Copper(II) complexes as superoxide dismutase mimics: Synthesis, characterization, crystal structure and bioactivity of copper(II) complexes

Two new copper(II) complexes of the type [Cu(L)X₂), where L = (E)-N-phenyl-2-[phenyl (pyridine-2-yl)methylene]hydrazinecarboxamide X = Cl/Br have been synthesized and characterized by elemental analyses, FAB (fast atomic bombardment) magnetic measurements, electronic absorption, conductivity measurements cyclic voltammetry (CV) and Electron paramagnetic resonance (epr) spectroscopy. The structures of these complexes determined by single crystal X-ray crystallography show a distorted square based pyramidal (DSBP) geometry around copper(II) metal center. The distorted CuN₂OX (X = Cl/Br) basal plane in them is comprised of two nitrogen and one oxygen atoms of the meridionally coordinated ligand and a chloride or bromide ion and axial position is occupied by other halide ion. The epr spectra of these complexes in frozen solutions of DMSO showed a signal at g ca. 2. The trend in g-value (g_|| > g_⊥ > 2.00) suggest that the unpaired electron on copper(II) has d_x²-y² character. Biological activities in terms of superoxide dismutase (SOD) and antimicrobial properties of copper(II) complexes have also been measured. The superoxide dismutase activity reveals that these two complexes catalyze the fast disproportionation of superoxide in DMSO solution.     

Platinum(II) triammine antitumour complexes: structure-activity relationship with guanosine 5′-monophosphate (5′-GMP)

The multinuclear (¹H, ¹⁵N, ³¹P and ¹⁹⁵Pt) NMR spectroscopies, ES-MS and HPLC have been employed to investigate the structure-activity relationship for the reactions between guanosine 5′-monophosphate (5′-GMP) and the platinum(II)-triamine complexes of the general formulation cis-[Pt(NH₃)₂(Am)Cl]NO₃ (where Am represents a substituted pyridine). The order of reaction rate of the reactions was found to be: 3-phpy > 4-phpy > py > 4-mepy > 3-mepy > 2-mepy. The two basic factors, steric and electronic, were attributed to the order of the binding rate constants. A possible mechanism of the reaction of cis-[Pt(NH₃)₂(Am)Cl]⁺ with 5′-GMP suggested that the reactions proceed via direct nucleophilic attack and no loss of ammonia. cis-[Pt(NH₃)₂(Am)Cl]⁺ binds to the N7 nitrogen of the guanine residue of 5′-GMP to form a coordinate bond with the Pt metal centre. This mechanism is apparently different from that of cisplatin. The pK_a value of cis-[Pt(NH₃)₂(4-mepy)(H₂O)](NO₃)₂ (5.63) has been determined at 298 K by the use of distortionless enhancement by polarization transfer (DEPT) ¹⁵N NMR spectroscopy and compared to the pK_a value of cis-[PtCl(H₂O)(NH₃)₂]⁺.     

Affinities of methylphenidate derivatives for dopamine,norepinephrine and serotonin transporters

We have synthesized several derivatives of dl-threo-methylphenidate (Ritalin) bearing substituents on the phenyl ring. IC₅₀ values for binding of these compounds to rat brain monoamine transporters were assessed using [³H]WIN 35,428 (striatal membranes, dopamine transporters, DAT), [³H]nisoxetine (frontal cortex membranes, norepinephrine transporters, NET) and [³H]paroxetine (brain stem membranes, 5HT transporters, 5HTT). Affinities (1/Ki) decreased in the order: DAT > NET ⪢ 5HTT. Substitution at the para position of dl-threo-methylphenidate generally led to retained or increased affinity for the dopamine transporter (bromo > iodo > methoxy > hydroxy). Substitution at the meta position also increased affinity for the DAT (m-bromo > methylphenidate; m-iodo-p-hydroxy > p-hydroxy). Substitution at the ortho position with bromine considerably decreased affinity. Similar IC₅₀ values for binding of o-bromomethylphenidate to the dopamine transporter were measured at 0, 22 and 37 degrees. N-Methylation of the piperidine ring of methylphenidate also considerably reduced affinity. The dl-erythro isomer of obromomethylphenidate did not bind to the DAT (IC₅₀ > 50,000 nM). Affinities at the dopamine and norepinephrine transporters for substituted methylphenidate derivatives were well correlated (r² = 0.90). Abilities of several methylphenidate derivatives to inhibit [³H]dopamine uptake in striatal synaptosomes corresponded well with inhibition of [³H]WIN 35, 428 binding. None of the compounds examined exhibited significant affinity to dopamine D1 or D2 receptors (IC₅₀ > 500 or 5,000 nM, respectively), as assessed by inhibition of binding of [³H]SCH 23390 or [¹²³I]epidepride, respectively, to striatal membranes.     

Transport of ions across peritoneal membrane

The electrical conductance of ions across the peritoneal membrane of young buffalo (approximately 18-24 months old) has been recorded. Aqueous solutions of NaF, NaNO₃, NaCl, Na₂SO₄, KF, KNO₃, KCl, K₂SO₄, MgCl₂, CaCl₂, CrCl₃, MnCl₂, FeCl₃, CoCl₂, and CuCl₂ were used. The conductance values have been found to increase with increase in concentration as well as with temperature (15 to 35 °C) in these cases. The slope of plots of specific conductance, κ, versus concentration exhibits a decrease in its values at relatively higher concentrations compared to those in extremely dilute solutions. Also, such slopes keep on increasing with increase in temperature. In addition, the conductance also attains a maximum limiting value at higher concentrations in the said cases. This may be attributed to a progressive accumulation of ionic species within the membrane. The κ values of electrolytes follow the sequence for the anions: SO₄²⁻>Cl⁻>NO₃⁻>F⁻ while that for the cations: K⁺>Na⁺>Ca²⁺>Mn²⁺>Co²⁺>Cu²⁺>Mg²⁺>Cr³⁺>Fe³⁺. In addition, the diffusion of ions depends upon the charge on the membrane and its porosity. The membrane porosity in relation to the size of the hydrated species diffusing through the membrane appears to determine the above sequence. As the diffusional paths in the membrane become more difficult in aqueous solutions, the mobility of large hydrated ions gets impeded by the membrane framework and the interaction with the fixed charge groups on the membrane matrix. Consequently, the membrane pores reduce the conductance of small ions, which are much hydrated. An increase in conductance with increase in temperature may be due to the state of hydration, which implies that the energy of activation for the ionic transport across the membrane follows the sequence of crystallographic radii of ions accordingly. The Eyring's equation, κ=(RT/Nh)exp[−ΔH*/RT]exp[ΔS*/R], has been found suitable for explaining the temperature dependence of conductance in the said cases. This is apparent from the linear plots of log[κNh/RT] versus 1/T. The results indicate that the permeation of ions through the membrane giving negative values of ΔS* suggest that there may be formation of either covalent linkage between the penetrating ions and the membrane material or else the permeation may not be the rate-determining step. On the one hand, a high ΔS* value associated with the high value of energy of activation, E_a, for diffusion may suggest the existence of either a large zone of activation or loosening of more chain segments of the membrane. On the other hand, low value of ΔS* implies that converse is true in such cases, i.e., either a small zone of activation or no loosening of the membrane structure upon permeation.     

Capturing the spin state diversity of iron(III)-aryl porphyrins: OLYP is better than TPSSh

DFT calculations with a variety of exchange-correlation functionals, including PW91, OLYP, TPSSh, B3LYP and B3LYP*, have been carried out on the low-energy spin states of chloroiron(III) porphyrin and four aryliron(III) porphyrins, viz. Fe^III(P)Ph (S = 1/2), Fe^III(P)C₆F₅ (S = 5/2), Fe^III(P)(3,4,5-C₆F₃H₂) (S = 1/2), Fe^III(P)(2,4,6-C₆F₃H₂) (S = 5/2), where the expected spin states have been indicated within parentheses. Qualitatively, OLYP reproduces all the expected ground spin states. B3LYP appears to have some difficulty yielding the observed sextet ground states. B3LYP*, TPSSh and PW91 all fail to reproduce the sextet ground states, the latter two by rather large margins of energy. As far as this study is concerned, the overall performance of the functionals appears to be OLYP/OPBE > B3LYP > B3LYP* >> TPSSh > PW91/BLYP/BP86/TPSS.     

Hymenolepis diminuta: Chloride fluxes and membrane potentials associated with sodium-coupled glucose transport

Glucose transport by Hymenolepis diminuta was inhibited when Cl^? in the bathing medium was replaced with acetate (C₂H₃O₂^Post?), but was unaffected when Cl^? was replaced with SCN^?. The relative effectiveness of the anions to inhibit influx of 7.4 mM Cl^? in the presence of 1 mM glucose was SCN^? > Cl^? > C₂H₃O₂^Post?. Glucose stimulated the influxes of 120 mM Cl^? and SCN^?, but had little effect on 120 mM C₂H₃O₂^Post? influx. While the diffusion rates of the anions were C₂H₃O₂^Post? > SCN^? = Cl^?, the preference of the glucose transport system for the anions was SCN^? > Cl^? > C₂H₃O₂^Post?. Efflux of Cl^? was not affected by the rate of glucose influx. Finally, microelectrode recordings of worms anesthetized with 2 mM arecoline revealed a transmembrane potential (TMP) of ?45 ± 3.6 mV (inside negative). Three to four minutes after addition of glucose (5 mM) there was a progressive hyperpolarization of the TMP to ?58 mV. A revised model of the glucose transport system that is consistent with previous observations on this organism is proposed.     

TASSER_WT: a protein structure prediction algorithm with accurate predicted contact restraints for difficult protein targets

To improve the prediction accuracy in the regime where template alignment quality is poor, an updated version of TASSER_2.0, namely TASSER_WT, was developed. TASSER_WT incorporates more accurate contact restraints from a new method, COMBCON. COMBCON uses confidence-weighted contacts from PROSPECTOR_3.5, the latest version, PROSPECTOR_4, and a new local structural fragment-based threading algorithm, STITCH, implemented in two variants depending on expected fragment prediction accuracy. TASSER_WT is tested on 622 Hard proteins, the most difficult targets (incorrect alignments and/or templates and incorrect side-chain contact restraints) in a comprehensive benchmark of 2591 nonhomologous, single domain proteins ≤200 residues that cover the PDB at 35% pairwise sequence identity. For 454 of 622 Hard targets, COMBCON provides contact restraints with higher accuracy and number of contacts per residue. As contact coverage with confidence weight ≥3 (F^wt≥3_cov) increases, the more improved are TASSER_WT models. When F^wt≥3_cov > 1.0 and > 0.4, the average root mean-square deviation of TASSER_WT (TASSER_2.0) models is 4.11 Å (6.72 Å) and 5.03 Å (6.40 Å), respectively. Regarding a structure prediction as successful when a model has a TM-score to the native structure ≥0.4, when F^wt≥3_cov > 1.0 and > 0.4, the success rate of TASSER_WT (TASSER_2.0) is 98.8% (76.2%) and 93.7% (81.1%), respectively.     

Two luminescent alkali-silver heterometallic sulfonates

Systematic survey on the reactions of silver nitrate with H₃Ssal ligand (H₃Ssal = 5-sulfo-salicylic acid) accompanying with different alkali metal ions under alkali conditions leads to two novel luminescent alkali-silver heterometallic sulfonates, [KAg(HSsal)(H₂O)₃]_n (1) with a 3-D structure constructed by [K₂O₁₀] binuclear units linking 1-D_∞¹[Ag₂(HSsal)₂] double chains, [NaAg(HSsal)(H₂O)₃]_n (2) with a 3-D structure constructed by the [Ag₂(HSsal)₂] dimers connecting 1-D_∞¹[NaO₆] chains, and also reveals the coordination strength of the alkali metal cations and Ag⁺ towards the sulfonate groups with K⁺ > Na⁺ > Ag⁺ > Li⁺ order. The two compounds show intense blue emissions in both solid state and water solution.     

Specific cation effect on quenching reactions of excited tris(α,α-diimine)ruthenium(II) and tris(2,2-bipyridine)chromium(III) by tris(oxalato)- and tris(malnato)chromates(III) in aqueous solutions

Specific salt effects were studied on the quenching reaction of excited [Ru(N-N)₃]²⁺ (N-N=2,2^′-bipyridine (bpy), 1,10-phenanthrorine (phen)) and [Cr(bpy)₃]³⁺ by [Cr(ox)₃]³⁻ (ox=oxalate ion) and [Cr(mal)₃]³⁻ (mal=malonate ion) in aqueous solutions as a function of alkali metal ions which were added for adjustment of ionic strength. The value of k_q, quenching rate constants, and k₁, energy transfer rate constant in encounter complex, is changed by the cations as the order of Li⁺ > Na⁺ > K⁺ ≈ Rb⁺ ? Cs⁺, although diffusion rate constants are not changed by the co-existing cations. Among the quenching reactions investigated in this work, a ratio of k₁ values in the aqueous solutions whose ionic strength was adjusted with LiCl and KCl, k₁^LiCl/k₁^KCl, is larger for quenching systems of closely approached donor-acceptor pair than loosely bounded pair. These results indicate that co-existing alkali cation tunes the distance between donor and acceptor in encounter complex where energy transfer occurs.     

Quantum chemical study of silanediols as metal binding groups for metalloprotease inhibitors

DFT (B3LYP and M06L) as well as ab initio (MP2) methods with Dunning cc-pVnZ (n?=?2,3) basis sets are employed for the study of the binding ability of the new class of protease inhibitors, i.e., silanediols, in comparison to the well-known and well-studied class of inhibitors with hydroxamic functionality (HAM). Active sites of metalloproteases are modeled by [R₃M-OH₂]²⁺ complexes, where R stands for ammonia or imidazole molecules and M is a divalent cation, namely zinc, iron or nickel (in their different spin states). The inhibiting activity is estimated by calculating Gibbs free energies of the water displacement by metal binding groups (MBGs) according to: [R₃M-OH₂]²⁺ + MBG → [R₃M-MBG]²⁺ + H₂O. The binding energy of silanediol is only a few kcal mol^?1 inferior to that of HAM for zinc and iron complexes and is even slightly higher for the triplet state of the (NH₃)₃Ni²⁺ complex. For both MBGs studied in the ammonia model the binding ability is nearly the same, i.e., Fe²⁺(t) > Ni²⁺(t) > Fe²⁺(q) > Ni²⁺(s) > Zn²⁺. However, for the imidazole model the order is slightly different, i.e., Ni²⁺(t) > Fe²⁺(t) > Fe²⁺(q) > Ni²⁺(s) ≥ Zn²⁺. Equilibrium structures of the R₃Zn ²⁺ complexes with both HAM and silanediol are characterized by the monodentate binding, but the bidentate character of binding increases on going to iron and nickel complexes. Two types of intermediates of the water displacement reactions for [(NH₃)₃M-OH₂]²⁺ complexes were found which differ by the direction of the attack of the MBG. Hexacoordinated complexes exhibit bidentate bonding of MBGs and are lower in energy for M=Ni and Fe. For Zn penta- and hexacoordinated complexes have nearly the same energy. Intermediate complexes with imidazole ligands have only octahedral structures with bidentate bonding of both HAM and dimethylsilanediol molecules.     

Identifying key factors of groundwater chemistry in three diverse Landscapes of Central Mexico

In order to understand the hydrogeochemical pattern, ground (n = 23) and surface water (n = 2) samples were collected from three different landscapes (mountain, plain, valley) of Hidalgo State, Central Mexico. Physicochemical characteristics (pH, electrical conductivity, total hardness, alkalinity, total acidity, total solids, total dissolved solids, total suspended solids, CO₂; cations (Ca²⁺, Mg²⁺, Na⁺), anions (NO₃^?, Cl^?, PO₄^3?and SO₄^2?) and dissolved geochemical elements (Fe, Mn, Cr, Cu, Ni, Co, Pb, Zn, Cd and As) were analyzed. Results illustrated they are neutral to slightly alkaline due to the dissolution of carbonates. Average concentrations of anions and cations presented an order of (in mg/l): Na⁺ (273) > Ca²⁺ (206) > SO₄^2? (181) > Cl^? (163) > Mg²⁺ (115) > NO₃^? (11.07) > PO₄^3? (0.12) revealing the local geogenic and anthropogenic influences. High mean concentrations of dissolved trace metals (0.052 mg/l) in the mountains is attributed to their diverse geochemical environment of the terrain and climatic variability. Concentrations of Cr, Cu, Ni and Zn were below the permissible limits set forth by WHO and the Mexican Government. Piper trilinear diagram revealed that they are mainly of Ca-Mg-HCO₃ and Ca-Mg-SO₄ type. Sodium Absorption Ratio (SAR) indicated that nearly 96% are of excellent quality, while Magnesium Adsorption Ratio (MAR) showed that 68% of them are unsuitable for irrigation purposes.     

Soluble and chloroplast malate dehydrogenase isoenzymes of Triticum aestivum

Three isoenzymes of malate dehydrogenase have been isolated from 9-day-old wheat shoots. The microbody (peroxisome) and chloroplast MDH are similar in their electrophoretic behaviour. The mitochondrial MDH, soluble MDH and chloroplast MDH differ in K_m values for malate and NAD. The activity of MDH isoenzymes with NAD⁺-analogues as substrate was in the order 3-AP-NAD⁺ > 3-AP-deam NAD⁺ > NAD⁺ > TN-NAD⁺ and deam NAD⁺. The thermal stabilities of the isoenzymes were significantly different: C-MDH > m-MDH > S-MDH.     

Synthesis of rhodium and iridium boryl complexes via oxidative addition of haloboranes

B-Chlorocatecholborane undergoes oxidative addition to M(PR₃)₃Cl (M = Rh, R = Me; M = Ir, R = Me, Et) yielding six-coordinate complexes of general formula mer,cis-(PR₃)₃Cl₂M(BO₂C₆H₄). The same M(PR₃)₃Cl complexes also react with B-bromocatecholborane to give a mixture of metal boryl homo- and heterodihalides (PR₃)₃X¹X²M(BO₂C₆H₄) (X¹, X² = Cl, Br), and the observed disproportionation is believed to involve the formation of a heteronuclear halide-bridged intermediate. The alkene 4-vinylanisole failed to react with the six-coordinate, 18-electron (PR₃)₃Cl₂M(BO₂C₆H₄) complexes at ambient temperatures.     

Reactions of [PtCl(dien)]Cl with glutathione,oxidized glutathione and S-methyl glutathione. Formation of an S-bridged dinuclear unit

The reaction of the monofunctional platinum compound [PtCl(dien)]Cl with the tripeptide glutathione (GSH), oxidized glutathione (GSSG) and S-methyl glutathione (GS-Me) has been investigated by ¹H, ¹³C and ¹⁹⁵Pt magnetic resonance spectroscopy and by potentiometric titrations. It appears that platinum binds with a high degree of specificity to the GSH sulfhydryl group. The reaction of platinum with GSH proceeds in two steps. In the first step only one platinum binds to the sulfur atom and, in the second step, another [Pt(dien)]²⁺ unit binds to [Pt(dien)GS]⁺ forming an S-bridged dinuclear unit [{Pt(dien)}₂GS]³⁺. The rate of the first binding step is pH-dependent, whereas the rate of the second step is not. At pH < 7 the rate of the first binding step is slow compared to the rate of the second binding step. At pH > 10, on the other hand, the rate of the first binding step is faster than the rate of the second binding step. Consequently, at pH < 7 one can only isolate the [{Pt(dien)}₂GS]³⁺ complex. In the presence of free GSH, at pH > 7, one [Pt(dien)]²⁺ unit of [{Pt(dien)}₂GS]³⁺ dissociates forming [Pt(dien)GS]⁺. The mechanism of the pH-dependent rate of the first platinum binding step and the ligand-exchange reaction are discussed. GSSG reacts with [Pt(dien)]²⁺, also forming the S-bridged dinuclear unit [{Pt(dien)}₂GS]³⁺, probably through a redox disproportionation reaction with a catalytic function of [PtCl(dien)]Cl. GS-Me reacts with [Pt(dien)]²⁺ forming the S-coordinated [Pt(dien)GS-Me]²⁺. [Pt(dien)GS-Me]²⁺ exists as a pair of diastereomers due to different configurations about sulfur. The rate of the inversion of configuration at the coordinated sulfur atom is slow on the NMR time-scale.     

Properties of [3H] prostaglandin F2α binding to dispersed bovine luteal cells

Intact viable bovine luteal cell suspensions prepared by collagenase digestion of luteal tissue were used in studying the selected properties of [³H] prostaglandin (PG) F_2α binding and compared with those observed in plasma membranes. [³H]PGF_2α specific binding to luteal cells was a rapid (K₁ = 8.4 × 10⁴M^?12αS^?1), reversible (K_?1 = 1.8 × 10^?4S^?1) and saturable process at 24°. There was a single class of receptors with an apparent dissociation constant of 10.6 nM and 1.8 × 10⁵ receptors per cell. The presence of increasing amounts of unlabeled PGs inhibited [³H]PGF_2α binding in a dose-dependent manner. The potency order for this inhibition was: (15S) 15-methyl-PGF_2α methyl ester > ICI-80,996 > PGF_2α > ICI-81,008 > PGF_1α > PGE₂, (15S) 15-methyl-PGE₂ methyl ester > PGF_2α metabolites > other PGs, PGF_1α metabolites and PGE metabolites. Other than the homegeneous nature of binding and a greater association rate in cells, the rest of the [³H]PGF_2α binding properties in cells were in good agreement with those observed in plasma membranes.     

Quantification of cis and trans influences in [PtX(PPh3)3] complexes. A P NMR study

In [PtX(PPh₃)₃]⁺ complexes (X = F, Cl, Br, I, AcO, NO₃, NO₂, H, Me) the mutual cis and trans influences of the PPh₃ groups can be considered constants in the first place, therefore the one bond Pt-P coupling constants of P_(cis) and P_(trans) reflect the cis and trans influences of X. The compounds [PtBr(PPh₃)₃](BF₄) (2), [PtI(PPh₃)₃](BF₄) (3), [Pt(AcO)(PPh₃)₃](BF₄) (4), [Pt(NO₃)(PPh₃)₃](BF₄) (5), and the two isomers [Pt(NO₂-O)(PPh₃)₃](BF₄) (6a) and [Pt(NO₂-N)(PPh₃)₃](BF₄) (6b) have been newly synthesised and the crystal structures of 2 and 4·CH₂Cl₂·0.25C₃H₆O have been determined. From the ¹J_PtP values of all compounds we have deduced the series: I > Br > Cl > NO₃ > ONO > F > AcO > NO₂ > H > Me (cis influence) and Me > H > NO₂ > AcO > I > ONO > Br > Cl > F > NO₃ (trans influence). These sequences are like those obtained for the (neutral) cis- and trans-[PtClX(PPh₃)₂] derivatives, showing that there is no dependence on the charge of the complexes. On the contrary, the weights of both influences, relative to those of X = Cl, were found to depend on the charge and nature of the complex.     

Uptake,incorporation and calcium-ionophore-stimulated mobilization of arachidonic,eicosapentaenoic and docosahexaenoic acids by leucocytes of the rainbow trout,Salmo gairdneri

Rainbow trout leucocytes contain high levels of neutral lipid (about 70% of total lipid on a wt% basis) consisting of mostly triacylglycerol, free sterols and sterol esters (25%, 15% and 52% of neutral lipid, respectively). The phospholipids, separated by thin-layer chromatography, consisted predominantly of phosphatidylcholine, phosphatidylethanolamine and phosphatidylserine, each present at about 30% of the total phospholipid. Radiolabelling of the leucocytes for 1 h with 1 μCi (approx. 6 μM) [1−¹⁴C]20:4(n−6), [1−¹⁴C]20:5(n−3) or [1−¹⁴C]22:6(n−3) each gave similar uptake values (approx. 1 · 10⁵ cpm/10⁷ leucocytes). The incorporation into total phospholipids was highest for 22:6(n−3) and lowest for 20:4(n−6). A higher percentage of radiolabel from [1−¹⁴C]22:6(n − 3) was found incorporated into phosphatidylcholine and phosphatidylethanolamine as compared to that from [1−¹⁴C]20:4(n − 6) and [1−^14C]20:5(n−3), while the reverse situation was found with phosphatidylinositol and phosphatidylserine. The relative rates of incorporation into the different phospholipid classes for all three fatty acids were in the order phosphatidylinositol > sphingomyelin > diphosphatidylglycerol > phosphatidylcholine > phosphatidylethanolamine > phosphatidylserine. Calcium ionophore-challenge did not significantly alter the pattern of phospholipid radiolabel. Ionophore-challenge released large amounts of radiolabel, much of which was recovered after high-performance liquid chromatographic separation as free fatty acid/monohydroxy fatty acids, although only approx. 0.3% was recovered in leukotriene B₄ and leukotriene B₅ for the [1−¹⁴C]20:4(n−6) and [1−¹⁴C]20:5(n−3) labelled leucocytes, respectively. Other lipoxygenase products were also radiolabelled and tentatively identified as 20-carboxy-LTB₄, 20-hydroxy-LTB₄, 6-trans-LTB₄, 6-trans-12-epi-LTB₄, 6-trans-8-cis-12-epi-LTB₄ and the corresponding LTB₅ structures. No ‘6-series’ leukotrienes were produced from [1−¹⁴C]22:6(n−3), nor was there any evidence for the synthesis of ‘5-series’ leukotrienes via retroconversion of 22:6(n−3) to 20:5(n−3). This latter finding shows that, despite the preponderance of 22:6(n−3) in the membranes of trout leucocytes, this fatty acid is not a substrate for leukotriene generation.     

Salt effects on β-glucosidase: pH-profile narrowing

Salts inhibit the activity of sweet almond β-glucosidase. For cations (Cl⁻ salts) the effectiveness follows the series: Cu⁺², Fe⁺² > Zn⁺² > Li⁺ > Ca⁺² > Mg⁺² > Cs⁺ > NH₄⁺ > Rb⁺ > K⁺ > Na⁺ and for anions (Na⁺ salts) the series is: I⁻ > ClO₄⁻ > ⁻SCN > Br⁻ ≈ NO₃⁻ > Cl⁻ ≈ ⁻OAc > F⁻ ≈ SO₄^− 2. The activity of the enzyme, like that of most glycohydrolases, depends on a deprotonated carboxylate (nucleophile) and a protonated carboxylic acid for optimal activity. The resulting pH-profile of k_cat/K_m for the β-glucosidase-catalyzed hydrolysis of p-nitrophenyl glucoside is characterized by a width at half height that is strongly sensitive to the nature and concentration of the salt. Most of the inhibition is due to a shift in the enzymic pK_as and not to an effect on the pH-independent second-order rate constant, (k_cat/K_m)^lim. For example, as the NaCl concentration is increased from 0.01 M to 1.0 M the apparent pK_a1 increases (from 3.7 to 4.9) and the apparent pK_a2 decreases (from 7.2 to 5.9). With p-nitrophenyl glucoside, the value of the pH-independent (k_cat/K_m)^lim (= 9 × 10⁴ M^− 1 s^− 1) is reduced by less than 4% as the NaCl concentration is increased. There is a similar shift in the pK_as when the LiCl concentration is increased to 1.0 M. The results of these salt-induced pK_a shifts rule out a significant contribution of reverse protonation to the catalytic efficiency of the enzyme. At low salt concentration, the fraction of the catalytically active monoprotonated enzyme in the reverse protonated form (i.e., proton on the group with a pK_a of 3.7 and dissociated from the group with a pK_a of 7.2) is very small (≈ 0.03%). At higher salt concentrations, where the two pK_as become closer, the fraction of the monoprotonated enzyme in the reverse protonated form increases over 300-fold. However, there is no increase in the intrinsic reactivity, (k_cat/K_m)^lim, of the monoprotonated species. For other enzymes which may show such salt-induced pK_a shifts, this provides a convenient test for the role of reverse protonation.     

Modeling the Zn and Cd metalation mechanism in mammalian metallothionein 1a

Mammalian metallothioneins (MTs) are a family of small cysteine rich proteins believed to have a number of physiological functions, including both metal ion homeostasis and toxic metal detoxification. Mammalian MTs bind 7 Zn²⁺ or Cd²⁺ ions into two distinct domains: an N-terminal β-domain that binds 3 Zn²⁺ or Cd²⁺, and a C-terminal α-domain that binds 4 Zn²⁺ or Cd²⁺. Although stepwise metalation to the saturated M₇-MT (where M = Zn²⁺ or Cd²⁺) species would be expected to take place via a noncooperative mechanism involving the 20 cysteine thiolate ligands, literature reports suggest a cooperative mechanism involving cluster formation prior to saturation of the protein. Electrospray ionization mass spectrometry (ESI-MS) provides this sensitivity through delineation of all species (M_n-MT, n = 0-7) coexisting at each step in the metalation process. We report modeled ESI-mass spectral data for the stepwise metalation of human recombinant MT 1a (rhMT) and its two isolated fractions for three mechanistic conditions: cooperative (where the binding affinities are: K₁ < K₂ < K₃ < ··· < K₇), weakly cooperative (where K₁ = K₂ = K₃ = ··· = K₇), and noncooperative, (where K₁ > K₂ > K₃ > ··· > K₇). Detailed ESI-MS metalation data of human recombinant MT 1a by Zn²⁺ and Cd²⁺ are also reported. Comparison of the experimental data with the predicted mass spectral data provides conclusive evidence that metalation occurs in a noncooperative fashion for Zn²⁺ and Cd²⁺ binding to rhMT 1a.     

Thin-layer chromatography of thiosulfonate anions

Organic thiosulfonates of the form RS(O₂)S^? can be separated from the characteristic contaminants in synthetic preparations by thin-layer chromatography on silica gel. Thiosulfonates and sulfinates can both be visualized, and differentiated, by treatment of plates with a FeCl₃ reagent in acetone. Data are presented for seven aromatic and six aliphatic thiosulfonate anions and the corresponding sulfonates and sulfinates. With exception of the series related to cysteic acid, R_f values are always in the order RS(O)₂S^? > RSO₃^? > RSO₂^?.