female posterior midgut. Next, carbonic anhydrase 9 was analyzed in larvae and adults using confocal microscopy and was detected in the midgut regions. According to our analyses, carbonic anhydrase 9 is a soluble cytoplasmic enzyme found in the alimentary canal of larvae and adults and is expressed throughout the life cycle of the mosquito. Based on previous physiological analyses of adults and larvae, it appears AeCA9 is one of the major carbonic anhydrases involved in producing bicarbonate/carbonate which is involved in pH regulation and ion transport processes in the alimentary canal. Detailed understanding of the molecular bases of ion homeostasis in mosquitoes will provide targets for novel mosquito control strategies into the new millennium.

Purification and Characterization of Carbonic Anhydrase from Ağrı Balık Lake Trout Gill (Salmo trutta labrax) and Effects of Sulfonamides on Enzyme Activity

Carbonic anhydrase (CA) was purified from Ağrı Balık Lake trout gill (fCA) by affinity chromatography on a sepharose 4B-tyrosine-sulfanilamide column. The fCA enzyme was purified with about a 303.9 purification factor, a specific activity 4130.4 EU (mg-protein)(-1), and a yield of 79.3 by using sepharose-4B-L tyrosine-sulfanilamide affinity gel chromatography. The molecular weight determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was found to be about 29.9 kDa. The kinetic parameters, K(M) and V(max) were determined for the 4-nitrophenyl acetate hydrolysis reaction. Some sulfonamides were tested as inhibitors against the purified CA enzymes. The Ki constants for mafenide (1), p-toluenesulfonamide (2), 2-bromo-benzene sulfonamide (3), 4-chlorobenzene sulfonamide (4), 4-amino-6-chloro-1-3 benzenedisulfonamide (5), sulfamethazine (6), sulfaguanidine (7), sulfadiazine (8), and acetozazolamide (9) were in the range of 7.5-108.75 μM.

Characterization of carbonic anhydrase from Turkish native “Gerze” chicken and influences of metal ions on enzyme activity

Carbonic anhydrase was purified and characterized from erythrocytes of the Turkish native chicken, Gerze, for the first time. The enzyme was purified 57.65-fold with a yield of 52%, and a specific activity of 954.08 U/mg proteins having optimum pH at 8.0; optimum temperature at 30 °C; optimum ionic strength at 10 mM and stable pH at 8.0. The purified enzyme had apparent KM and Vmax values of 0.73 mM and 0.236 μmol × min−1, respectively. Al+3, Hg+2, Cu+2, Pb+2, and Cd+2 showed inhibitory effects on the enzyme. Pb+2 exhibited the strongest inhibitory action. Cd+2 and Hg+2 were moderate inhibitor, whereas Al+3 and Cu+2 showed weaker actions. All tested metals inhibited the enzyme in competitive manner. Our findings indicate that these metals inhibit the chicken enzyme in a similar manner to other α-CAs from mammals investigated earlier, but susceptibility to various metals differ between the native chicken and other mammalian enzymes.

Bio Remediation of CO2 and Characterization of Carbonic Anhydrase from Mangrove Bacteria

Bio Remediation of CO2 and Characterization of Carbonic Anhydrase from Mangrove Bacteria

IMMOBILIZATION OF CARBONIC ANHYDRASE ENZYME PURIFIED FROM Bacillus subtilis VSG-4 AND ITS APPLICATION AS CO2 SEQUESTERER

The purification, immobilization, and characterization of carbonic anhydrase (CA) secreted by Bacillus subtilis VSG-4 isolated from tropical soil have been investigated in this work. Carbonic anhydrase was purified using ammonium sulfate precipitation, Sephadex-G-75 column chromatography, and DEAE-cellulose chromatography, achieving a 24.6-fold purification. The apparent molecular mass of purified CA obtained by SDS-PAGE was found to be 37 kD. The purified CA was entrapped within a chitosan–alginate polyelectrolyte complex (C-A PEC) hydrogel for potential use as an immobilized enzyme. The optimum pH and temperature for both free and immobilized enzymes were 8.2 and 37°C, respectively. The immobilized enzyme had a much higher storage stability than the free enzyme. Certain metal ions, namely, Co2+, Cu2+, and Fe3+, increased the enzyme activity, whereas CA activity was inhibited by Pb2+, Hg2+, ethylenediamine tetraacetic acid (EDTA), 5,5′-dithiobis-(2-nitrobenzoic acid (DTNB), and acetazolamide. Free and immobilized CAs were tested further for the targeted application of the carbonation reaction to convert CO2 to CaCO3. The maximum CO2 sequestration potential was achieved with immobilized CA (480 mg CaCO3/mg protein). These properties suggest that immobilized VSG-4 carbonic anhydrase has the potential to be used for biomimetic CO2 sequestration.

Purification and characterization of carbonic anhydrase from sheep kidney and effects of sulfonamides on enzyme activity

Carbonic anhydrase (CA, EC: 4.2.1.1) was purified from sheep kidney by affinity chromatography on a Sepharose 4B-tyrosine-sulfanilamide column. By means of two consecutive procedures, the enzyme (sCA) was purified 227.61-fold with a yield of 60.75%, and a specific activity of 838.89U/mg proteins. The optimum temperature, ionic strength and pH were determined to be 35°C, 20mM and 8.5, respectively. The molecular weight determined by SDS–PAGE was found to be 29kDa. The kinetic parameters, KM and Vmax values were determined for the 4-nitrophenyl acetate (p-NpA) hydrolysis reaction. Some sulfonamides were tested as inhibitors against the purified CAs enzyme. The Ki constants for benzenesulfonamide (1), sulfanilamide (2), mafenide (3), 4-(2-aminoethyl) benzenesulfonamide (4), 4-methyl-benzenesulfonamide (5), 2-bromo-benzenesulfonamide (6), naphthalene-2-sulfonamide (7), 4-amino-6-chlorobenzene-1,3-disulfonamide (8) and saccharin (9) were in the range 1.348–69.31μM.

Purification and characterization of carbonic anhydrase from the teleost fish Dicentrarchus labrax (European seabass) liver and toxicological effects of metals on enzyme activity

Carbonic anhydrase (EC 4.2.1.1; CA) was purified and characterized from the liver of the teleost fish Dicentrarchus labrax (European seabass) for the first time. The purification procedure consisted of a single step affinity chromatography on Sepharose 4B-tyrosine-sulfanilamide. The enzyme was purified 78.8-fold with a yield of 46%, and a specific activity of 751.72 U/mg proteins. It has an optimum pH at 7.5; an optimum temperature at 25 °C; an optimum ionic strength at 10 mM and a stable pH at 8.5. The kinetic parameters of this enzyme were determined for its esterase activity, with 4-nitrophenyl acetate (NPA) as substrate and the purified enzyme had an apparent K M and V max values of 0.44 mM and 0.249 μmol x min −1, respectively. The following metals, Al +3, Cu +2, Pb +2, Co +3, Ag +1, Zn +2 and Hg +2 showed inhibitory effects on the enzyme. Al +3 and Cu +2 exhibited the strongest inhibitory action. Pb +2 was moderate inhibitor, whereas other metals showed weaker actions. All tested metals inhibited the enzyme in a competitive manner. Our findings indicate that these metals inhibit the fish enzyme in a similar manner to other α-CAs from mammals investigated earlier, but the susceptibility to various metals differ between the fish and mammalian enzymes. Our results also demonstrate that these metals might be dangerous at low micromolar concentrations for fish CA enzymes.

Characterization of carbonic anhydrase from diversified genus for biomimetic carbon-dioxide sequestration.

Diversified group of bacteria were screened for carbonic anhydrase (CA) activity. Significant CA activity was found in crude enzyme extracts of Enterobacter and Aeromonas isolates while minimal or negligible CA activity was observed in case of Shigella and Klebsiella spp. Optimization and characterization study of potent CA producing isolates revealed that the maximum enzyme activity of 3.86 EU/ml was observed in E. taylorae and the optimum pH range for enzyme stability was found to be 7.5-9.0 along with an optimum temperature range of 35-50 °C. The molecular mass of CA was 29-kDa indicating α-type with periplasmic and cytosolic location. Present investigation for the first time reports CA in diversified genus and optimized parameters for enhanced production of CA in Enterobacter sp. & Aeromonas sp. from fresh water bodies that inturn lay down grounds for exploitation of CA from E. taylorae as an efficient catalyst for CO(2) sequestration within a bioreactor.

Isolation and characterization of carbonic anhydrases from the marine diatom Phaeodactylum tricornutum

Carbonic anhydrase (CA) isozymes were identified and isolated from three strains of Phaeodactylum tricornutum [University of Texas Culture Collection (UTEX 640), North Eastern Pacific Culture Collection at the University of British Columbia B31 and Culture Collection of Algae and Protozoa 1052/1A]. External (CAext) and internal CA activity was detected by potentiometric assay of intact cells and cell homogenates of air and high CO2‐grown cells. CAext was detected only in UTEX 640 grown under CO2‐limited conditions and present in trace amounts in cells grown on high CO2. CA isozymes in cells extracts were separated by cellulose acetate electrophoresis and by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis. All three strains had two CA bands in common, while UTEX 640 had a third, faster‐running band which was absent from extracts of high CO2‐grown cells and thus was the external isozyme. The internal CA isoforms of the UTEX 640 strain were shown to have molecular masses of 28 and 25 kDa, and the external 24 kDa. A fourth CAext isozyme with a molecular weight of 23.5 kDa was later detected using a polyclonal CA antibody. The CA isozymes were low‐CO2‐inducible proteins because Western blot analysis, using a polyclonal antibody, indicated that CA expression was repressed in high CO2‐grown cells. CA localization, using both immunofluorescence and immunogold techniques, with air‐grown cells indicated that the CAext was located in the periplasmic space and on the cell membrane, whereas in high CO2‐grown cells only internal CA was detected.

Purification and characterization of carbonic anhydrase from bovine stomach and effects of some known inhibitors on enzyme activity

Carbonic anhydrase (CA) was purified from four different cell localisation (outer peripheral, cytosolic, inner peripheral and integral) in bovine stomach using affinity chromatography with Sepharose-4B-l-tyrosine sulphanilamide. During the purification steps, the activity of the enzyme was measured using p-nitrophenyl acetate at pH 7.4. Optimum pH and optimum temperature values for all CA samples were determined, and their Km and Vmax values for the same substrate by Lineweaver–Burk graphics. The extent of purification for all CA localizations was controlled by SDS-PAGE. The Km values at optimum pH and 20°C were 0.625 mM, 0.541 mM, 0.785 mM and 0.862 mM with p-nitro phenyl acetate, for all CA localizations. The respective Vmax values at optimum pH and 20°C were 0.875 μmol/L min, 0.186 μmol/L min, 0.214 μmol/L min and 0.253 μmol/L min with the same substrate. The Ki and I50 values for the inhibitors sulphanilamide, KSCN, NaN3 and acetazolamide were determined for all the CA localizations.

762 poster Characterization of carbonic anhydrase 9 (CA9) as an endogenous hypoxia marker in live tumor cells

762 poster Characterization of carbonic anhydrase 9 (CA9) as an endogenous hypoxia marker in live tumor cells

96 Characterization of carbonic anhydrase 9 (CA9) overexpression: endogenous hypoxia marker and potential tumor-specific target

96 Characterization of carbonic anhydrase 9 (CA9) overexpression: endogenous hypoxia marker and potential tumor-specific target

Characterization of carbonic anhydrases from Riftia pachyptila, a symbiotic invertebrate from deep-sea hydrothermal vents.

The symbiotic hydrothermal vent tubeworm Riftia pachyptila needs to supply its internal bacterial symbionts with carbon dioxide, their inorganic carbon source. Our aim in this study was to characterize the carbonic anhydrase (CA) involved in CO(2) transport and conversion at various steps in the plume and the symbiotic tissue, the trophosome. A complete 1209 kb cDNA has been sequenced from the trophosome and identified as a putative alpha-CA based on BLAST analysis and the similarities of total deduced amino-acid sequence with those from the GenBank database. In the plume, the putative CA sequence obtained from cDNA library screening was 90% identical to the trophosome CA, except in the first 77 nucleotides downstream from the initiation site identified on trophosome CA. A phylogenetic analysis showed that the annelidan Riftia CA (CARp) emerges clustered with invertebrate CAs, the arthropodan Drosophila CA and the cnidarian Anthopleura CA. This invertebrate cluster appeared as a sister group of the cluster comprising mitochondrial and cytosolic isoforms in vertebrates: CAV, CAI II and III, and CAVII. However, amino acid sequence alignment showed that Riftia CA was closer to cytosolic CA than to mitochondrial CA. Combined biochemical approaches revealed two cytosolic CAs with different molecular weights and pI's in the plume and the trophosome, and the occurrence of a membrane-bound CA isoform in addition to the cytosolic one in the trophosome. The physiologic roles of cytosolic CA in both tissues and supplementary membrane-bound CA isoform in the trophosome in the optimization of CO(2) transport and conversion are discussed.

Characterization of carbonic anhydrase from Neisseria gonorrhoeae

Characterization of carbonic anhydrase from Neisseria gonorrhoeae

Purification and Characterization of Carbonic Anhydrase from Bovine Erythrocyte Plasma Membrane

ABSTRACT Carbonic anhydrase (CA) was purified from bovine erythrocyte plasma membrane and characterized in this study. For this purpose, the blood taken from young animals was hemolysed, the membrane fraction was separated, and this fraction was repeatedly washed. The enzyme (CA) was removed from the membrane with buffered TritonX-100 (1%); it could be purified at a factor of 22.8 by affinity chromatography.

Characterization of carbonic anhydrase and anion exchange in the erythrocytes of bowfin ( Amia calva), a primitive air-breathing fish

The purpose of this study was to investigate the characteristics of carbonic anhydrase (CA) and the Cl −/HCO 3 − exchanger (Band 3; AE1) in the erythrocytes of bowfin ( Amia calva), a primitive air-breathing fish, in order to further understand the strategies of blood CO 2 transport in lower vertebrates and gain insights into the evolution of the vertebrate erythrocyte proteins, CA and Band 3. A significant amount of CA activity was measured in the erythrocytes of bowfin (70 mmol CO 2 min −1 ml −1), although it appeared to be lower than that in the erythrocytes of teleost fish. The turnover number ( K cat) of bowfin erythrocyte CA was intermediate between that of the slow type I CA isozyme in agnathans and elasmobranchs and the fast type II CA in the erythrocytes of the more recent teleost fishes, but the inhibition properties of bowfin erythrocyte CA were similar to the fast mammalian CA isozyme, CA II. In contrast to previous findings, a plasma CA inhibitor was found to be present in the blood of bowfin. Bowfin erythrocytes were also found to possess a high rate of Cl −/HCO 3 − exchange (6 nmol HCO 3 − s −1 cm −2) that was sensitive to DIDS. Visualization of erythrocyte membrane proteins by SDS-PAGE revealed a major band in the 100 kDa range for the trout, which would be consistent with the anion exchanger. In contrast, the closest major band for the membranes of bowfin erythrocytes was around the 140 kDa range. Taken together, these results suggest that the strategy for blood CO 2 transport in bowfin is probably similar to that in most other vertebrates despite several unique characteristics of erythrocyte CA and Band 3 in these primitive fish.

Purification and characterization of carbonic anhydrase from the saliva of the rat.

Carbonic anhydrase purified from the saliva of the rat had kinetic properties identical with those of carbonic anhydrase II from rat red cells, but its molecular properties were distinctly different from the type II isozyme. Kinetic parameters were measured under steady state conditions by stopped-flow spectrophotometry and under equilibrium conditions by an 18O exchange method. The turnover number kcat for hydration of CO2 was 6.5 X 10(4) s-1 and the Michaelis constant was 4.2 mM at pH 7.5 and 25 degrees C, values which are equal to the steady state constants for red cell carbonic anhydrase II from the rat. Inhibition of the salivary isozyme by sulfanilamide (Ki = 3.7 microM) was nearly as efficient as inhibition of the erythrocyte isozyme II (Ki = 1.1 microM). The molecular weight for the salivary isozyme was 46,000 and the isoelectric point was 5.5. Salivary carbonic anhydrase had high mannose oligosaccharide components as measured by concanavalin A binding. The amino acid composition for the salivary isozyme was not similar to rat type II, but it was similar to that reported for membrane-bound carbonic anhydrase from bovine lung (Whitney, P.L., and Briggle, T.V. (1982) J. Biol. Chem. 257, 12056-12059). These observations suggest to us that salivary carbonic anhydrase is a secretory product.

Isolation and partial characterization of carbonic anhydrase from erythrocytes of Meleagris gallopavo.

A soluble enzyme with carbonic anhydrase activity has been isolated from domestic turkey(Meleagris gallopavo) erythrocytes and purified by chloroformethanol precipitation, ammonium sulfate fractionation and gel filtration on a Sephadex G-75 column. Analytical polyacrylamide gel disc electrophoresis showed one major and two minor bands. The specific activity for the CO2 hydration reaction was approximately 2000 Wilbur-Anderson units/mg protein at 0°C. The presence of the reducing agents 2-mercaptoethanol or dithioerythritol was required throughout the procedure. Upon removal of the 2-mercaptoethanol by dialysis the activity was lost but could be restored by addition of the reducing agent. The enzymatic activity was inhibited by acetazolamide,p-chloromercuribenzoate ando-iodosobenzoate. Esterase activity was detected withp-nitrophenylacetate as the substrate. The molecular weight of the enzyme was determined as 31,000 by gel filtration and 34,000 ± 2000 with analytical ultracentrifugation. Atomic absorption spectroscopy indicated the presence of zinc in the ratio of one mole of zinc per one mole of enzyme.