Triethylenetetraminehexaacetic Acid Research Articles

Dual-Responsive Hydrogels for Mercury Ion Detection and Removal from Wastewater.

This study describes the development of a fast and cost-effective method for the detection and removal of Hg2+ ions from aqueous media, consisting of hydrogels incorporating chelating agents and a rhodamine derivative (to afford a qualitative evaluation of the heavy metal entrapment inside the 3D polymeric matrix). These hydrogels, designed for the simultaneous detection and entrapment of mercury, were obtained through the photopolymerization of 2-acrylamido-2-methyl-1-propanesulfonic acid (AMPSA) and N-vinyl-2-pyrrolidone (NVP), utilizing N,N'-methylenebisacrylamide (MBA) as crosslinker, in the presence of polyvinyl alcohol (PVA), a rhodamine B derivative, and one of the following chelating agents: phytic acid, 1,3-diamino-2-hydroxypropane-tetraacetic acid, triethylenetetramine-hexaacetic acid, or ethylenediaminetetraacetic acid disodium salt. The rhodamine derivative had a dual purpose in this study: firstly, it was incorporated into the hydrogel to allow the qualitative evaluation of mercury entrapment through its fluorogenic switch-off abilities when sensing Hg2+ ions; secondly, it was used to quantitatively evaluate the level of residual mercury from the decontaminated aqueous solutions, via the UV-Vis technique. The ICP-MS analysis of the hydrogels also confirmed the successful entrapment of mercury inside the hydrogels and a good correlation with the UV-Vis method.

Photoresponsive oxidase-like phosphorescent carbon dots in colorimetric Hg2+ detection

Developing novel photoresponsive oxidase mimics is highly useful for environmental pollution monitoring and biological sensing. Herein, long-life room-temperature phosphorescent nitrogen-doped carbon quantum dots (P-NCDs) were synthesized from triethylenetetramine hexaacetic acid via a simple one-step hydrothermal method. The P-NCDs showed high photoresponsive oxidase-like activity. On this basis, a P-NCD-based photostimulated colorimetric sensing system was developed and used to detect Hg2+ in environmental and biological samples. P-NCDs under 365 nm UV lamp irradiation converted dissolved oxygen, via triplet excited state (T1) exciton transfer, to singlet oxygen (1O2), which then oxidized 3,3′,5,5′-tetramethylbenzidine (TMB), leading to a color changing reaction. Cysteine can suppress the catalysis of P-NCDs, and its specific complexation with Hg2+ can recover the oxidation activity of P-NCDs. Hence, efficient colorimetric Hg2+ detection with a linear range of 0.01–14 μM and a detection limit of 3.1 nM was achieved by detecting the color change of TMB. The feasibility of this strategy was validated through real sample analysis. Our study broadens the application scope of phosphorescent nanomaterials into colorimetric sensing.

Biochemical characterization of a hemolymph phenoloxidase and its endogenous inhibitor in the larvae of an invasive moth, Cydalima perspectalis Walker (Lepidoptera: Crambidae)

• A phenoloxidase was purified from the hemolymph of C. perspectalis with the molecular weight of ~ 78.7. • The optimal values of 7 and 30–35 °C were evaluated for pH and temperature dependency of the purified PO. • Syntehtic and endogenous inhibitors significantly affected PO activity both in vitro and in vivo. Phenoloxidase system is a crucial component of insect innate immunity which contribute to oxidize phenols to quinones and to generate reactive oxygen and nitrogen intermediates. In the current study, a phenoloxidase (PO) was extracted by hemocyte lysate preparation and purified through ammonium sulfate precipitation, Sepharyl G-100, and DEAE-Cellulose fast flow columns. At the end of the purification process, an enzyme was purified with a specific activity of 0.462 U/mg protein, recovery of 40.47%, purification fold of 14.43 and molecular weight of ~78.7 kDa. The optimal activity was recorded at pH 7 while the optimal temperature was recorded at 30–35 °C, 35 °C and 25–35 °C, using L-dihydroxyphenylalanine, hydroquinone, and pyrocatechol, respectively. The highest V max of PO was obtained using L-dopa while the lowest K m value was gained using hydroquinone. Among used synthetic inhibitors of ethylenediaminetetraacetic acid (EDTA), diethyldithiocarbamate (DTC), N, N,N0,N0-tetraacetic acid (EGTA) and triethylenetetramine hexaacetic acid (TTHA), EDTA and DTC inhibited more than 60% of the enzyme activity. Moreover, an endogenous phenoloxidase inhibitor (POI) was purified by twice processing of Sepharyl G-100 chromatography with the molecular weight of ~52 kDa. The IC 50 of POI was found 31.3 mg against the purified PO of C. perspectalis and led to a higher value of K m . Finally, larval injection by DTC and POI demonstrated significant inhibition of PO over the time of exposure. A comprehensive understanding of insect’s POs may better clarify the ways of their survival within infected areas and to potentially target them by specific and selective compounds.

Kinetics and equilibrium studies on the uptake of Nd3+ by Zn–Al layered double hydroxide intercalated with triethylenetetramine-hexaacetic acid

A Zn–Al layered double hydroxide intercalated with triethylenetetramine-hexaacetic acid (TTHA·Zn−Al LDH) was able to take up Nd3+ from an aqueous solution. To determine the reaction mechanism of this Nd3+ uptake by TTHA·Zn−Al LDH, we conducted kinetics and equilibrium studies. The uptake was more adequately described by the mass-transfer-controlled shrinking core model than the surface-reaction-controlled model. The apparent activation energy was 15.0 kJ mol−1, confirming that the reaction involved in the uptake of Nd3+ by TTHA·Zn−Al LDH proceeds under mass transfer control. This reaction could also be expressed by Langmuir-type adsorption, suggesting the formation of a chelate complex between Nd3+ and TTHA ions in the TTHA·Zn−Al LDH interlayer. The equilibrium adsorption constant was 2.6, and the maximum adsorption amount was 0.3 mmol g−1.

Brightly Luminescent and Kinetically Inert Lanthanide Bioprobes Based on Linear and Preorganized Chelators

The synthesis, photophysical properties, and kinetic stability of a series of water-soluble, highly emissive Tb(III) and Eu(III) complexes featuring triethylenetetraamine hexaacetic acid (TTHA) and cyclohexyl triethylenetetraamine hexaacetic acid (cyTTHA) chelator scaffolds and carbostyril sensitizers are reported. The unique and modular design of the chelators gives rise to striking quantum yields of emission in aqueous solutions (up to 54%) as well as the characteristic lanthanides' photophysical properties (long excited-state lifetimes, large effective Stokes shifts, and narrow emission peaks). Furthermore, the preorganized chelators (L3, L4, and L6) bind metal within minutes at ambient temperature yet exhibit substantial resistance to transchelation in the presence of a challenge solution (EDTA, 1 mM). Moreover, the Eu(III) complex of L4 remains stably luminescent in HeLa cells over hours, demonstrating the suitability of these compounds for live-cell imaging applications. Representative chelators suitable for derivatization and protein bioconjugation were also prepared that were functionalized with clickable azide and alkyne moieties, biotin, and trimethoprim (TMP). With exceptional long-wavelength brightness, enhanced kinetic inertness, and an adaptable synthetic route, the reported lanthanide complexes are promising probes and labels for time-gated bioanalysis, biosensing, and optical microscopy.

Uptake of Nd3+ and Sr2+ by Li[sbnd]Al layered double hydroxides intercalated with ethylenediaminetetraacetate

A LiAl layered double hydroxide intercalated with ethylenediaminetetraacetate (EDTA·LiAl LDH) was prepared by the drop-wise addition of an Al solution to a Li-EDTA solution at a constant pH of 8.0. EDTA·LiAl LDH was found to take up Nd3+ and Sr2+ ions from aqueous solutions, which can be attributed to the metal-chelating functions of the EDTA ions in the interlayers of EDTA·LiAl LDH. However, EDTA·LiAl LDH was found to be inferior to LiAl LDH intercalated with triethylenetetramine-hexaacetic acid (TTHA·LiAl LDH) for both Nd3+ and Sr2+ uptake. This may be explained by the fact that the Nd-EDTA and Sr-EDTA complexes are less stable than the Nd-TTHA and Sr-TTHA complexes, respectively, leading to the lower uptake of Nd3+and Sr2+, respectively. Further, EDTA·LiAl LDH was found to take up Nd3+ ions preferentially over Sr2+ ions from aqueous solutions, which may be caused by the fact that the Nd-EDTA complex is more stable than the Sr-EDTA complex. It should be noted that the Nd3+/Sr2+ uptake selectivity in the case of EDTA·LiAl LDH was higher than that in the case of TTHA·LiAl LDH. In other words, EDTA·LiAl LDH was found to be superior to TTHA·LiAl LDH in selectively taking up metal ions.

Crystal structure of di-aqua-(μ2-tri-ethyl-ene-tetra-minehexa-acetato)-dizinc tetra-hydrate.

The reaction of ZnO and tri­ethyl­ene­tetra­minehexaacetic acid (H6TTHA) in aqueous solution after refluxing yields the binuclear title compound, [Zn2(C18H26N4O12)(H2O)2]·4H2O. There is a centre of symmetry in the [Zn2(H2TTHA)(H2O)2] mol­ecule in the crystalline state. Both ZnII ions are octahedrally surrounded and bound by an N2O3 donor set from the H2TTHA4− anion and a water mol­ecule; the N atoms are cis and the water mol­ecule is trans to an N atom. The Zn⋯Zn separation is 7.562 (1) Å. An intra­molecular C—H⋯O inter­action is observed and both carboxyl­ate H atoms are disordered over two adjacent sites. In the crystal, the components are linked by O—H⋯O and C—H⋯O hydrogen bonds generating a three-dimensonal network.

Uptake of Nd3+ and Sr2+ by Li–Al layered double hydroxide intercalated with triethylenetetramine-hexaacetic acid: kinetic and equilibrium studies

Li–Al layered double hydroxide intercalated with triethylenetetramine-hexaacetic acid (TTHA) was found to take up Nd3+ ions preferentially over Sr2+ ions from solutions, due to the difference in the stabilities of the Nd–TTHA and Sr–TTHA complexes.

Purification and characterization of hemocyte phenoloxidases in Chilo suppressalis walker (Lepidoptera: Crambidae)

In the current study, two phenoloxidases (POs) from the larvae of Chilo suppressalis Walker were extracted and purified by column chromatography using Sepharyl G-100 and DEAE-Cellulose fast flow column. Two proteins possessing PO activity, named as POI and POII, were extracted by purification, 5.08- and 5.62-fold, respectively, with 8.94% and 7.31% recoveries, respectively. Also, the specific activities of POI and POII were 0.478 and 0.529 U/mg protein, respectively. Finally, the molecular weights of POI and POII were calculated as 94.6 and 95.7 kDa, respectively. Kinetic parameters of the purified phenoloxidases by Lineweaver-Burk analysis were Vmax of 2.27 and 1.11 U/mg protein and Km of 15.51 and 17.31 mM for POI and POII, respectively. Mg2+ and Cu2+ significantly increased the PO activities. Ca2+ decreased the activity of POI and showed no statistical effects on POII activity. EDTA and DTC significantly inhibited the activities of the purified enzymes, while triethylenetetramine hexaacetic acid (TTHA) and RGTA showed no significant effects on enzymatic activities.

Preparation of Zn–Al layered double hydroxide intercalated with triethylenetetramine-hexaacetic acid by coprecipitation: uptake of rare-earth metal ions from aqueous solutions

A Zn–Al layered double hydroxide intercalated with triethylenetetramine-hexaacetic acid (TTHA·Zn–Al LDH) was prepared by the dropwise addition of a Zn–Al nitrate solution to a TTHA solution at a constant pH value of 10.0. The TTHA·Zn–Al LDH could uptake rare-earth metal ions such as Nd3+ ions from aqueous solutions. This can be attributed to the chelating ability of the TTHA ions in the interlayer, i.e., Nd–TTHA complexes could be formed in the interlayer. However, the TTHA·Zn–Al LDH could hardly uptake Sr2+ ions from aqueous solutions, indicating that the TTHA ions in the interlayer did not function as effective chelating agents for Sr2+ ions. Therefore, TTHA·Zn–Al LDH could uptake Nd3+ ions selectively from an aqueous mixture of Nd3+ and Sr2+ ions. This was confirmed by the fact that the uptake amount after 120 min was 0.078 and 0.007 mmol g−1 for Nd3+ and Sr2+, respectively, when TTHA·Zn–Al LDH was added to a 1 : 1 mixed nitrate solution of Nd3+ and Sr2+ ions. In this case, the degree of selectivity, i.e., the Nd/Sr molar ratio, was 10.7. Furthermore, TTHA·Zn–Al LDH was superior to ethylenediaminetetraacetate (EDTA)·Zn–Al LDH for the uptake of Nd3+ ions, which can be attributed to the order of the stability of the metal–chelate complexes: Nd–TTHA complex > Nd–EDTA complex.

Synthesis of silver nanoparticles in aqueous aminopolycarboxylic acid solutions via γ-irradiation and hydrogen reduction

Formation of silver nanoparticles in the presence of various aminipolycarboxylic acids (APCAs) such as iminodiacetic acid (IDA), nitrilotriacetic acid (NTA), ethylenediaminetetraacetic acid (EDTA), N-(2-hydroxyethyl) ethylenediaminetriacetic acid (HEDTA), diethylenetriaminepentaacetic acid (DTPA), and N,N,N′,N″,N‴,N‴-triethylenetetraminehexaacetic acid (TTHA) was studied. Monodispersed silver nanoparticles capped by carboxylate groups of APCAs were prepared by the reduction of Ag+ ions induced by either γ-radiolytic or H2 reduction at room temperature. APCAs act as stabilizer to avoid the aggregation of silver nanoparticles. It has been shown that H2-induced reduction of Ag+ is possible at room temperature in the presence of APCAs. TTHA stabilized γ-reduced particles showed reactivity towards oxygen. The formed particles were characterized by UV–vis absorption, X-ray diffraction, zetasizer and transmission electron microscopy.

Digestive glucosidases in the midgut of Apodiphus amygdali Germar (Hemiptera: Pentatomidae)

Apodiphus amygdali or stink bug of fruit trees is one of the polyphagous species from pentatomid bugs that attack many of fruit trees and ornamental trees. In the current study, activities of α- and β-glucosidases were measured in the midgut of A. amygdali adults. It was found the higher activity of β-glucosidase than α-glucosidase in addition to different enzymatic properties of the enzymes. Optimal pHs for enzymatic activities were found to be 5 and 7 for α- and β-glucosidases, respectively. Values regarding optimal temperatures were obtained at 30 °C for both α- and β-glucosidases. Among ions used on α-glucosidase activity, K+ and Ca2+ significantly increased enzymatic activity, Na+ had no effect, and Cu2+, Fe2+ and Mg2+ had the significant negative effects on the enzyme activity. Ca2+ and Fe2+ increased β-glucosidase activity in the midgut of A. amygdali, Na+ had no effect, and other ions significantly decreased the enzyme activity. Ethylene glycol-bis (β-aminoethylether) N,N,N−,N-tetraacetic acid (EGTA), citric acid, ethylenediamide tetraacetic acid (EDTA) and sodium dodecylsulfate (SDS) significantly decreased α-glucosidase activity but EGTA, triethylenetetramine hexaacetic acid (TTHA), EDTA and SDS decreased β-glucosidase activity in the midgut of A. amygdali. Characterisation of digestive enzymes, especially the effect of inhibitors on enzyme activity, could be useful for better understanding of enzyme roles in nutritional physiology of insects in addition to reach safe and useful controls of insect pests.

Three cobalt (II) complexes with triethylenetetraaminehexaacetic acid: From binuclear complex to 3d-4f coordination polymers

The cobalt(II)–lanthanide(III) heteronuclear complexes [Ln(H2O)4Co2(TTHA)(SCN)2]·H3O+ (H6TTHA = triethylenetetraaminehexaacetic acid, Ln = La(2), Ce(3)) have been synthesized based on a binuclear building block of [Co2(H2TTHA)(H2O)2] in [Co2(H2TTHA)(H2O)2]·4H2O (1). Single-crystal structures show that complex 1 is a binuclear complex, containing the [Co2(H2TTHA)(H2O)2] unit as a useful building block. Adding the La3 + and Ce3 + ions to this synthesis system, two new 3d-4f mixed complexes 2 and 3 were obtained. Complexes 2 and 3 show 3D framework, comprising an infinite 1D (one-dimensional) chain based on heterometallic Ln2Co2 (Ln = La(2), Ce(3)). Further investigations such as IR spectra, UV–vis spectra, TGA, XRD and magnetic properties were studied.

Total and Specific Solubility and Activity Coefficients of Neutral Species of (CH2)2i−2Ni(CH2COOH)i+2Complexons in Aqueous NaCl Solutions at Different Ionic Strengths, (0 ≤I≤ 5) mol·L−1, and 298.15 K

The total solubility ST of (CH2)2i−2Ni(CH2COOH)i+2 complexons (i = 1, nitrilotriacetic acid (NTA); i = 3, diethylenetriaminepentaacetic acid (DTPA); i = 4, triethylene tetraaminehexaacetic acid (TTHA)) in aqueous NaCl solutions at different ionic strengths, (0 ≤ I ≤ 5) mol·L−1, and at 298.15 K are determined and are reported in both molar (mol·L−1) and molal (mol·kg−1) concentration scales, together with the protonation constants. The total solubility in pure water S0T is (8.1, 8.9, and 4.5) mmol·L−1 for NTA, DTPA, and TTHA, respectively. Increasing the ionic strength from (0.1 to 5) mol·L−1 (NaCl), the total solubility decreases for NTA, while the opposite trend is observed for DTPA and TTHA. From total solubility and protonation constant data, the solubility of the neutral species S0 and the solubility products KS0 are calculated. The dependence on ionic strength is analyzed, and the Setschenow (km) coefficients are obtained, together with the solubility of the neutral species in pure water S00 [S00 = (...

Fluorescence and conformation in water-soluble bis(pyrenyl amide) receptors derived from polyaminopolycarboxylic acids

Fluorescent responses and conformational changes against pH were studied on four new water-soluble 1-pyrene and 1-methylpyrene bichromophores, (ttha1py)H 4, (edta1mpy)H 2, (dtpa1mpy)H 3 and (ttha1mpy)H 4, with the objective of modifying pH–fluorescence profiles and other solution properties of 1-pyrene bichromophores, (edta1py)H 2 and (dtpa1py)H 3, developed previously by our group; the abbreviations with acidic protons stand for pyrenyl moieties interlinked by an EDTA, DTPA (diethylenetriaminepentacetic acid) or TTHA (triethylenetetraminehexaacetic acid) chain through amide linkages. The new derivatives exhibit emission bands of monomeric pyrene and an intense structureless excimer band; the latter responds to pH sensitively. The pH dependence of the emission intensity of the DTPA and TTHA derivatives is well correlated with species distribution determined by potentiometry. In every derivative, the completely deprotonated species L n− is the most efficient for excimer emission, followed by the corresponding monoprotonated species LH ( n−1)− . In the latter, the acidic hydrogen is located on the central amine, as confirmed by 1H NMR. The resulting hydrogen bond between amino nitrogen atoms makes the aliphatic chain rigid. Further protonation stretches the interlinking chain due to electrostatic repulsion. These conformational changes with protonation result in fluorescent on–off cycles against pH window. The switching cycles of the methylpyrene derivatives are reverse to those of the corresponding pyrene derivatives, as the CH 2 spacer between the amide and aromatic groups defines the orientation of pyrene rings. The sensitiveness of amide group to environment leads to a sharp change in excimer emission above pH ∼11. The combined functions of amide and amino groups lead to the novel pH-responses.

Analyzing several chelating agents and their effect on elemental composition of Lolium perenne and two growth media by capillary zone electrophoresis and inductively coupled plasma optical emission spectrometry

Capillary zone electrophoresis (CZE) along with ICP-OES was used for the determination of several chelating agents in clay and black soil, and in plants (Lolium perenne) grown in these matrices. Elemental levels in all the samples (Al, As, Ca, Cd, Cu, Fe, K, Mg, Mn, Pb, S and Zn) were investigated by inductively coupled plasma optical emission spectrometry (ICP-OES). Compared to previous methods, much shorter total analysis times were accomplished by the validation of an internal standard method for CZE analysis. The solubility of elements increased noticeably in the soils treated by the chelating agents before cultivation, and this phenomenon was strictly metal complex stability constant dependent. The most persistent chelating agents were triethylenetetraaminehexaacetic acid (TTHA) and 1,3-diaminopropanetetraacetic acid (PDTA). Both agents were bound to the growth medium and taken up by ryegrass, while only small proportions of them were degraded during cultivation. Diethylenetriaminepentaacetic acid (DTPA), ethylenediaminetetraacetic acid (EDTA) and nitrilotriamineacetic acid (NTA), mainly bound to the clay matrix during the 19 days cultivation, but after 40 days cultivation in black soil they were degraded to smaller acids. The results clearly show uptake of chelating agents by plants and are a clear indication of the degradation of chelating agents.

Minocycline‐Based Europium(III) Chelate Complexes: Synthesis, Luminescent Properties, and Labeling to Streptavidin

AbstractTwo chelate ligands for europium(III) having minocycline (=(4S,4aS,5aR,12aS)‐4,7‐bis(dimethylamino)‐1,4,4a,5,5a,6,11,12a‐octahydro‐3,10,12,12a‐tetrahydroxy‐1,11‐dioxonaphthacene‐2‐carboxamide; 5) as a VIS‐light‐absorbing group were synthesized as possible VIS‐light‐excitable stable Eu3+ complexes for protein labeling. The 9‐amino derivative 7 of minocycline was treated with H6TTHA (=triethylenetetraminehexaacetic acid=3,6,9,12‐tetrakis(carboxymethyl)‐3,6,9,12‐tetraazatetradecanedioic acid) or H5DTPA (=diethylenetriaminepentaacetic acid=N,N‐bis{2‐[bis(carboxymethyl)amino]ethyl}glycine) to link the polycarboxylic acids to minocycline. One of the Eu3+ chelates, [Eu3+(minocycline‐TTHA)] (13), is moderately luminescent in H2O by excitation at 395 nm, whereas [Eu3+(minocycline‐DTPA)] (9) was not luminescent by excitation at the same wavelength. The luminescence and the excitation spectra of [Eu3+(minocycline‐TTHA)] (13) showed that, different from other luminescent EuIII chelate complexes, the emission at 615 nm is caused via direct excitation of the Eu3+ ion, and the chelate ligand is not involved in the excitation of Eu3+. However, the ligand seems to act for the prevention of quenching of the Eu3+ emission by H2O. The fact that the excitation spectrum of [Eu3+(minocycline‐TTHA)] is almost identical with the absorption spectrum of Eu3+ aqua ion supports such an excitation mechanism. The high stability of the complexes of [Eu3+(minocycline‐DTPA)] (9) and [Eu3+(minocycline‐TTHA)] (13) was confirmed by UV‐absorption semi‐quantitative titrations of H4(minocycline‐DTPA) (8) and H5(minocycline‐TTHA) (12) with Eu3+. The titrations suggested also that an 1 : 1 ligand Eu3+ complex is formed from 12, whereas an 1 : 2 complex was formed from 8 minocycline‐DTPA. The H5(minocycline‐TTHA) (12) was successfully conjugated to streptavidin (SA) (Scheme 5), and thus the applicability of the corresponding Eu3+ complex to label a protein was established.

Effect of ligand on the redox reactions of thallium metal clusters

Pulse radiolysis of an aqueous solution of mono-valent thallium ion and mixed solutions of Tl+/Ag+ in the presence of various amino polycarboxylic acids such as trans-1,2-diaminocyclohexane-N,N,N′,N′-tetraacetic acid (DCTA), diethylenetriaminepentaacetic acid (DTPA), N-(2-hydroxyethyl)ethylenediaminetriacetic acid (HEDTA) and triethylenetetraminehexaacetic acid (TTHA) has been carried out. Abnormal valence states of Tl ions were generated. It is concluded that DCTA, DTPA, HEDTA and TTHA decrease the redox potential of Tl ions in aqueous solutions. It was observed that the electron transfer from complexed Tl2+ to Ag+ varied in the range 7.5 × 108 to 1.0 × 109, depending on the type of complexing ligand. Electron transfer from Tl2+ to Ag+ lead to the formation of silver atoms, which agglomerate further to form silver colloid.

Purification and characterization of hemolymph prophenoloxidase from Ostrinia furnacalis (Lepidoptera: Pyralidae) larvae

Prophenoloxidase (PPO) was isolated from the hemolymph of Ostrinia furnacalis larvae and purified to homogeneity. A 369.85-fold purification and 35.34% recovery of activity were achieved by employing ammonium sulfate precipitation, Blue Sepharose CL-6B chromatography and Phenyl Sepharose CL-4B chromatography. The purified enzyme exhibits a band with a molecular mass of 158 kDa on native PAGE and two spots with a molecular mass of 80 kDa and a pI of 5.70, and a molecular mass of 78 kDa and a pI of 6.50, respectively, on two-dimensional gel electrophoresis. The N-terminal amino acid sequences of two subunits are as follows: PPO1, FGEEPGVQTTELKPLANPPQFRRASQLPRD; PPO2, FGDDAGERIPLQNLSQVPQFRVPSQLPTD. The amino acid composition of purified PPO was similar to that from Galleria mellonella. The enzyme kinetic property of the purified protein showed that the affinity of the enzyme for dopamine was higher than that for l-DOPA and N-acetyldopamine. The phenoloxidase (PO) reaction was strongly inhibited by phenylthiourea, thiourea, dithiothreitol and ethylene diamine tetraacetic acid (EDTA), but poorly inhibited by diethyldithiocarbamate (DTC) and triethylenetetramine hexaacetic acid (THAA), and was not inhibited by o-phenanthroline and ethylene glycol-bis (β-aminoethylether) N, N, N′, N′-tetraacetic acid (EGTA). Both Mg 2+ and Cu 2+ stimulated PO activity when compared with controls. The β-sheet content of PPO treated with Mg 2+ and Cu 2+ increased significantly ( P < 0.05). The purified PPO has magnesium level of 5.674 ± 2.294 µg/mg and copper level of 1.257 ± 0.921 µg/mg as determined with ICP-MS, suggesting that the purified PPO is a metalloprotein.

Synthesis, crystal structure and magnetic properties of bimetallic complex of Ni2Co(TTHA)·12H2O

The bimetallic complex of Ni2Co(TTHA)·12H2O (TTHA = triethylene tetraminehexaacetic acid) was synthesized and characterized structurally and magnetically. The title complex crystallizes in the triclinic space group P ī with a = 0.7316(2), b = 0.8624(2), c = 1.5041(4) nm; α = 73.38(2), β = 83.97(2), γ = 70.50(2)°. The crystal structure is built up of [Ni2(TTHA)(H2O)2]2−, Co(H2O)62+ and water molecules. The variable magnetic measurement shows that there is strong antiferromagnetic interaction between two Ni(II) ions in [Ni2(TTHA) (H2O)2]2− with JNi−Ni = −141.64 cm−1, gNi = 2.21 and that the constant of spin-orbit coupling of Co(II) ion is −134.8 cm−1.