Mechanism of an insect glutathione S-transferase: kinetic analysis supporting a rapid equilibrium random sequential mechanism with housefly I1 isoform

The steady-state kinetics of glutathione S-transferase I1 (GST I1) from housefly Musca domestica expressed in Escherichia coli were investigated with glutathione (GSH) and 1-chloro-2,4-dinitrobenzene (CDNB). Concentrations of the varied substrates were from 0.03 to 1 mM for GSH and 0.05 to 1 mM for CDNB. Within this range, Michaelis-Menten behaviour was observed and convergent straight lines in double reciprocal plots excluded a ping-pong kinetic mechanism. Instead, data were consistent either with rapid-equilibrium random or with steady-state ordered sequential mechanisms because of abscissa convergence. Discrimination was achieved by studying the reaction with another electrophilic partner, p-nitrophenyl-acetate (PNPA). Concentrations of PNPA and GSH varied within the ranges 0.5 to 10 mM and 0.03 to 0.6 mM, respectively. The complete set of data supports the proposal of a rapid-equilibrium random-sequential model with strictly independent sites for GSH and CDNB or PNPA. Kinetic parameters are thus true dissociation equilibrium constants with values of 0.15 mM for GSH, 0.15 mM for CDNB, and 7 mM for PNPA. Analysis of the inhibition by the product (S-(2,4-dinitrophenyl)-glutathione, 10 to 100 microM), on the coupling reaction between GSH and CDNB with either GSH (0.05 to 0.5 mM, CDNB 0.2 mM) or CDNB (0.05 to 0.5 mM, GSH 0.2 mM) varied, consistent with the proposed mechanism. Binding of product to the free enzyme excludes GSH (competitive inhibition pattern with Kp = 12 microM) but only slightly hinders binding of CDNB. Binding free energies, together with the inhibition pattern, suggest that the non-peptidic moiety of product interacts with an alternative sub-site within the large open pocket accommodating the various electrophilic substrates. These results lead us to propose a model for intra-pocket shifting of the non-peptidic moiety upon product formation which contributes to the product release.     

Effects of trivalent antimony on human erythrocyte glutathione-S-transferases

Trivalent antimony (SB3+) in the form of potassium antimony tartrate was found to be an inhibitor of glutathione-S-transferases (GST) from human erythrocytes with a 50% inhibition concentration (IC50) of 0.05 mM. The inhibition was, however, incomplete with 15-20% of the GST activity remaining unaffected. In comparison, ethacrynic acid, a known inhibitor of GST, was tenfold more potent and affected close to 100% inhibition. Pentavalent antimony (SB5+) in the form of sodium stibogluconate had no effect on GST. Group V metalloids such as arsenite was slightly inhibitory, and arsenate was noninhibitory. When compared with five heavy metals, the inhibitory potency followed the order of SB3+ > Hg2+, Cu2+ > Cd 2+ > Cr3+ > Fe2+ x SB3+ inhibition of GST was competitive against the substrate 1-chloro-2,4-dinitrobenzene (CDNB) with an apparent Ki of 0.018 mM. Increasing the glutathione (GSH) concentration, however, produced a biphasic response: at concentrations below 1 mM, GSH was noncompetitive against SB3+, but at 1 mM and higher it was apparently competitive. A concurrent study of interactions between GSH, CDNB, and SB3+ showed that there was a significant nonenzymatic conjugation of CDNB at high GSH concentrations, which was suppressed by SB3+. The presence of albumin (500 mg/dL), or up to 5 mM N-acetylcysteine, cysteine, or ethylenediamine tetraacetic acid (EDTA) did not protect GST from the inhibitory effect of SB3+. The ability of erythrocyte GST to conjugate CDNB, which was measured directly by the formation of dinitrophenyl-glutathione (DNP-glutathione), was reduced by approximately 20 and 33%, respectively, in the presence of 2 and 10 mM SB3+, and nearly abolished with the addition of 0.2 mM ethacrynic acid. Based on these inhibition characteristics and the preferential accumulation of SB3+ in mammalian erythrocytes, it may be deduced that in the case of high antimonial intake, for example, during therapeutic treatment of Leishmaniasis, SB3+ levels in erythrocytes may be high enough to depress GST activity, which might compromise the ability of erythrocytes to detoxify electrophilic xenotbiotics.     

The inhibition characteristics of human placental glutathione S-transferase-π by tricyclic antidepressants: amitriptyline and clomipramine

Tricyclic antidepressants (TCAs) are the non-selective amine re-uptake inhibitors, well absorbed from small intestine, cross the blood-brain barrier, distributed in the brain, and are bound to glutathione S-transferase-π (GST-π). TCAs can pass through placenta, accumulate in utero baby, and cause congenital malformations. Thus, the study of the interaction of GST-π with antidepressants is crucial. In this study, the interaction of GST-π with amitriptyline and clomipramine was investigated. The K (m) values for glutathione (GSH) and 1-chloro-2,4-dinitrobenzene (CDNB) were found to be 0.16 ± 0.04 and 3.60 ± 1.67 mM, respectively. The V (m) values were varying according to the fixed substrate; [CDNB] fixed, 53 ± 3 and [GSH] fixed 182 ± 63 U/mg protein. At variable [GSH] and variable [CDNB], the k (cat) values of 7.0 × 10(6) and 1.42 × 10(7) s(-1) and the k (cat)/K (m) values of 4.38 × 10(10) and 3.94 × 10(9 )M(-1 )s(-1) were obtained, respectively. At fixed [CDNB] and variable [GSH], amitriptyline (K (s) = 0.16 ± 0.03 mM; α = 2.08; and K (i) = 1.75 ± 0.37 mM) and clomipramine (K (s) = 0.24 ± 0.05 mM; α = 1.57; and K (i) = 3.90 ± 2.26 mM) showed linear mixed-type inhibition whereas when the varied substrate is CDNB, amitriptyline (K (i) = 4.90 ± 0.68 mM) and clomipramine (K (i) = 3.37 ± 0.39 mM) inhibition were noncompetitive. The inhibition of GST-π by TCAs means the destruction of its protective role against toxic electrophiles. The effect of antidepressants on fetus will be much severe, thus, the antidepressant therapy of pregnant women should be done with caution.     

A rapid equilibrium random sequential bi-bi mechanism for human placental glutathione S-transferase

Double-reciprocal plots of initial-rate data for the conjugation of 1-chloro-2,4-dinitrobenzene (CDNB) and GSH by human placental GSH S-transferase pi were linear for both substrates. Computer modelling of the initial-rate data using nonlinear least-squares regression analysis favoured a rapid equilibrium random sequential bi-bi mechanism, over a steady-state random sequential mechanism or a steady-state or rapid equilibrium ordered mechanism. KGSH was calculated as 0.125 +/- 0.006 mM, KCDNB was 0.87 +/- 0.07 mM and alpha was 2.1 +/- 0.3 for the rapid equilibrium random model. The product, S-(2,4-dinitrophenyl)glutathione, was a competitive inhibitor with respect to GSH, and a mixed-type inhibitor toward CDNB (KP = 18 +/- 3 microM). The observed pattern of inhibition is consistent with a rapid equilibrium random mechanism, with a dead-end enzyme.CDNB.product complex, but inconsistent with the inhibition patterns of other bireactant mechanisms. Since rat liver GSH S-transferase 3-3 acts via a steady-state random sequential mechanism [1], while human placental GSH S-transferase and perhaps also rat liver GSH S-transferase 1-1 [2] exhibit rapid equilibrium random mechanisms, we conclude that the kinetic mechanism of the GSH S-transferases is isoenzyme-dependent.     

Cadmium(II)-stimulated enzyme activation of Arabidopsis thaliana phytochelatin synthase 1

Phytochelatin (PC), a class of heavy metal-binding peptides, is synthesized from the tripeptide glutathione (GSH) and/or previously synthesized PC in a reaction mediated by PC synthase (PCS). In the present study, the PC production rate catalyzed by recombinant Arabidopsis PCS1 (rAtPCS1) in the presence of a constant free Cd(II) level increased steadily and the kinetic parameters were approximated using a substituted-enzyme mechanism in which GSH and bis(glutathionato)cadmium acted as co-substrates. In contrast, the PC production rate as a function of GSH concentration at a constant total Cd(II) concentration reached a maximum, which shifted toward higher GSH concentrations as the concentration of Cd(II) was increased. These observations are consistent with the suggestion that rAtPCS1 possesses a Cd(II) binding site where Cd(II) binds to activate the enzyme. The affinity constant, optimized using a one-site mathematical model, successfully simulated the experimental data for the assay system using lower concentrations of Cd(II) (5 or 10 μM) but not for the assay using higher concentrations (50 or 500 μM), where a sigmoidal increase in PCS activity was evident. Furthermore, the PCS activity determined at a constant GSH concentration as a function of Cd(II) concentration also reached a maximum. These findings demonstrate that rAtPCS1 also possesses a second Cd(II) binding site where Cd(II) binds to induce an inhibitory effect. A two-site mathematical model was applied successfully to account for the observed phenomena, supporting the suggestion that rAtPCS1 possesses two Cd(II) binding sites.     

Comparative insight into the Zn(II)-, Cd(II)- and Cu(I)-binding features of the protozoan Tetrahymena pyriformis MT1 metallothionein

Tetrahymena pyriformis MT1 (TpyMT1) is a model among ciliate metallothioneins (MTs). Here, we report on the analytic (ICP-AES, GC-FPD), spectroscopic (CD, UV-Vis, Raman) and spectrometric (ESI-MS) characterization of its recombinant Cd(II)-, Zn(II)- and Cu(I)-complexes, and of those formed during in vitro Zn/Cd and Zn/Cu replacement. In the presence of Cd(II), TpyMT1 renders a major Cd 11-TpyMT1 species, which is also the final step reached in the in vitro Zn/Cd exchange process in Zn 11-TpyMT1. Spectroscopic data supports a different folding of the isostoichiometric Cd 11- and Zn 11-TpyMT1 complexes. Unexpectedly, TpyMT1 biosynthesis in Zn(II)-rich cultures was sensitive to the aeration degree, so that high oxygenation rendered undermetalated, partially-oxidized, complexes (Zn9-TpyMT1). Biosynthesis in Cu(I)-rich media rendered extremely heterogeneous mixtures of CuxZny-species (x+y=8-20), where the higher the aeration, the higher the Zn(II) content. The complexity of these samples was reproduced during the Zn/Cu replacement, as the number of generated species increased gradually with the addition of copper to Zn(11)-TpyMT1. According to our results, a clear preference of TpyMT1 for Cd(II) binding, rather than for Zn(II), and especially Cu(I) can be postulated. This character is totally consistent with the induction pattern of the TpyMT1 gene and the postulated role of TpyMT1 in Cd-detoxification.     

Concurrent sorption of Zn(II), Cu(II) and Co(II) by Oscillatoria angustissima as a function of pH in binary and ternary metal solutions

This paper reports biosorption of Zn(II), Cu(II) and Co(II) onto O. angustissima biomass from single, binary and ternary metal solutions, as a function of pH and metal concentrations via Central Composite Design generated by statistical software package Design Expert 6.0. The experimental design revealed that metal interactions could be best studied at lower pH range i.e. 4.0-5.0, which facilitates adequate availability of all the metal ions. The sorption capacities for single metal decreased in the order Zn(II)>Co(II)>Cu(II). In absence of any interfering metals, at pH 4.0 and an initial metal concentration of 0.5 mM in the solution, the adsorption capacities were 0.33 mmol/g Zn(II), 0.26 mmol/g Co(II) and 0.12 mmol/g Cu(II). In a binary system, copper inhibited both Zn(II) and Co(II) sorption but the extent of inhibition of former was greater than the latter; sorption values being 0.14 mmol/g Zn(II) and 0.27 mmol/g Co(II) at initial Zn(II) and Co(II) concentration of 1.5 mM each, pH 4.0 and 1mM Cu(II) as the interfering metal. Zn(II) and Co(II) were equally antagonistic to each others sorption; Zn(II) and Co(II) sorption being 0.23 and 0.24 mmol/g, respectively, at initial metal concentration of 1.5 mM each, pH 4.0 and 1mM interfering metal concentration. In contrast, Cu(II) sorption remained almost unaffected at lower concentrations of the competing metals. Thus, in binary system inhibition dominance observed was Cu(II)>Zn(II), Cu(II)>Co(II) and Zn(II) approximately Co(II), due to this the biosorbent exhibited net preference/affinity for Cu(II) sorption over Zn(II) or Co(II). Hence, the affinity series showed a trend of Cu(II)>Co(II)>Zn(II). In a ternary system, increasing Co(II) concentration exhibited protection against the inhibitory effect of Cu(II) on Zn(II) sorption. On the other hand, the inhibitory effect of Zn(II) and Cu(II) on Co(II) sorption was additive. The model equation for metal interactions was found to be valid within the design space.     

Homoserine kinase of Escherichia coli: kinetic mechanism and inhibition by L-aspartate semialdehyde

The kinetic mechanism of homoserine kinase, purified to homogeneity from Escherichia coli, was examined by initial velocity techniques at pH 7.6. Whereas ATP displayed normal Michaelis-Menten saturation kinetics (Km = 0.2 mM), L-homoserine showed hyperbolic saturation kinetics only up to a concentration of 0.75 mM (Km = 0.15 mM). Above this concentration, L-homoserine caused marked but partial inhibition (Ki approximately 2 mM). The kinetic data indicated that the addition of substrates to homoserine kinase occurs by a preferred order random mechanism, with ATP preferentially binding before L-homoserine. When the ATP concentration was varied at several fixed inhibitory concentrations of L-homoserine, the resulting inhibition pattern indicated hyperbolic mixed inhibition. This suggested a second binding site for L-homoserine. L-Aspartate semialdehyde, an amino acid analog of L-homoserine, proved to be an alternative substrate of homoserine kinase (Km = 0.68 mM), and was subsequently used as a probe of its kinetic mechanism. In aqueous solution, at pH 7.5, this analog was found to exist predominantly (ca 85%) as its hydrated species. When examined as an inhibitor of the physiological reaction, L-aspartate semialdehyde showed mixed inhibition versus both L-homoserine and ATP. Although the pH profiles for the binding of L-homoserine as a substrate (Km) and as an inhibitor (Ki) were identical, the kinetic data were best fit to a two-site model, with separate catalytic and inhibitory sites for L-homoserine.     

Binding of cadmium(II) and zinc(II) to human and dog serum albumins. An equilibrium dialysis and 113Cd-NMR study.

The binding of Cd(II) and Zn(II) to human serum albumin (HSA) and dog serum albumin (DSA) has been studied by equilibrium dialysis and 113Cd(II)-NMR techniques at physiological pH. Scatchard analysis of the equilibrium dialysis data indicate the presence of at least two classes of binding sites for Cd(II) and Zn(II). On analysis of the high-affinity class of sites, HSA is shown to bind 2.08 +/- 0.09 (log K = 5.3 +/- 0.6) and 1.07 +/- 0.12 (log K = 6.4 +/- 0.8) moles of Cd(II) and Zn(II) per mole of protein, respectively. DSA bound 2.02 +/- 0.19 (log K = 5.1 +/- 0.8), and 1.06 +/- 0.15 (log K = 6.0 +/- 0.2) moles of Cd(II) and Zn(II) per mole of protein, respectively. Competition studies indicate the presence of one high-affinity Cd(II) site on both HSA and DSA that is not affected by Zn(II) or Cu(II), and one high-affinity Zn(II) site on both HSA and DSA that is not affected by Cd(II) or Cu(II). 113Cadmium-HSA spectra display three resonances corresponding to three different sites of complexation. In site I, Cd(II) is most probably coordinated to two or three histidyl residues, site II to one histidyl residue and three oxygen ligands (carboxylate), while for the most upfield site III, four oxygens are likely to be involved in the binding of the metal ion. The 113Cd(II)-DSA spectra display only two resonances corresponding to two different sites of complexation. The environment around Cd(II) at sites I and II on DSA is similar to sites I and II, respectively, on HSA. No additional resonances are observed in any of these experiments and in particular in the low field region where sulfur coordination occurs. Overall, our results are consistent with the proposal that the physiologically important high-affinity Zn(II) and Cd(II) binding sites of albumins are located not at the Cu(II)-specific NH2-terminal site, but at internal sites, involving mostly nitrogen and oxygen ligands and no sulphur ligand.     

Sorption of Cu(II) and Cd(II) by extracellular polymeric substances (EPS) from Aspergillus fumigatus

Sorption of Cu(II) and Cd(II) onto the extracellular polymeric substances (EPS) produced by Aspergillus fumigatus was investigated for the initial pH of the solution, EPS concentrations, contact time, NaCl concentration, initial metal ion concentration and the presence of other ions in the solution. The results showed that the adsorption of metal ions was significantly affected by pH, EPS concentrations, initial metal concentration, NaCl concentration and co-ions. The sorption of Cu(II) and Cd(II) increased with increasing pH and initial metal ion concentration but decreased with an increase in the NaCl concentration. The maximum sorption capacities of A. fumigatus EPS calculated from the Langmuir model were 40 mg g⁻¹ EPS and 85.5 mg g⁻¹ EPS for Cu(II) and Cd(II), respectively. The binary metal sorption experiments showed a selective metal binding affinity in the order of Cu(II) > Pb(II) > Cd(II). Both the Freundlich and Langmuir adsorption models described the sorption of Cu(II) and Cd(II) by the EPS of A. fumigatus adequately. Fourier transform infrared spectroscopy (FTIR) analysis revealed that carboxyl, amide and hydroxyl functional groups were mainly correlated with the sorption of Cu(II) and Cd(II). Energy dispersive X-ray (EDX) system analysis revealed that the ion-exchange was an important mechanism involved in the Cu(II) and Cd(II) sorption process taking place on EPS.     

人胎盘谷胱甘肽S-转移酶动力学及抑制剂的研究

研究了人胎盘型谷胱甘肽S-转移酶(GST-π)的动力学。底物GSH和1-氯-2,4-二硝基苯(CDNB)的km分别为0.109和0.870mmol/L。苯唑青霉素和先锋霉素Ⅰ能抑制GST—π,以先锋霉素较明显,属非竞争性抑制。溴磺酜对CDNB也是非竞争作用,但胆红素则对CDNB竞争而对GSH非竞争地抑制酶活力。S-正辛烷和S-正已烷谷胱甘肽与GSH竞争而与CDNB非竞争地抑制GST-π。已充分证明GST-π所催化的双底物反应属随机顺序机制。化学修饰实验发现:巯基、胍基、氨基、羧基和吲哚基可能参与酶活性中心的组成。     

Redox-changes associated with the glutathione-dependent ability of the Cu(II)-GSSG complex to generate superoxide

The intracellularly-occurring Cu(I)-glutathione complex (Cu(I)-[GSH](2)) has the ability to reduce molecular oxygen into superoxide. Removal of such radicals leads to the irreversible conversion of Cu(I)-[GSH](2) into the redox-inactive Cu(II)-GSSG complex. The present study addressed the potential of reduced glutathione, ascorbate and superoxide to reductively regenerate Cu(I)-[GSH](2) from Cu(II)-GSSG, and investigated the redox changes involved in such process. Results show that: (i) among the three tested reductants, only GSH is able to reduce the Cu(II) bound to GSSG; (ii) during the reduction of Cu(II)-GSSG, a Cu(I)-GSSG intermediate would be formed (supported here by Cu(I) and GSSG recovery data and by NMR studies); (iii) when GSH is present in a molar excess equal or greater than 1:3, the reduction of Cu(II)-GSSG into Cu(I)-[GSH](2) is quantitative and complete. Under such conditions, the Cu(II)-GSSG complex acquires a superoxide-generating capacity which is identical to that seen with the Cu(I)-[GSH](2) complex. Within cells, the concentrations of GSH are at least 2- to 3-fold order of magnitude higher than those expected for the Cu(II)-GSSG complex. Thus, we postulate that the interaction between GSH and Cu(II)-GSSG could be seen as a potential mechanism to regenerate continuously the Cu(I)-[GSH](2) complex and thereby affect the ability of the latter to generate superoxide.     

Reactivity of Cd7-metallothionein with Cu(II) ions: evidence for a cooperative formation of Cd3,Cu(I)5-metallothionein

Reaction of Cd7-metallothionein-2 (MT) with Cu(II) ions has been studied by a variety of spectroscopic techniques including UV-absorption, circular dichroism (CD) and luminescence spectroscopy. The addition of up to 5 Cu(II) equivalents to Cd7-MT resulted in a cooperative formation of the monomeric Cd3,Cu5-MT form, as revealed by the analytical data and the presence of isosbestic or isodichroic points in the respective UV and CD spectra. The presence of Cu(I) luminescence and the absence of Cu(II) EPR signal indicated that copper is bound in the Cu(I) oxidation state, i.e., Cd3,Cu(I)5-MT. Consequently, the reduction of Cu(II) ions is accompanied by the oxidation of thiolate ligands of the protein. The absorption features and the luminescence data at 77 K are consistent with the presence of an air-stable Cu(I)-cluster in Cd3,Cu(I)5-MT. The participation of other ligands, besides cysteine thiolates, in metal coordination cannot be ruled out. With more than 5 Cu(II) equivalents added a mixture of unstable MT metalloforms were formed. The concomitant reduction and binding of copper ions by metallated MT represent a new aspect of the MT structure.     

Paradoxical inhibition of rat glutathione transferase 4-4 by indomethacin explained by substrate-inhibitor-enzyme complexes in a random-order sequential mechanism.

Under standard assay conditions, with 1-chloro-2,4-dinitrobenzene (CDNB) as electrophilic substrate, rat glutathione transferase 4-4 is strongly inhibited (I50 = 1 microM) by indomethacin. No other glutathione transferase investigated is significantly inhibited by micromolar concentrations of indomethacin. Paradoxically, the strong inhibition of glutathione transferase 4-4 was dependent on high (millimolar) concentrations of CDNB; at low concentrations of this substrate or with other substrates the effect of indomethacin on the enzyme was similar to the moderate inhibition noted for other glutathione transferases. In general, the inhibition of glutathione transferases can be explained by a random-order sequential mechanism, in which indomethacin acts as a competitive inhibitor with respect to the electrophilic substrate. In the specific case of glutathione transferase 4-4 with CDNB as substrate, indomethacin binds to enzyme-CDNB and enzyme-CDNB-GSH complexes with an even greater affinity than to the corresponding complexes lacking CDNB. Under presumed physiological conditions with low concentrations of electrophilic substrates, indomethacin is not specific for glutathione transferase 4-4 and may inhibit all forms of glutathione transferase.     

Two glutathione S-transferase isoenzymes purified from Bulinus truncatus (Gastropoda: Planorbidae)

We purified and characterized two major glutathione S-transferase isoenzymes (GST2 and GST3) from snail Bulinus truncatus (Mollusca, Gastropoda, Planorbidae) tissue. The Km with respect to 1-chloro-2, 4-dinitrobenzene (CDNB) for both isoenzymes was increased as the pH decreased. Km of both isoenzymes with respect to glutathione (GSH) doubled when the pH was increased from 6.0 to 6.5. Acid inactivated GST2 and GST3 and the two enzymes were almost inactive at pH 3.5. However, they retain the full activity for at least 20 h when incubated at pH between 6.0 and 9.0. The optimum temperature was 45 degrees C for GST2 and 50 degrees C for GST3. The half lifetime at 50 degrees C was 70 min and 45 min for GST2 and GST3 isoenzymes, respectively. Addition of 5 mM GSH to the incubation buffer increased the half life of both isoenzymes more than fourfold. The activation energy for catalyzing the conjugation of CDNB was 1.826 and 3.435 kcal/mol for GST2 and GST3, respectively. I50 values for Cibacron blue, bromosulphophthalein, indocyanine green, hematin and ethacrynic acid were 0.76 microM, 47.9 microM, 7.59 microM, 0.03 microM and 0.79 microM for GST2, and 0.479 microM, 79.4 microM, 89.1 microM, 32.4 microM and 1.15 microM for GST3, respectively. Cibacron blue and indocyanine green were non-competitive inhibitors, while hematin was a mixed inhibitor. Bromosulphophthalein was found to be a competitive inhibitor for GST2 and a mixed inhibitor for GST3.     

Copper (II) induced polymerization of human albumin, and its depolymerization by diglycyl-L-histidine: a pH static and ultracentrifugation study.

Copper (II) ions successively induce dimers and tetramers of human serum albumin (L) when the Cu (II) concentration is extended beyond that of 200 muM. This is shown by emf titrations and by ultracentrifugation experiments. The emf titrations, which involve a new pH static method, were performed at 25 degrees, in a 0.5 M NaCIO4 medium at pH 6.59, using glass and copper amalgam electrodes. The total concentration of Cu(II) varied from 0.14 to 2.2 mM and the albumin concentration from 0.05 to 0.7 mM. In order to evaluate the formula of the main complexes, without using any a priori assumptions regarding their compositions, a detailed graphic procedure was used. The results, in the form of equilibrium constants for the main species, were refined by the use of a general least squares computer program. The experimental data are found to be consistent with the formation of the monomeric CuL, Cu5L, and Cu6L species and the dimeric Cu3L2, Cu4L, Cu6L, and Cu8L2 species. In addition, there is some indication for a minor species, most probably the Cu12L4 tetramer. The pH static results qualitatively agree with the findings obtained by ultracentrifugation. As indicated by distinct bands and their S-values, ultracentrifugation experiments show not only monomeric and dimeric species of albumin, but also tetrameric species. The polymerization of the albumin is reversible, since diglycyl-L-histidine, a peptide designed to mimic the Cu (II) transport site of albumin, depolymerizes the Cu (II)-albumin polymers.     

Biosorption of cadmium(II), lead (II) and copper(II) with the filamentous fungus Phanerochaete chrysosporium

The biosorption from artificial wastewaters of heavy metals (Cd(II), Pb(II) and Cu(II)) onto the dry fungal biomass of Phanerochaete chryosporium was studied in the concentration range of 5-500 mg l(-1). The maximum absorption of different heavy metal ions on the fungal biomass was obtained at pH 6.0 and the biosorption equilibrium was established after about 6 h. The experimental biosorption data for Cd(II), Pb(II) and Cu(II) ions were in good agreement with those calculated by the Langmuir model.     

Plant phenols as in vitro inhibitors of glutathione S-transferase(s)

Ellagic acid, a commonly occurring plant phenol, was shown to be a potent in vitro inhibitor of GSH-transferase(s) activity. Other plant phenols such as ferrulic acid, caffeic acid and chlorogenic acid also showed a concentration dependent inhibition of GSH-transferase(s) activity. The I50 values of ellagic acid, caffeic acid, chlorogenic acid and ferrulic acid were 8.3 X 10(-5)M, 14.0 X 10(-5)M, 20.0 X 10(-5)M and 22.0 X 10(-5)M respectively, suggesting that ellagic acid is the most potent inhibitor of all the four studied plant phenols. At 55 microM concentration of ellagic acid, a significant inhibition (35-47%) was observed on GSH-transferase activity towards CDNB, p-nitrobenzyl chloride and 1,2-epoxy-3-(p-nitrophenoxy)propane as substrates. Ellagic acid inhibited GSH-transferase(s) activity in a non-competitive manner with respect to CDNB while with respect to GSH it inhibited the enzyme activity in a competitive manner. Other phenolic compounds purpurogallin , quercetin, alizarin and monolactone also showed a concentration dependent inhibition of the enzyme activity with a I50 of 0.8 X 10(-5)M, 1.0 X 10(-5)M, 8.0 X 10(-5)M and 16.0 X 10(-5)M respectively. These inhibitors of GSH-transferase(s) activity should be useful in studying the in vitro enzyme mediated reactions of exogenous and endogenous compounds.     

Competitive adsorption of Cu(II) and Cd(II) ions by chitosan crosslinked with epichlorohydrin-triphosphate

In this study, chitosan (CTS) was crosslinked with both epichlorohydrin (ECH) and triphosphate (TPP), by covalent and ionic crosslinking reactions, respectively. The resulting adsorbent (CTS-ECH-TPP) was characterized by SEM, CHN, EDS, FT-IR and TGA analyses, and tested for metal adsorption. The adsorbent was used in batch experiments to evaluate the adsorption of Cu(II) and Cd(II) ions in single and binary metal solutions. In single metal solutions the maximum adsorption capacities for Cu(II) and Cd(II) ions, obtained by Langmuir model, were 130.72 and 83.75 mg g?1, respectively. Adsorption isotherms for binary solutions showed that the presence of Cu(II) decreased Cd(II) adsorption due to a significant competition effect, that is, the adsorbent was selective towards Cu(II) rather than Cd(II).