Permeant Anions Control Gating of Calcium-dependent Chloride Channels

The effects of external anions (SCN⁻, NO₃⁻, I⁻, Br⁻, F⁻, glutamate, and aspartate) on gating of Ca²⁺-dependent Cl⁻ channels from rat parotid acinar cells were studied using the whole-cell configuration of the patch-clamp technique. Shifts in the reversal potential of the current induced by replacement of external Cl⁻ with foreign anions, gave the following selectivity sequence based on permeability ratios (P_x/P_Cl): SCN⁻>I⁻>NO₃⁻>Br⁻>Cl⁻>F⁻>aspartate>glutamate. Using a continuum electrostatic model we calculated that this lyotropic sequence resulted from the interaction between anions and a polarizable tunnel with an effective dielectric constant of ∼23. Our data revealed that anions with P_x/P_Cl > 1 accelerated activation kinetics in a voltage-independent manner and slowed deactivation kinetics. Moreover, permeant anions enhanced whole-cell conductance (g, an index of the apparent open probability) in a voltage-dependent manner, and shifted leftward the membrane potential-g curves. All of these effects were produced by the anions with an effectiveness that followed the selectivity sequence. To explain the effects of permeant anions on activation kinetics and g_Cl we propose that there are 2 different anion-binding sites in the channel. One site is located outside the electrical field and controls channel activation kinetics, while a second site is located within the pore and controls whole-cell conductance. Thus, interactions of permeant anions with these two sites hinder the closing mechanism and stabilize the channel in the open state.This revised version was published online in August 2005 with a corrected cover date.     

Characterization of inhibitory effects of NH2OH and its N-methyl derivatives on the O2-evolving complex of Photosystem II

Inorganic cofactors (Mn, Ca²⁺ and Cl^-) are essential for oxidation of H₂O to O₂ by Photosystem II. The Mn reductants NH₂OH and its N-methyl derivatives have been employed as probes to further examine the interactions between these species and Mn at the active site of H₂O oxidation. Results of these studies show that the size of a hydroxylamine derivative regulates its ability to inactivate O₂ evolution activity, and that this size-dependent inhibition behavior arises from the protein structure of Photosystem II. A set of anions (Cl^-, F^- and SO₄ ^2-) is able to slow NH₂OH and CH₃NHOH inactivation of intact Photosystem II membranes by exerting a stabilizing influence on the extrinsic 23 and 17 kDa polypeptides. In contrast to this non-specific anion effect, only Cl^- is capable of attenuating CH₃NHOH and (CH₃)₂NOH inhibition in salt-washed preparations lacking the 23 and 17 kDa polypeptides. However, Cl^- fails to protect against NH₂OH inhibition in salt-washed membranes. These results indicate that the attack by NH₂OH and its N-methyl derivatives on Mn occurs at different sites in the O₂-evolving complex. The small reductant NH₂OH acts at a Cl^--insensitive site whereas the inhibitions by CH₃NHOH and (CH₃)₂NOH involve a site that is Cl^- sensitive. These findings are consistent with earlier studies showing that the size of primary amines controls the Cl^- sensitivity of their binding to Mn in the O₂-evolving complex.Abbreviation MES 4-morpholinoethanesulfonic acid - PS II Photosystem II     

Microfluorometric analysis of Cl permeability and its relation to oscillatory Ca signalling in glucose-stimulated pancreatic β-cells

The cytoplasmic concentrations of Cl⁻([Cl⁻]_i) and Ca²⁺ ([Ca²⁺]_i) were measured with the fluorescent indicators N-(ethoxycarbonylmethyl)-6-methoxyquinilinum bromide (MQAE) and fura-2 in pancreatic β-cells isolated from ob/ob mice. Steady-state [Cl⁻]_i in unstimulated β-cells was 34 mM, which is higher than expected from a passive distribution. Increase of the glucose concentration from 3 to 20 mM resulted in an accelerated entry of Cl⁻ into β-cells depleted of this ion. The exposure to 20 mM glucose did not affect steady-state [Cl⁻]_i either in the absence or presence of furosemide inhibition of Na⁺, K⁺, 2 Cl⁻ co-transport. Glucose-induced oscillations of [Ca²⁺]_i were transformed into sustained elevation in the presence of 4,4′ diisothiocyanato-dihydrostilbene-2,2′-disulfonic acid (H₂DIDS). A similar effect was noted when replacing 25% of extracellular Cl⁻ with the more easily permeating anions SCN⁻, I⁻, NO₃⁻ or Br⁻. It is concluded that glucose stimulation of the β-cells is coupled to an increase in their Cl⁻ permeability and that the oscillatory Ca²⁺ signalling is critically dependent on transmembrane Cl⁻ fluxes.     

Thermoluminescence properties of the S2-state in chloride-depleted water oxidizing complexes after reconstituting treatments with various monovalent anions

Under conditions that assured rebinding of the extrinsic 17 and 23 kDa polypeptides, Cl^--depleted Photosystem II membranes isolated from spinach chloroplasts were subjected to reconstituting treatments in media containing NaF, NaCl, NaBr, NaI or NaNO₃, or they were kept in a medium without any added salt other than the buffer. After removing most of the unbound reconstituting anions by washing, the O₂-evolution activities and thermoluminescence properties of the membranes were compared. While the temperature of maximal thermoluminescence emission was lowest for membranes treated with Cl^-, no uniform correlation was evident between the temperature profile of the thermoluminescence emission and the apparent activating effectiveness of the anions in the membranes' water oxidizing machinery. However, the differences between the thermoluminescence features did conform to a trend according to which the emission temperatures were upshifted as the size of the activating anion increased, and its hydration energy decreased, i.e. Cl^-<Br^-<NO₃ ^-<I^-. The inactive F^- anions were not well retained by the membranes. To explain the experimental data it is suggested that the structural environment of the charge accumulating Mn-center is influenced by the ionic conditions encountered by the Photosystem II membranes after Cl^- removal, further enforced by the binding of compatible anions, and then stabilized by the 17 and 23 kDa extrinsic polypeptides. If, as some concepts imply, the anion binding sites are located at or near the functional Mn, only very exceptional characteristics of the water-oxidizing mechanism may account for the observation that the potentially electron-donating I^- anion can serve as activator and that it stabilizes rather than destabilizes the S₂-state.Abbreviations Chl chlorophyll - Hepes 4-(2-hydroxyethyl)-1-piperazine-ethane sulfonic acid - Mes 2-(N-morpholino)ethane sulfonic acid - Pheo the pheophytin a of the Photosystem II reaction center - PS photosystem     

The effect of chloride on the thermal inactivation of oxygen evolution

The effect of Cl depletion on the sensitivity of the oxygen-evolving complex of Photosystem II (PS II) to heat treatment was examined by a parallel study of the Hill activity (H₂O2,6-dichlorophenolindophenol), Cl^- binding (by ³⁵Cl-NMR) and Mn release (by EPR). The extent of thermal inactivation in spinach thylakoids was found to depend on the degree of Cl^- depletion in the sample. In partially Cl^--depleted thylakoids, mild heating (38°C, 3 min) was found to eliminate inflections in plots of both Hill activity versus [Cl^-] (at low light intensity) and excess ³⁵Cl-NMR linewidth versus [Cl^-] (in the dark). In PS II membranes, the same treatment reduced the differences between the linewidth maxima and minima, particularly in the region of 0.3 mM and 7.0 mM Cl^-, as compared to unheated membranes. These results indicate that mild heating affects the Cl^--binding domains within the oxygen-evolving complex, OEC, EPR measurements of the temperature dependence of Mn release from heated thylakoids show that Mn release begins to correlate with the loss of Hill activity only at higher temperatures, where the OEC is already substantially inactivated. We conclude from these studies that the Cl^--binding domains of the OEC constitute a principal site of damage by heat treatment.     

The high-affinity binding site for manganese on the oxidizing side of Photosystem II

Electron donation to Photosystem II (PS II) by diphenylcarbazide (DPC) is interrupted by the presence of endogenous Mn in PS II particles. Removal of this Mn by Tris treatment greatly stimulates the electron transport with DPC as donor. Binding of low concentration of exogenous Mn(II) to Tris-treated PS II particles inhibits DPC photooxidation competitively with DPC. This phenomenon was used to locate a highly specific Mn(II) binding site on the oxidizing side of Photosystem II with dissociation constant about 0.15 μM. The binding of Mn(II) is electrostatic in nature. Its affinity depends not only on the ionic strength, but also on the anion species of the salt in the medium. The effectiveness in decreasing the affinity follows the order F⁻ > SO²⁻₄ > CH₃COO⁻ > CI⁻ > Br⁻ > NO₃⁻. This observation is interpreted as follows: smaller ions, like F⁻, CH₃COO⁻, and larger ions, like SO²⁻₄, have inhibitory effects on Mn(II) binding, whereas ions with optimal size, like Cl⁻, Br⁻ and NO₃⁻, can stabilize the binding, resembling the anion requirement for reactivation of Cl⁻-depleted chloroplasts. We suggest that the binding site for Mn(II) we observed is the site for the endogenous Mn in the O₂-evolving complex of PS II. This site remains after Tris treatment, which removes all the endogenous Mn as well as the three extrinsic proteins, indicating that it is on the intrinsic component(s) of PS II reaction centers. Furthermore, the Cl⁻ requirement for O₂ evolution may be attributed, at least partly to its stabilizing effect on Mn binding.     

Effect of inorganic salts on hemochromogen aggregation

The absorbance and difference absorbance spectra of pyridine-hemochromogen at different concentrations showed the presence of two different types of pyridine-hemochromogens, one unstable form (compound III) at low concentration and a more stable form (compound II) at higher concentrations, suggesting that there is an interaction between hemochromogen molecules at high concentration. In the presence of inorganic salts, the unstable compound III is converted to stable compound II. The effect of various inorganic ions on the formation of compound II has been studied. The order of increasing effectiveness of the anions on compound II formation was HPO₄²⁻SO₄²⁻F⁻Cl⁻Br⁻NO₃⁻SCN⁻ and that of cation was Li⁺Na⁺K⁺. The results are discussed on the basis of the effect of these ions on the structure of water. It appears compound II is an aggregate of compound III and the aggregation is due to hydrophobic interaction.     

Laser raman studies of molecular interactions with phosphatidylcholine multilayers. II. Effects of mono- and divalent ions on bilayer structure

Laser Raman spectroscopy was applied to characterize structural behavior of dipalmitoyl phosphatidylcholine multibilayer systems in the presence of several cations (K⁺, Na⁺, Cs⁺, Rb⁺, Ca²⁺, Mg²⁺, Cd²⁺, Ba²⁺) and anions (Cl⁻, Br⁻, I⁻, NO₃⁻, SO₃²⁻, SO₄²⁻, S₂O₃²⁻, S₂O₈²⁻). To evaluate the Raman-spectroscopical data quantitatively, characteristic intensity ratios, lateral and trans order parameters were used and compared. It was shown that the different trans order parameters are rather sensitive to ion-polar head group interactions and thus, they cannot give unequivocal information on the trans-gauche isomerization of hydrocarbon chains of phospholipids. The observed effects of ions on Raman spectra of phospholipid multilayers could not be explained simply on the basis of electrostatic interactions. The possible involvement of other factors (changes in polarizability, hydrogen bonds, etc.) is also discussed. It was demonstrated that the order parameters defined in different ways may result in different effectiveness sequences of ions. Of monopositive ions Na⁺ was found to be the most effective to influence the bilayer structure. For dipositive ions, of which Ca²⁺ proved to be the most effective, concentration-dependent effectiveness sequences were obtained. A plausible interpretation and some consequences of the concentration-dependent two-step binding of divalent cations were also outlined. Bilayered phospholipid structures turned out to be more responsive to anions than to most cations investigated. Interdependent actions of cations and anions, as well as the possible relevance of the charge distribution on anions are postulated.     

Studies on photosynthetic oxygen-evolving complex by means of Fourier transform infrared spectroscopy: calcium and chloride cofactors

Structural roles of functional Ca²⁺ and Cl⁻ ions in photosynthetic oxygen-evolving complexes (OEC) were studied using low- (640–350 cm⁻¹) and mid- (1800–1200 cm⁻¹) frequency S₂/S₁ Fourier transform infrared (FTIR) difference spectroscopy. Studies using highly active Photosystem (PS) II core particles from spinach enabled the detection of subtle spectral changes. Ca²⁺-depleted and Ca²⁺-reconstituted particles produced very similar mid- and low-frequency spectra. The mid-frequency spectrum was not affected by reconstitution with ⁴⁴Ca isotope. In contrast, Sr²⁺-substituted particles showed unique spectral changes in the low-frequency Mn–O–Mn mode at 606 cm⁻¹ as well as in the mid-frequency carboxylate stretching modes. The mid-frequency spectrum of Cl⁻-depleted OEC exhibited marked changes in the carboxylate stretching modes and the suppression of protein modes compared with that of Cl⁻-reconstituted OEC. However, Cl⁻-depletion did not exert significant effects on the low-frequency spectrum.     

Cl−, Na+, and H+ fluxes during the acidification of rabbit reticulocyte endocytic vesicles

The ionic fluxes associated with the ATP-dependent acidification of endocytic vesicles were studied in a preparation isolated from rabbit reticulocytes enriched for transferrin-transferrin receptor complexes. No vesicle acidification was observed in the absence of intra- and extravesicular ions (sucrose_in/sucrose_out), while maximal acidification was observed with NaCl_in/KCl_out·K _in ⁺ was a poor substitute for Na _in ⁺ , and Cl _out ^– could be replaced by other anions with the following efficacy of acidification: Cl^–>Br^–>I^–>PO ₄ ^3– >gluconate>SO ₄ ^2– . Flux studies using³⁶Cl^– and²²Na⁺ showed that the vesicles had a permeability for Cl^– and Na⁺, and that ATP-dependent H⁺ pumping was accompanied by a net influx of Cl^– and a net efflux of Na⁺ provided that there was a Na⁺ concentration gradient. After 3 mins, the time necessary to maximal acidification, the electrical charge generated by the entrance of H⁺ was countered to about 45% by the Cl^– influx and to about 42% by the Na⁺ efflux. These studies demonstrated that both Cl^– and Na⁺ fluxes are necessary for optimal endocytic vesicle acidification.     

Structure and function of the PsbP protein of Photosystem II from higher plants

PsbP is a membrane extrinsic subunit of Photosystem II (PS II), which is involved in retaining Ca²⁺ and Cl⁻, two inorganic cofactors for the water-splitting reaction. In this study, we re-investigated the role of N-terminal region of PsbP on the basis of its three-dimensional structure. In previous paper [Ifuku and Sato (2002) Plant Cell Physiol 43: 1244–1249], a truncated PsbP lacking 19 N-terminal residues (Δ19) was found to bind to NaCl-washed PS II lacking PsbP and PsbQ without activation of oxygen evolution at all. Three-dimensional (3D) structure of PsbP suggests that deletion of 19 N-terminal residues would destabilize its protein structure, as indicated by the high sensitivity of Δ19 to trypsin digestion. Thus, a truncated PsbP lacking 15 N-terminal residues (Δ15), which retained core PsbP structure, was produced. Whereas Δ15 was resistant to trypsin digestion and bound to NaCl-washed PS II membranes, it did not show the activation of oxygen evolution. This result indicated that the interaction of 15-residue N-terminal flexible region of PsbP with PS II was important for Ca²⁺ and Cl⁻ retention in PS II, although the 15 N-terminal residues were not essential for the binding of PsbP to PS II. The possible N-terminal residues of PsbP that would be involved in this interaction are discussed.     

Ecophysiological responses of the euhalophyte Suaeda salsa to the interactive effects of salinity and nitrate availability

Effects of salinity and nitrate nitrogen (NO₃⁻-N) on ion accumulation and chlorophyll fluorescence were monitored for two populations of Suaeda salsa grown from seeds in a greenhouse experiment. One population inhabits the intertidal zone and the other occurs on inland saline soils. Ion contents in soils and in leaves of the two populations were also investigated in field. In the greenhouse, seedlings were exposed to a NaCl concentration of 0.6 and 35.1 ppt, with 0.1 or 5 mM NO₃⁻-N treatments for 20 days. The contents of Na⁺ and Cl⁻ were higher, but NO₃⁻ was lower in soils of the intertidal zone than at the inland site. In the field, ion concentrations and the estimated contribution of these ions to osmotic potential in leaves showed no difference between the two populations, except that the estimated contribution of Na⁺ to osmotic potential in leaves of the intertidal population was lower than that in the inland population. In the greenhouse, in contrast, the concentration of Cl⁻ was lower, but NO₃⁻ concentration and the estimated contribution of NO₃⁻ to osmotic potential were higher, in the leaves of plants from the intertidal zone. Salinity had no effect on the maximal efficiency of PSII photochemistry (Fv/Fm) and the actual PSII efficiency (ΦPSII). The results indicated that S. salsa from the intertidal zone was better able to regulate Cl⁻ to a lower level, and accumulate NO₃⁻ even with low soil NO₃⁻ concentrations. Tolerance of the PSII machinery to high salinity stress may be an important characteristic for the studied species supporting growth in highly saline environments.     

Simulation analysis of intracellular Na and Cl homeostasis during β1-adrenergic stimulation of cardiac myocyte

To quantitatively understand intracellular Na⁺ and Cl⁻ homeostasis as well as roles of Na⁺/K⁺ pump and cystic fibrosis transmembrane conductance regulator Cl⁻ channel (I_CFTR) during the β1-adrenergic stimulation in cardiac myocyte, we constructed a computer model of β1-adrenergic signaling and implemented it into an excitation-contraction coupling model of the guinea-pig ventricular cell, which can reproduce membrane excitation, intracellular ion changes (Na⁺, K⁺, Ca²⁺ and Cl⁻), contraction, cell volume, and oxidative phosphorylation. An application of isoproterenol to the model cell resulted in the shortening of action potential duration (APD) after a transient prolongation, the increases in both Ca²⁺ transient and cell shortening, and the decreases in both Cl⁻ concentration and cell volume. These results are consistent with experimental data. Increasing the density of I_CFTR shortened APD and augmented the peak amplitudes of the L-type Ca²⁺ current (I_CaL) and the Ca²⁺ transient during the β1-adrenergic stimulation. This indirect inotropic effect was elucidated by the increase in the driving force of I_CaL via a decrease in plateau potential. Our model reproduced the experimental data demonstrating the decrease in intracellular Na⁺ during the β-adrenergic stimulation at 0 or 0.5 Hz electrical stimulation. The decrease is attributable to the increase in Na⁺ affinity of Na⁺/K⁺ pump by protein kinase A. However it was predicted that Na⁺ increases at higher beating rate because of larger Na⁺ influx through forward Na⁺/Ca²⁺ exchange. It was demonstrated that dynamic changes in Na⁺ and Cl⁻ fluxes remarkably affect the inotropic action of isoproterenol in the ventricular myocytes.     

Studies of the slowly exchanging chloride in Photosystem II of higher plants

³⁶Cl^- was used to study the slow exchange of chloride at a binding site associated with Photosystem II (PS II). When PS II membranes were labeled with different concentrations of ³⁶Cl^-, saturation of binding at about I chloride/PS II was observed. The rate of binding showed a clear dependence on the concentration of chloride approaching a limiting value of about 3·10^-4 s^-1 at high concentrations, similar to the rate of release of chloride from labeled membranes. These rates were close to that found earlier for the release of chloride from PS II membranes isolated from spinach grown on ³⁶Cl^-, which suggests that we are observing the same site for chloride binding. The similarity between the limiting rate of binding and the rate of release of chloride suggests that the exchange of chloride with the surrounding medium is controlled by an intramolecular process. The binding of chloride showed a pH-dependence with an apparent pK_a of 7.5 and was very sensitive to the presence of the extrinsic polypeptides at the PS II donor side. The binding of chloride was competitively inhibited by a few other anions, notably Br^- and NO₃ ^-. The slowly exchanging Cl^- did not show any significant correlation with oxygen evolution rate or yield of EPR signals from the S₂ state. Our studies indicate that removal of the slowly exchanging chloride lowers the stability of PS II as indicated by the loss of oxygen evolution activity and S₂ state EPR signals.Abbreviations Chl chlorophyll - EPR electron paramagnetic resonance - Hepes 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid - Mes 4-morpholineethanesulfonic acid - MWCO molecular weight cut off - PPBQ phenyl-p-benzoquinone - PS II Photosystem II     

Can elevated CO(2) improve salt tolerance in olive trees?

We compared growth, leaf gas exchange characteristics, water relations, chlorophyll fluorescence, and Na⁺ and Cl⁻ concentration of two cultivars (‘Koroneiki’ and ‘Picual’) of olive (Olea europaea L.) trees in response to high salinity (NaCl 100 mM) and elevated CO₂ (eCO₂) concentration (700 μL L⁻¹). The cultivar ‘Koroneiki’ is considered to be more salt sensitive than the relatively salt-tolerant ‘Picual’. After 3 months of treatment, the 9-month-old cuttings of ‘Koroneiki’ had significantly greater shoot growth, and net CO₂ assimilation (A_CO2) at eCO₂ than at ambient CO₂, but this difference disappeared under salt stress. Growth and A_CO2 of ‘Picual’ did not respond to eCO₂ regardless of salinity treatment. Stomatal conductance (g_s) and leaf transpiration were decreased at eCO₂ such that leaf water use efficiency (WUE) increased in both cultivars regardless of saline treatment. Salt stress increased leaf Na⁺ and Cl⁻ concentration, reduced growth and leaf osmotic potential, but increased leaf turgor compared with non-salinized control plants of both cultivars. Salinity decreased A_CO2, g_s, and WUE, but internal CO₂ concentrations in the mesophyll were not affected. eCO₂ increased the sensitivity of PSII and chlorophyll concentration to salinity. eCO₂ did not affect leaf or root Na⁺ or Cl⁻ concentrations in salt-tolerant ‘Picual’, but eCO₂ decreased leaf and root Na⁺ concentration and root Cl⁻ concentration in the more salt-sensitive ‘Koroneiki’. Na⁺ and Cl⁻ accumulation was associated with the lower water use in ‘Koroneiki’ but not in ‘Picual’. Although eCO₂ increased WUE in salinized leaves and decreased salt ion uptake in the relatively salt-tolerant ‘Koroneiki’, growth of these young olive trees was not affected by eCO₂.     

Basis of Chloride Transport in Ciliary Epithelium

The aqueous humor is formed by the bilayered ciliary epithelium. The pigmented ciliary epithelium (PE) faces the stroma and the nonpigmented ciliary epithelium (NPE) contacts the aqueous humor. Cl⁻ secretion likely limits the rate of aqueous humor formation. Many transport components underlying Cl⁻ secretion are known. Cl⁻ is taken up from the stroma into PE cells by electroneutral transporters, diffuses to the NPE cells through gap junctions and is released largely through Cl⁻ channels. Recent work suggests that significant Cl⁻ recycling occurs at both surfaces of the ciliary epithelium, providing the basis for modulation of net secretion. The PE-NPE cell couplet likely forms the fundamental unit of secretion; gap junctions within the PE and NPE cell layers are inadequate to maintain constancy of ionic composition throughout the epithelium under certain conditions. Although many hormones, drugs and signaling cascades are known to have effects, a persuasive model of the regulation of aqueous humor formation has not yet been developed. cAMP likely plays a central role, potentially both enhancing and reducing secretion by actions at both surfaces of the ciliary epithelium. Among other hormone receptors, A₃ adenosine receptors likely alter intraocular pressure by regulating NPE-cell Cl⁻ channel activity. Recently, functional evidence for the regional variation in ciliary epithelial secretion has been demonstrated; the physiologic and pathophysiologic implications of this regional variation remain to be addressed.This revised version was published online in June 2005 with a corrected cover date.     

Further evidence for local osmotic coupling in the rabbit cornea

1. 1. The present experiments measure net fluxes of fluid, Cl⁻ and HCO₃⁻ across de-epithelialised rabbit corneas clamped between half chambers and bathed in Ringer solutions.

2. 2. Net fluxes of HCO₃⁻ and fluid occurred together across the cornea from stroma to aqueous when HCO₃⁻ and CO₂ were present in the bathing solution.

3. 3. No net trans-corneal Cl⁻ flux was found

4. 4. The initiation of fluid flow in the presence of HCO₃⁻ and CO₂ cannot be accounted for by bulk-phase osmotic flow across the cornea.

Magnetic nanoparticles supported ionic liquids for lipase immobilization: Enzyme activity in catalyzing esterification

Candida rugosa lipase was immobilized on magnetic nanoparticles supported ionic liquids having different cation chain length (C₁, C₄ and C₈) and anions (Cl⁻, BF₄⁻ and PF₆⁻). Magnetic nanoparticles supported ionic liquids were obtained by covalent bonding of ionic liquids–silane on magnetic silica nanoparticles. The particles are superparamagnetic with diameter of about 55 nm. Large amount of lipase (63.89 mg/(100 mg carrier)) was loaded on the support through ionic adsorption. Activity of the immobilized lipase was examined by the catalysis of esterification between oleic acid and butanol. The activity of bound lipase was 118.3% compared to that of the native lipase. Immobilized lipase maintained 60% of its initial activity even when the temperature was up to 80 °C. In addition, immobilized lipase retained 60% of its initial activity after 8 repeated batches reaction, while no activity was detected after 6 cycles for the free enzyme.     

Synthesis, characterisation of a complex of Cu(II) with a multidentate macrocyclic ligand and its interactions with anions

A macrocyclic ligand possessing a donor set of {N₃S₂} synthesised via Cs⁺-templation, 4-(pyridin-2-ylmethyl)-1,7-dithia-4,10-diazacyclododecane (L) and its Cu(II) complex, [CuL(NCMe)]²⁺ (6), are described. This Cu(II) complex interacts with a range of anions, F⁻, Cl⁻, Br⁻, I⁻, HCOO⁻, AcO⁻, CO₃²⁻, NO₃⁻, C₂O₄²⁻, H₂PO₄⁻, SCN⁻, CN⁻, BF₄⁻. Of the investigated anions, I⁻, SCN⁻, and CN⁻, show strong interaction with the Cu(II) centre as indicated by their spectral variations. The iodide particularly demonstrates distinct change in colour. This change originates from a newly appeared band at 471 nm upon iodide binding, which arises from the ligand (I⁻) to Cu(II) charge transfer (LMCT) in the iodide-substituted Cu(II) complex, [CuLI]⁺ (7). All organic compounds are characterised by NMR spectroscopy and/or microanalysis. The identities of the two Cu(II) complexes are confirmed by using microanalysis and the complex 6 is crystallographically analysed.     

The structures of bis(1H,5H-S-methylisothiocarbonohydrazidium) di-μ-chloro-octachlorodibismuthate(III) tetrahydrate and tris(1H-S-methylisothiocarbonohydrazidium) esachlorobismuthate(III)

The structures of bis(1H⁺,5H⁺-S-methylisothiocarbonohydrazidium) di-μ-chlorooctachlorodibismuthate(III) tetrahydrate: (C₂H₁₀N₄S)₂(Bi₂Cl₁₀)· 4H₂O (compound [I]) and of tris(1H⁺-S-methylisothiocarbonohydrazidium) esachlorobismuthate(III): (C₂H₉N₄S)₃(BiCl_5.67I_0.33) (compound [II]) were determined from single crystal X-ray diffractometer data. Both compounds crystallize as triclinic (P ); crystals [I] with Z = 1 formula unit in a cell of constants: A = 10.621(3), B = 9.989(5), C = 7.439(3) Å, α = 88.31(2), β = 84.51(2), γ = 68.88(2)°, final R = 0.0427 for 2229 unique reflections with I 2σ(I); crystals [II] with Z = 2 and cell dimensions: A = 14.109(4), B = 12.209(9), C = 8.206(7) Å, α = 103.54(3), β = 104.95(2), γ = 81.96(2)°, final R = 0.0411 for 3637 unique reflections (1 2σ(I)). The structure of [I] is built up of diprotonated organic cations, water molecules and dinuclear centrosymmetric [Bi₂Cl₁₀]⁴⁻ anions held together by N-HCl, N-HO, O-HCl hydrogen bonds and Van der Waals interactions. The [Bi₂Cl₁₀]⁴⁻ complex consists of two edge-sharing octahedra in which three pairs of bonds of similar length are observed (Bi-Cl_av = 2.602(5), 2.712(4), 2.855(5) Å). The structure of [II] consists of monoprotonated cations and [BiCl_5.67I_0.33]³⁻ anions held together by a tridimensional network of hydrogen bonds. Each bismuth atom is octahedrally surrounded by six chlorine atoms, one of which is statistically substituted by a iodine atom.