Oktay Arslan

Carbonic anhydrase inhibitors: Inhibition of the β-class enzyme from the yeast Saccharomyces cerevisiae with sulfonamides and sulfamates

The protein encoded by the Nce103 gene of Saccharomyces cerevisiae, a beta-carbonic anhydrase (CA, EC 4.2.1.1) designated as scCA, has been cloned, purified, characterized kinetically and investigated for its inhibition with a series of sulfonamides and one sulfamate. The enzyme showed high CO(2) hydrase activity, with a k(cat) of 9.4x10(5)s(-1), and k(cat)/K(M) of 9.8x10(7)M(-1)s(-1). Simple benzenesulfonamides substituted in 2-, 4- and 3,4-positions of the benzene ring with amino, alkyl, halogeno and hydroxyalkyl moieties were weak scCA inhibitors with K(I)s in the range of 0.976-18.45 microM. Better inhibition (K(I)s in the range of 154-654 nM) was observed for benzenesulfonamides incorporating aminoalkyl/carboxyalkyl moieties or halogenosulfanilamides; benzene-1,3-disulfonamides; simple heterocyclic sulfonamides and sulfanilyl-sulfonamides. The clinically used sulfonamides/sulfamate (acetazolamide, ethoxzolamide, methazolamide, dorzolamide, topiramate, celecoxib, etc.) generally showed effective scCA inhibitory activity, with K(I)s in the range of 82.6-133 nM. The best inhibitor (K(I) of 15.1 nM) was 4-(2-amino-pyrimidin-4-yl)-benzenesulfonamide. These inhibitors may be useful to better understand the physiological role of beta-CAs in yeast and some pathogenic fungi which encode orthologues of the yeast enzyme and eventually for designing novel antifungal therapies.

A New Method for Purification of Carbonic Anhydrase Isozymes by Affinity Chromatography

A new affinity gel for purification of carbonic anhydrase isozymes was prepared using EUPERGITR C-250L derivatized with p-aminobenzenesulfonamide, an inhibitor of carbonic anhydrase. The binding capacity of the affinity gel was determined at different temperatures, pH values, elution buffers, and ionic strengths. Human carbonic anhydrase isozymes (HCA I and HCA II) and bovine carbonic anhydrase (BCA) were purified in high yields from erythrocytes.

Carbonic anhydrase inhibitors. Inhibition of the β-class enzyme from the yeast Saccharomyces cerevisiae with anions

The protein encoded by the Nce103 gene of Saccharomyces cerevisiae, a beta-carbonic anhydrase (CA, EC 4.2.1.1) designated as scCA, has been cloned, purified, characterized kinetically, and investigated for its inhibition with a series simple, inorganic anions such as halogenides, pseudohalogenides, bicarbonate, carbonate, nitrate, nitrite, hydrogen sulfide, bisulfite, perchlorate, sulfate, and some of its isosteric species. The enzyme showed high CO(2) hydrase activity, with a k(cat) of 9.4x10(5) s(-1) and k(cat)/K(m) of 9.8x10(7) M(-1) s(-1). scCA was weakly inhibited by metal poisons (cyanide, azide, cyanate, thiocyanate, K(I)s of 16.8-55.6 mM) and strongly inhibited by bromide, iodide, and sulfamide (K(I)s of 8.7-10.8 microM). The other investigated anions showed inhibition constants in the low millimolar range.

Purification human PON1Q192 and PON1R192 isoenzymes by hydrophobic interaction chromatography and investigation of the inhibition by metals

In this study, a new purification strategy for human PON1 enzyme was developed using two-step procedures, namely ammonium sulfate precipitation and sepharose-4B-L-tyrosine-9-aminophenantrene hydrophobic interaction chromatography. SDS polyacrylamide gel electrophoresis of the enzyme indicates a single band with an apparent MW of 43kDa. Overall purification rate of our method was found 901-fold for R isoenzyme and 453-fold for Q isoenzyme. The V(max) and K(M) of the purified enzyme were determined for Q isoenzyme 55 EU and 0.599 mM and for R isoenzyme 50 EU and 0.492 mM, respectively. The in vitro effects of some heavy metals (Hg, Cd, Cu, Mn and Ni) were investigated on the purified human serum PON1Q and R isoenzyme, using paraoxon as substrate. Metals were more effective inhibitors on purified human serum PON1(R192) activity than PON1(Q192) activity. The kinetics of interaction of metals with the purified human serum PON1(R192) and PON1(Q192) indicated a different inhibition pattern. Kinetic constants K(M), V(max), and inhibition type were determined.

Synthesis and carbonic anhydrase inhibitory properties of novel coumarin derivatives

A newly series of water-soluble 1-alkyl-3-(4-methyl-7, 8-dihydroxy-2H-chromen-2-one) benzimidazolium chloride salts (3a-j) were synthesized and their inhibitory effects on the activity of purified human carbonic anhydrase (hCA) I and II were evaluated. hCA I and II from human erythrocytes were purified by a simple one step procedure by using Sepharose 4B-L-tyrosine-sulphanilamide affinity column. The result showed that all the synthesized compounds were inhibited the CA isoenzymes activity. Among them, 3g and 3j were found to be most active (IC50 = 22.09 µM and 20.33 µM) for hCA I and hCA II, respectively.

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

Abstract 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.

Evaluation of in vitro effects of some analgesic drugs on erythrocyte and recombinant carbonic anhydrase I and II

The in vitro effects of the injectable form of analgesic drugs, dexketoprofen trometamol, dexamethasone sodium phosphate, metamizole sodium, diclofenac sodium, thiocolchicoside, on the activity of purified human carbonic anhydrase I and II were evaluated. The effect of these drugs on erythrocyte hCA I and hCA II was compared to recombinant hCA I and hCA II expressed in Ecoli. IC50 values of the drugs that caused inhibition were determined by means of activity percentage diagrams. The IC50 concentrations of dexketoprofen trometamol and dexamethasone sodium phosphate on hCA I were 683 μM and 4250 μM and for hCA II 950 μM and 6200 μM respectively. Conversely, the enzyme activity was increased by diflofenac sodium. In addition, thiocolchicoside has not any affect on hCA I and hCA II. The effect of these drugs on erythrocyte hCA I and hCA II were consistent with the inhibition of recombinant enzymes.

Carbonic anhydrase activators: Activation of the β-carbonic anhydrase Nce103 from the yeast Saccharomyces cerevisiae with amines and amino acids

The protein encoded by the Nce103 gene of Saccharomyces cerevisiae, a beta-carbonic anhydrase (CA, EC 4.2.1.1) designated as scCA, was investigated for its activation with amines and amino acids. scCA was poorly activated by amino acids such as l-/d-His, Phe, DOPA, Trp (K(A)s of 82-90 microM) and more effectively activated by amines such as histamine, dopamine, serotonin, pyridyl-alkylamines, aminoethyl-piperazine/morpholine (K(A)s of 10.2-21.3 microM). The best activator was l-adrenaline, with an activation constant of 0.95 microM. This study may help to better understand the catalytic/activation mechanisms of the beta-CAs and eventually to design modulators of CA activity for similar enzymes present in pathogenic fungi, such as Candida albicans and Cryptococcus neoformans.

Synthesis of a novel affinity gel for the purification of carbonic anhydrases.

Abstract A new affinity gel was synthesized for the purification of carbonic anhydrase (CA, EC 4.2.1.1) isozymes from erythrocytes. The gel was prepared on a Sepharose 4B matrix on which a spacer arm based on ethylenediamine was covalently attached via CNBr activation, followed by reaction with the CA inhibitor 4-isothiocyanato-benzenesulfonamide. The derivatized gel incorporated thioureido-benzenesulfonamide moieties as CA ligand. The binding capacity of the new affinity gel was determined at different temperatures, pH values, ionic strengths and elution buffers. The maximum binding of various CAs was achieved at 25 °C with pH 8.5 and ionic strength around 0.4. The overall purifications for human (h) hCA I and hCA II were 672- and 580-fold, and with 62 and 43% yields, respectively. SDS–polyacrylamide gel electrophoresis showed single bands for each purified isozymes, corresponding to a molecular weight of approx. 29 kDa. This is an easily obtainable, efficient and robust affinity gel, useful for the purification of many other α-CAs.

Effects of some metals on paraoxonase activity from shark Scyliorhinus canicula

Paraoxonase (PON) is an organophosphate hydrolyser enzyme which also has antioxidant properties in metabolism. Due to its crucial functions, the inhibition of the enzyme is undesirable and very dangerous. PON enzyme activity should not be altered in any case. Inhibitory investigations of this enzyme are therefore important and useful. Metal toxicology of enzymes has become popular in the recent years. Here, we report the in vitro inhibitory effects of some metal ions, including Ni2+, Cd2+, Cu2+ and Hg2+, on the activity of shark serum PON (SPON). For this purpose, we first purified the enzyme from shark Scyliorhinus canicula (LINNAEUS, 1758) serum and analysed the alterations in the enzyme activity in the presence of metal ions. The KM and Vmax is 0.227 mM and 454.545 U/mL, respectively. The results show that metal ions exhibit inhibitory effects on SPON1 at low concentrations with IC50 values ranging from 0.29 to 2.00 mM. Copper was determined to be the most effective inhibitor with IC50 of 0.29 mM.