Two galactomannan preparations from seeds from Mimosa scabrella (bracatinga): Complexation with oxovanadium(IV/V) and cytotoxicity on HeLa cells

Two galactomannans, GALMAN-A and GALMAN-B, were isolated from seeds of Mimosa scabrella (bracatinga), with deactivation and exposure to native enzymes, respectively. They were treated with oxovanadium(IV) and oxovanadium(V), designated (VO²⁺/VO³⁺) to form GALMAN-A:VO²⁺/VO³⁺ and GALMAN-B:VO²⁺/VO³⁺ complexes, respectively. The potentiometric studies provided the binding constants for the complexes and the resulting complexed species were a function of pH. ⁵¹V NMR spectra of GALMAN-A:VO²⁺/VO³⁺ and GALMAN-B:VO²⁺/VO³⁺ at pH 7.8 and at 30 °C indicated the occurrence of two types of complexes formed by oxovanadium ions and galactomannans. GALMAN-A:VO²⁺/VO³⁺ and GALMAN-B:VO²⁺/VO³⁺ caused loss of HeLa cells viability at concentrations of 50-200 μg/mL. GALMAN-A:VO²⁺/VO³⁺ exhibited low toxicity for 24 h, although GALMAN-B:VO²⁺/VO³⁺ was extremely toxic, since 50 μg/mL was sufficient to decrease HeLa cell viability after 48 h by 60%. GALMAN-A gave rise to a slight increase in cell proliferation after 48 h at 100 μg/mL, whereas GALMAN-B promoted a slight decrease at concentrations of 50-100 μg/mL. GALMAN-A:VO²⁺/VO³⁺ and GALMAN-B:VO²⁺/VO³⁺ exhibited a significant decrease in cell proliferation after 48 h, each reaching 60% inhibition at 5-10 μg/mL. The complexes which caused this effect were at concentrations 10 times lower than the uncomplexed polymers.     

Aminoacid-derivatised picolinato-oxidovanadium(IV) complexes: Characterisation, speciation and ex vivo insulin-mimetic potential

The proligands PicMe-AaR (PicMe = methoxipicolyl-5-amide, where the amide substituent is an amino acid AaR = HisH, HisMe, IleH, IleMe, TrpH, TrpMe, HTyrEt, tBuTyrMe, HThrMe, tBuThrMe) and the complexes [VO(Pic-AaR)₂] have been synthesised and characterised. A detailed EPR study of the VO²⁺/Pic-His systems in water revealed the predominance of the complex [VO(Pic-His)H₂O] in the pH range 2-6, with tridentate coordination of Pic-His via the picolinate moiety and imidazole-Nδ. Speciation analyses of the binary systems VO²⁺/Pic-Aa (Aa = His, Ile, Trp) and the ternary systems VO²⁺/Pic-Aa/B (Aa = His, Ile; B = citrate (cit), lactate (lac), phosphate) showed a predominance of the ternary complexes [VO(Pic-Aa)(cit/lac)] and [VO(Pic-Aa)(cit/lac)OH]⁻ in the physiological pH regime. If, in addition, human serum albumin (HAS) and apotransferrin (Tf) are present, with all of the low and high molecular mass constituents in their blood serum concentrations, about two thirds of VO²⁺ is bound to the protein, while there is still a sizable amount of ternary complex [VO(Pic-Aa)(cit/lac)] present (about 1/4 for Pic-His and 1/3 for Pic-Ile) when the vanadium(IV) concentration is relatively high; at lower concentrations Tf is the predominant binder. Insulin-mimetic studies for VO²⁺/Pic-Aa (Aa = His, Ile, Tyr and Trp), based on a lipolysis assay with rat adipocytes, provided IC₅₀ values of 0.41(1) for VO²⁺/Pic-His and VO²⁺/Pic-Ile, which compares with 0.87(17) for VOSO₄.     

Complex Coacervation and Precipitation Between Soluble Pea Proteins and Apple Pectin

Complex formation (leading to either coacervation or precipitation) offers a tool to generate plant-based novel food structures and textures. This study investigated the formation of complexes between soluble pea proteins and apple pectin upon varying the protein-to-pectin ratio (r?=?2:1 to 10:1), pH (3–7), and temperature (25 and 85 °C) with a total biopolymer concentration set to 1% (w/w). The results showed that predominantly soluble biopolymer complexes were formed at pH 5, and at low ratio (r?=?2:1), whereas lowering the pH to more acidic condition, and to higher ratios (r?=?4:1–10:1) induced the formation of more insoluble biopolymer complexes. In general, the mean particle sizes of the biopolymer complexes ranged between approximately 20 and 100 μm. Upon heating to 85 °C, the amount of insoluble biopolymer complexes increased at pH 3–5 at all ratios, except at r?=?2:1. In addition, the complex sizes became somewhat larger at r?=?2:1 to 6:1 upon heat treatment, whereas only trivial size changes were observed at higher ratios (r?=?8:1 to 10:1). Overall, electrostatic and hydrophobic interactions played a major role in the complex formation between the soluble pea proteins and apple pectin. These findings are important for designing solely plant-based food structures.

     

Synthesis, structure and solution chemistry of dioxidovanadium(V) complexes with a family of hydrazone ligands. Evidence of formation of centrosymmetric dimers via H-bonds in the solid state

[V^IVO(acac)₂] reacts with the methanol solution of tridentate ONO donor hydrazone ligands (H₂L^1-4, general abbreviation H₂L; are derived from the condensation of benzoyl hydrazine with 2-hydroxyacetophenone and its 5-substituted derivatives) in presence of neutral monodentate alkyl amine bases having stronger basicity than pyridine e.g., ethylamine, diethylamine, triethylamine and piperidine (general abbreviation B) to produce BH⁺[VO₂L]⁻ (1-16) complexes. Five of these sixteen complexes are structurally characterized revealing that the vanadium is present in the anionic part of the molecule, [VO₂L]⁻ in a distorted square pyramidal environment. The complexes 5, 6, 15 and 16 containing two H-atoms associated with the amine-N atom in their cationic part (e.g., diethylammonium and piperidinium ion) are involved in H-bonding with a neighboring molecule resulting in the formation of centrosymmetric dimers while the complex 12 (containing only one hydrogen atom in the cationic part) exhibits normal H-bonding. The nature of the H-bonds in each of the four centrosymmetric dimeric complexes is different. These complexes have potential catalytic activity in the aerial oxidation of l-ascorbic acid and are converted into the [VO(L)(hq)] complexes containing VO³⁺ motif on reaction with equimolar amount of 8-hydroxyquinoline (Hhq) in methanol.     

Synthesis, structure and solution chemistry of mixed-ligand oxovanadium(IV) and oxovanadium(V) complexes incorporating tridentate ONO donor hydrazone ligands

In the title family, the ONO donor ligands are the acetylhydrazones of salicylaldehyde (H₂L¹) and 2-hydroxyacetophenone (H₂L²) (general abbreviation, H₂L). The reaction of bis(acetylacetonato)oxovanadium(IV) with a mixture of tridentate H₂L and a bidentate NN donor [e.g., 2,2′-bipyridine(bpy) or 1,10-phenanthroline(phen), hereafter B] ligands in equimolar ratio afforded the tetravalent complexes of the type [V^IVO(L)(B)]; complexes (1)-(4) whereas, if B is replaced by 8-hydroxyquinoline(Hhq) (which is a bidentate ON donor ligand), the above reaction mixture yielded the pentavalent complexes of the type [V^VO(L)(hq)]; complexes (5) and (6). Aerial oxygen is most likely the oxidant (for the oxidation of V^IV → V^V) in the synthesis of pentavalent complexes (5) and (6). [V^IVO(L)(B)] complexes are one electron paramagnetic and display axial EPR spectra, while the [V^VO(L)(hq)] complexes are diamagnetic. The X-ray structure of [V^VO(L²)(hq)] (6) indicates that H₂L² ligand is bonded with the vanadium meridionally in a tridentate dinegative fashion through its phenolic-O, enolic-O and imine-N atoms. The general bond length order is: oxo < phenolato < enolato. The V-O (enolato) bond is longer than V-O (phenolato) bond by ∼0.07 Å and is identical with V-O (carboxylate) bond. ¹H NMR spectrum of (6) in CDCl₃ solution indicates that the binding nature in the solid state is also retained in solution. Complexes (1)-(4) display two ligand-field transitions in the visible region near 820 and 480 nm in DMF solution and exhibit irreversible oxidation peak near +0.60 V versus SCE in DMSO solution, while complexes (5) and (6) exhibit only LMCT band near 535 nm and display quasi-reversible one electron reduction peak near −0.10 V versus SCE in CH₂Cl₂ solution. The VO³⁺-VO²⁺E_1/2 values shift considerably to more negative values when neutral NN donor is replaced by anionic ON donor species and it also provides better VO³⁺ binding via phenolato oxygen. For a given bidentate ligand, E_1/2 increases in the order: (L²)²⁻ < (L¹)²⁻.     

Entamoeba histolytica: Soluble and membrane-associated neutral sphingomyelinase-C and other unidentified esterase activity

Sphingomyelinase (SMase) activity was measured in Entamoeba histolytica particulate and soluble subcellular fractions. The effects on SMase of incubation time, total protein concentration, pH, and several divalent cations were determined. SMase-C and other unidentified esterase activity were detected in soluble and particulate fractions. SMase-C was 94.5-96.0% higher than the unidentified esterase activity. Soluble and insoluble SMase-C specific activities increased with protein dose and incubation time. Soluble and insoluble SMase-C activities were maximum at pH 7.5 and were dependent on Mg²⁺, Mn²⁺, or Co²⁺, and inhibited by Zn²⁺, Hg²⁺, Ca²⁺, and EDTA. SMase-C was active in the pH range of 3-10 and its maximum activity was at pH 7.5. The soluble and insoluble SMases have remarkably similar physicochemical properties, strongly suggesting that E. histolytica has just one isoform of neutral SMase-C that had not been described before and might be essential for E. histolytica metabolism or virulence.     

Interaction of VO ion with human serum transferrin and albumin

The complexation of VO²⁺ ion with the high molecular mass components of the blood serum, human serum transferrin (hTf) and albumin (HSA), has been re-examined using EPR spectroscopy. In the case of transferrin, the results confirm those previously obtained, showing that VO²⁺ ion occupies three different binding sites, A, B₁ and B₂, distinguishable in the X-band anisotropic spectrum recorded in D₂O. With albumin the results show that a dinuclear complex (VO)₂^dHSA is formed in equimolar aqueous solutions or with an excess of protein; in the presence of an excess of VO²⁺, the multinuclear complex (VO)_x^mHSA is the prevalent species, where x = 5-6 indicates the equivalents of metal ion coordinated by HSA. The structure of the dinuclear species is discussed and the donor atoms involved in the metal coordination are proposed on the basis of the measured EPR parameters. Two different binding modes of albumin can be distinguished varying the pH, with only one species being present at the physiological value. The results show that the previously named “strong” site is not the N-terminal copper binding site, and some hypothesis on the metal coordination is discussed, with the ⁵¹V A_z values for the proposed donor sets obtained by DFT (density functional theory) calculations. Finally, preliminary results obtained in the ternary system VO²⁺/hTf/HSA are shown in order to determine the different binding strength of the two proteins. Due to the low VO²⁺ concentration used, the recording of the EPR spectra through the repeated acquisition of the weak signals is essential to obtain a good signal to noise ratio in these systems.     

Characterization of phytase produced byAspergillus niger

The extracellular activity ofAspergillus niger phytase at the end of the growth phase was 132 nkat/mL in a laboratory bioreactor. The purified enzyme has molar mass approximately 100 kDa, pH optimum at 5.0, temperature optimum at 55°C and high pH and temperature stability. TheK _m for dodecasodium phytate, calcium phytate and 4-nitrophenyl phosphate are 0.44, 0.45 and 1.38 mmol/L, respectively. The enzyme is noncompetively inhibited by inorganic monophosphate (K _i=2.85 mmol/L) and by Cu²⁺, Zn²⁺, Hg²⁺, Sn²⁺, Cd²⁺ ions and strongly by F⁻ ones; it is activated by Ca²⁺, Mg²⁺ and Mn²⁺ ions. The substrate specificity of phytase is broad with the highest affinity to calcium phytate.     

Cyanoscorpionates: Co(II), Mn(II), and Ni(II) complexes coordinated only through the cyano group

New complexes have been synthesized of scorpionate ligands with cyano substituents in the 4-positions of the pyrazoles and tert-butyl substituents in the 3-positions of the pyrazoles. Reaction of Co²⁺, Mn²⁺, and Ni(cyclam)²⁺ (cyclam = 1,4,8,11-tetraazacyclotetradecane) with Tp^t-Bu,4CN in a 1:2 ratio produced new octahedral metal complexes of the form (Tp^t-Bu,4CN)₂ML₄ (L₄ = (H₂O)₄, (H₂O)₂(MeOH)₂, or cyclam). Unlike the sandwich complexes previously isolated with Tp^Ph,4CN, the crystal structures showed none of the pyrazole nitrogen atoms coordinated to the metal. Rather, the metal is coordinated to one CN nitrogen atom from each ligand, with two Tp anions coordinated trans to each other around the metal center. This leaves the Tp pyrazole nitrogen atoms open for another metal to coordinate, which could to lead to heterometallic complexes, new coordination polymers, as well as the framework for supramolecular complexes.     

Synthesis and characterization of new unsaturated macrobicyclic and bis(macrocyclic) copper(II) complexes containing N-CH2-N linkages

New copper(II) complexes [CuL²]²⁺ (L²=7,7,9-trimethyl-1,3,6,10,13-pentaazabicyclo[11,2,1^1.13]hexadec-9-ene) and [Cu₂(L³)(H₂O)₂]⁴⁺ have been prepared by the reaction of [CuL¹]²⁺ (L¹=5,5,7-trimethyl-1,4,8,11,14-pentaazatetradce-7-ene) and formaldehyde. The mononuclear complex [CuL²]²⁺ has a square-planar coordination geometry with a 5-6-5-6 chelate ring sequence and is relatively stable even in low pH at room temperature. The dinuclear complex [Cu₂(L³)(H₂O)₂]⁴⁺ consists of two unsaturated 15-membered pentaaza macrocyclic units (7,7,9-trimethyl-1,3,6,10,13-pentaazacyclopentadec-9-ene) that are linked together by a methylene group in a tilted face-to-face arrangement [Cu?Cu distance: 7.413(2) Å ]. Each macrocyclic unit of [Cu₂(L³)(H₂O)₂]⁴⁺ contains one four-membered chelate ring and has a severely distorted octahedral coordination polyhedron. The dinuclear complex is quite stable in aqueous solutions containing an excess of formaldehyde or in dry acetonitrile but is decomposed to [CuL¹]²⁺ and [CuL²]²⁺ in pure water.     

Binding of palladium(II) complexes to guanine, guanosine or guanosine 5-monophosphate in aqueous solution: potentiometric and NMR studies

The interaction of guanine, guanosine or 5^′-GMP (guanosine 5^′-monophosphate) with [Pd(en)(H₂O)₂](NO₃)₂ and [Pd(dapol)(H₂O)₂](NO₃)₂, where en is ethylenediamine and dapol is 2-hydroxy-1,3-propanediamine, were studied by UV-Vis, pH titration and ¹H NMR. The pH titration data show that both N1 and N7 can coordinate to [Pd(en)(H₂O)₂]²⁺ or [Pd(dapol)(H₂O)₂]²⁺. The pK_a of N1-H decreased to 3.7 upon coordination in guanosine and 5^′-GMP complexes, which is significantly lower than that of ∼9.3 in the free ligand. In strongly acidic solution where N1-H is still protonated, only N7 coordinates to the metal ion, but as the pH increases to pH ∼3, ¹H NMR shows that both N7-only and N1-only coordinated species exist. At pH 4-5, both N1-only and N1,N7-bridged coordination to Pd(II) complexes are found for guanosine and 5^′-GMP. The latter form cyclic tetrameric complexes, [Pd(diamine)(μ-N1,N7-Guo]₄⁴⁺ and [Pd(diamine)(μ-N1,N7-5^′-GMP)]₄H_x^(4−x)−, (x=2,1, or 0) with either [Pd(en)(H₂O)₂](NO₃)₂ or [Pd(dapol)(H₂O)₂](NO₃)₂. The pH titration data and ¹H NMR data agree well with the exception that the species distribution diagrams show the initial formation of the N1-only and N1,N7-bridged complexes to occur at somewhat higher pH than do the NMR data. This is due to a concentration difference in the two sets of data.     

Synthesis and pH-sensitive luminescence of bis-terpyridyl iridium(III) complexes incorporating pendent pyridyl groups

A series of iridium(III) bis-terpyridine complexes have been prepared which incorporate pendent pyridyl groups at the 4′-positions of one or both of the terpyridine (tpy) ligands. These include: three mutually isomeric homoleptic complexes, in which the nitrogen atom of the pendent pyridyl is para, meta or ortho to the C-C bond to the terpyridine; their heteroleptic analogues in which the second ligand is 4′-tolyl-terpyridine (ttpy); analogous complexes of the new ligand, 4′-(2,6-dimethylpyrid-4-yl)-terpyridine; and related complexes incorporating an additional phenyl ring interposed between the terpyridine and the pendent pyridyl group. All of the complexes are luminescent in air-equilibrated aqueous solution at room temperature. The homoleptic complexes display structured emission resembling that of unsubstituted [Ir(tpy)₂]³⁺, with luminescence lifetimes of around 1 μs under these conditions. The heteroleptic analogues give broader, red-shifted emission spectra, similar to that of [Ir(ttpy)₂]³⁺, indicating that emission in these complexes arises primarily from a lower-energy excited state associated with the 4′-tolyl-terpyridine ligand. A further red-shift for the complexes incorporating the additional phenyl ring suggests that the emissive state involves the more conjugated phenylpyridyl-appended ligand in these cases. The luminescence of all of the heteroleptic complexes investigated, except the meta-substituted system, is sensitive to the protonation state of the pendent pyridyl group, and the structure of the ligand can have a significant influence on both the magnitude of the response and the pH region over which it occurs.     

DNA binding and bending by dinuclear complexes comprising ruthenium polypyridyl centres linked by a bis(pyridylimine) ligand

The interaction of enantiomerically pure dinuclear complexes of the form [Ru₂(L-L)₄L¹]⁴⁺ (where L-L = 2,2^′-bipyridine (bpy) or 1,10-phenanthroline (phen) and L¹ = bis(pyridylimine) ligand ((C₅H₄N)CN(C₆H₄))₂CH₂)) with ct-DNA have been investigated by absorbance, circular dichroism, fluorescence displacement assays, thermal analysis, linear dichroism and gel electrophoresis. The complexes all bind more strongly to DNA than ethidium bromide, stabilise DNA and have a significant bending effect on DNA. The data for Δ,Δ-[Ru₂(bpy)₄L¹]⁴⁺ are consistent with it binding to DNA outside the grooves wrapping the DNA about it. By way of contrast the other complexes are groove-binders. The phen complexes provide a chemically and enantiomerically stable alternative to the DNA-coiling di-iron triple-helical cylinder previously studied. In contrast to the di-iron helicates, the phen complexes show DNA sequence effects with Δ,Δ-[Ru₂(phen)₄L¹]⁴⁺ binding preferentially to GC and Λ,Λ-[Ru₂(phen)₄L¹]⁴⁺ to AT.     

A novel vanadium transporter of the Nramp family expressed at the vacuole of vanadium-accumulating cells of the ascidian Ascidia sydneiensis samea

Background

Vanadium is an essential transition metal in biological systems. Several key proteins related to vanadium accumulation and its physiological function have been isolated, but no vanadium ion transporter has yet been identified.

Methods

We identified and cloned a member of the Nramp/DCT family of membrane metal transporters (AsNramp) from the ascidian Ascidia sydneiensis samea, which can accumulate extremely high levels of vanadium in the vacuoles of a type of blood cell called signet ring cells (also called vanadocytes). We performed immunological and biochemical experiments to examine its expression and transport function.

Results

Western blotting analysis showed that AsNramp was localized at the vacuolar membrane of vanadocytes. Using the Xenopus oocyte expression system, we showed that AsNramp transported VO²⁺ into the oocyte as pH-dependent manner above pH 6, while no significant activity was observed below pH 6. Kinetic parameters (K_m and V_max) of AsNramp-mediated VO²⁺ transport at pH 8.5 were 90 nM and 9.1 pmol/oocyte/h, respectively. A rat homolog, DCT1, did not transport VO²⁺ under the same conditions. Excess Fe²⁺, Cu²⁺, Mn²⁺, or Zn²⁺ inhibited the transport of VO²⁺. AsNramp was revealed to be a novel VO²⁺/H⁺ antiporter, and we propose that AsNramp mediates vanadium accumulation coupled with the electrochemical gradient generated by vacuolar H⁺-ATPase in vanadocytes.

General Significance

This is the first report of identification and functional analysis on a membrane transporter for vanadium ions.     

Vanadate inhibition of the Ca-ATPase from human red cell membranes

1. (1) VO₃⁻ combines with high affinity to the Ca²⁺-ATPase and fully inhibits Ca²⁺-ATPase and Ca²⁺-phosphatase activities. Inhibition is associated with a parallel decrease in the steady-state level of the Ca²⁺-dependent phosphoenzyme.

2. (2) VO₃⁻ blocks hydrolysis of ATP at the catalytic site. The sites for VO₃⁻ also exhibit negative interactions in affinity with the regulatory sites for ATP of the Ca²⁺-ATPase.

3. (3) The sites for VO₃⁻ show positive interactions in affinity with sites for Mg²⁺ and K⁺. This accounts for the dependence on Mg²⁺ and K⁺ of the inhibition by VO₃⁻. Although, with less effectiveness, Na⁺ substitutes for K⁺ whereas Li⁺ does not. The apparent affinities for Mg²⁺ and K⁺ for inhibition by VO₃⁻ seem to be less than those for activation of the Ca²⁺-ATPase.

4. (4) Inhibition by VO₃⁻ is independent of Ca²⁺ at concentrations up to 50 μM. Higher concentrations of Ca²⁺ lead to a progressive release of the inhibitory effect of VO₃⁻.

Approach to a metallacycling polymerization — reactions of [(η-C5H5)Co(PPh3)2] with α,β-diynes and Me3SiCCC6H4C6H4CCSiMe3

Reactions of [CpCo(PPh₃)₂](Cp=η⁵-cyclopentadienyl) with conjugated diacetylenes were investigated in terms of the synthesis of π-conjugated organometallic polymers. The reaction of an α,β-diyne, PhCC---CCPh, gave three geometric isomers of dialkynylcobaltacyclopentadienes, 1a-c, and an insoluble polymeric product, 1d. A 2,4-dialkynyl complex, 2, and a 2,5-dialkynyl complex, 3, were obtained solely from Me₃SiCC---CCSiMe₃ and MeCC---CCMe, respectively. 1,1′-Bis(trimethylsilylethynyl)-4,4′-biphenyl afforded two isomers of 1,3-dialkynylcyclobutadiene complexes, 4a and 4b. The stability of the one-electron oxidized forms of the cobalacyclopentadiene and cyclobutadiene complexes was examined by cyclic voltammetry.     

Mono and oligonuclear vanadium complexes as catalysts for alkane oxidation: synthesis, molecular structure, and catalytic potential

A series of mono- and oligonuclear vanadium(V) and vanadium(IV) complexes containing various chelating N,O-, N₃-, and O₂-ligands have been prepared. The biphasic reaction of an aqueous solution of ammonium vanadate and a dichloromethane solution of hexamethylphosphoramide (hmpa) and pyrazine-2-carboxylic acid (pcaH) or pyrazine-2,5-dicarboxylic acid (pdcaH₂) or pyridine-2,5-dicarboxylic acid (pycaH₂) yields yellow crystals of [VO₂(pca)(hmpa)] (1), [(VO₂)₂(pdca)(hmpa)₂] (2), and [VO₂(pycaH)(hmpa)] (3), respectively. The single-crystal X-ray structure analyses reveal 1 and 3 to be mononuclear vanadium(V) complexes, in which a VO₂ unit coordinates to one nitrogen and one oxygen atom of a pca or pycaH chelating ligand, and 2 to be a dinuclear vanadium(V) complex, in which two VO₂ units are coordinated through one nitrogen and one oxygen atom of a pdca bridging ligand; in the three complexes the vanadium atoms also coordinate to the oxygen atom of a hmpa ligand. The reaction of N,N,N^′,N^′-tetrakis(2-benzimidazolylmethyl)-2-hydroxo-1,3-diaminopropane (hptbH) and VOSO₄ in methanol gives the cationic complex [(VO)₄(hptb)₂(μ-O)]⁴⁺ (4), which can be crystallized as the perchlorate salt. In this tetranuclear complex, two dinuclear vanadium(IV) units are held together by a μ-oxo bridge. The known complex [VOCl₂(tmtacn)] (5) was synthesized from the reaction of 1,4,7-trimethyl-1,4,7-triazacyclononane (tmtacn) and VCl₃ in acetonitrile; the reaction of tetrabutylammonium vanadate with pyro-cathecol (catH₂) in acetonitrile gives the known anionic complex [V(cat)₃]⁻ (6), in which the vanadium(V) center is bonded to three cat chelating ligands through the oxygen atoms, obtained as the tetrabutylammonium salt. All compounds synthesized are highly efficient oxidation catalysts for the reaction of cyclohexane with air and hydrogen peroxide in the presence of four equivalents of pcaH per vanadium, although the catalytic activity of the complexes containing bulky chelating ligands 4 and 5 is somewhat lower in the initial period of the reaction. During this period the active species are formed from the complexes and final turnover numbers are high. The catecholate ligands of complex 6 may reduce from V(V) to V(IV) in the beginning of the process, thus providing very high initial oxidation rates.     

Purification and characterization of a heat-stable alkaline protease from Bacillus stearothermophilus F1

A thermophilic Bacillus stearothermophilus F1 that produced an extremely thermostable alkaline protease was isolated from decomposed oil palm branches. The isolated protease was purified to homogeneity by heat treatment, ultrafiltration and gel filtration chromatography with a 128-fold increase in specific activity and 75% recovery. The protease, which is a serine-type enzyme, has a relative molecular mass of 33 500 by sodium dodecyl sulphate-polyacrylamide gel electrophoresis but only 20 000 by gel-filtration chromatography. The enzyme was optimally active at pH 9.0 and was stable for 24 h at 70° C and in the pH range from 8.0 to 10.0. It was capable of hydrolysing many soluble and insoluble protein substrates but no esterase activity was detected. The enzyme activity was markedly inhibited by Co²⁺ and Hg²⁺, whereas Mg²⁺, Fe²⁺, Cu²⁺, Zn²⁺ and Sr²⁺ had little or no inhibitory effect. However, Mn²⁺ strongly activated the protease activity. The protease exhibited a high degree of thermostability [t _1/2 (85° C) = 4 h, (90° C) = 25 min]. The stability at higher temperatures (85° C and above) was shown to be dependent on the presence of Ca²⁺. Correspondence to: A. B. Salleh     

Erratum

Abstract

The condensation and the precipitation of rat liver chromatin upon addition of spermine⁴⁺, spermidine³⁺, hexamminecobalt(III)³⁺ and Mg²⁺ cations have been studied using solubility, fluorescence, circular dichroism, melting curves, electric dichroism and spermidine binding measurements, made on both soluble and precipitated complexes. The soluble complexes obtained with tetra- and trivalent cations were depleted from all histones and enriched in other proteins, particularly high mobility group proteins 1 and 2, which brings about an important enhancement of tryptophan fluorescence without modification of its two lifetimes 5.1 and 1.2 ns. In the precipitates the non-histone proteins are eliminated. Under precipitation by Mg²⁺ ions, the distribution of proteins remains practically unchanged. The electric dichroism and the melting curves indicate that the soluble complexes between polyamines and chromatin undergo important condensation and, at high ratios of cation over phosphate, are constituted by heterogeneous assemblies of non-histone proteins and DNA. On the contrary, the insoluble complexes seem to retain the main features of original chromatin. Precipitation by Mg²⁺ ions reveal much less drastic changes than those produced by polyamines. Precipitation by spermidine occurs when one cation is bound per eight nucleotides, which in addition to the histone positive charges brings about a complete neutralization of chromatin phosphates.