Mass spectrometry studies of demetallation of haemin by recombinant horse L chain apoferritin and its mutant (E 53,56,57,60 Q)

An essential difference between eukaryotic ferritins and bacterioferritins is that the latter contain naturally, in vivo haem as Fe-protoporphyrin IX. This haem is located in a hydrophobic pocket along the 2-fold symmetry axes and is liganded by two Met 52. However, in in vivo studies, a cofactor has been isolated in horse spleen apoferritin similar to protoporphyrin IX; in in vitro experiments, it has been shown that horse spleen apoferritin is able to interact with haem. Studies of haemin (Fe(III)-PPIX) incorporation into horse spleen apoferritin have been carried out, which show that the metal free porphyrin is found in a corresponding pocket to haem in bacterioferritins [Précigoux, G., Yariv, J., Gallois, B., Dautant, A., Courseille, C. and Langlois, d'Estaintot B. (1994) A crystallographic study of haem binding to ferritin. Acta Cryst. D 50, 739-743]. A mechanism of demetallation of haemin by L-chain apoferritin was proposed [Crichton, R.R., Soruco, J.A., Roland, F., Michaux, M.A., Gallois, B., Précigoux, G., Mahy, J.P. and Mansuy. (1997) Remarkable ability of horse spleen apoferritin to demetallate hemin and to metallate protoporphyrin IX as a function of pH. J. P. Biochem. 36, 49, 15049-15054]: this involved four Glu residues (53,56,57,60) situated at the entrance of the hydrophobic pocket and appeared to be favoured by acidic conditions. To verify this mechanism, we have mutated these four Glu to Gln and examined demetallation in both acidic and basic conditions. In this paper, we report the mass spectrometry studies of L-chain apoferritin and its mutant incubated with haemin and analysed after different times of incubation: 15 days, 2 months, 6 months, 9 months and 12 months. These studies show that the recombinant L-chain apoferritin and its mutant are able to demetallate haemin to give a hydroxyethyl protoporphyrin IX derivative in a dimeric form [Macieira, S., Martins, B. M. and Huber, R. (2003) Oxygen-dependent coproporphyrinogen IX oxidase from Escherichia coli: one-step purification and biochemical characterization. FEMS. Microbiology Letters 226, 31-37].     

EPR spectroscopic study of a dinuclear copper(II) complex of tolfenamic acid

The copper(II) complex with tolfenamic acid [Cu(tolf)₂(H₂O)]₂ was studied by X-band and K-band EPR spectroscopies in the temperature range from 90 to 300 K. The Cu²⁺ ions in dinuclear complex show a strong antiferromagnetic exchange interaction with |J| = 292 cm⁻¹. The EPR spectra, which were observed for [Cu(tolf)₂(H₂O)]₂, are typical powder spectra of the copper pairs. The spectra exhibit the hyperfine structure in low temperature range. The values of the spin-Hamiltonian parameters were determined on the basis of the best fit for the simulated spectra at both K-band (0.75 cm⁻¹) at T = 298 K and X-band (0.3 cm⁻¹) at T = 93 K as compared with the experimentally observed spectra. These values show that the local environment around the copper species is distorted tetragonal pyramid. This EPR evidence is consistent with the crystallographic data.     

EPR investigation of Cu(II)-complexes with nitrogen derivatives of dialdehyde starch

Dialdehyde starch obtained by periodate oxidation from potato starch was converted into its disemicarbazone (DSC), dithiosemicarbazone (DTSC), dihydrazone (DHZ) and dioxime (DOX). The Cu(II) complexes of these compounds were prepared and characterized by Raman and EPR spectra, as well as by the measurements of magnetic susceptibility. EPR investigations showed that two types of complexes with different surroundings of copper centres existed in each starch derivative. Besides nitrogen atoms of the CN moiety and sulphur atoms of the CS moiety, also oxygen atoms from starch hydroxyl groups and/or water molecule were proposed as the coordination sites for the central copper ions.     

Site-specific interactions of copper(II) ions with heparin revealed with complementary (SRCD, NMR, FTIR and EPR) spectroscopic techniques

The interactions between Cu(II) ions and heparin were investigated using several complementary spectroscopic techniques. NMR indicated an initial binding phase involving specific coordination to four points in the structure that recur in slightly different environments throughout the heparin chain; the carboxylic acid group and the ring oxygen of iduronate-2-O-sulfate, the glycosidic oxygen between this residue and the adjacent (towards the reducing end) glucosamine and the 6-O-sulfate group. In contrast, the later binding phase showed little structural specificity. One- and two-dimensional correlated FTIR revealed that complex out of phase (asynchronous) conformational changes also occurred during the titration of Cu(II) ions into heparin, involving the CO and N-H stretches. EPR demonstrated that the environments of the Cu(II) ions in the initial binding phase were tetragonal (with slightly varied geometry), while the later non-specific phases exhibited conventional coordination. Visible spectroscopy confirmed a shift of the absorbance maximum. Titration of Cu(II) ions into a solution of heparin indicated (both by analysis of FTIR and EPR spectra) that the initial binding phase was complete by 15-20 Cu(II) ions per chain; thereafter the ions bound in the non-specific mode. Hetero-correlation spectroscopy (FTIR-CD) improved resolution and assisted assignment of the broad CD features from the FTIR spectra and indicated both in-phase and more complex out of phase (synchronous and asynchronous, respectively) changes in interactions within the heparin molecule during the titration of Cu(II) ions.     

Structural, IR, and EPR studies of the bis(methoxyacetato)diaquo-copper(II) complex

The structure, stability, and the IR, and EPR spectroscopic properties of bis(methoxyacetato)diaquo-copper(II) were studied both experimentally using FT-IR and theoretically using B3LYP/6-31G**, B3LYP/6-311G, BWP91/6-31G** methods. The same approaches were used to calculate the harmonic frequencies and to compare them to the experimental solid state values. The g-tensors are calculated using the NMR/GIAO computational method.     

Tautomeric changes in guanfacine and its copper(II) complex as revealed by X-ray crystallography; synthesis and EPR

Obtention and crystal structure of guanfacine (guaH) together with synthesis and crystal structure of its copper complex [Cu^II(gua)₂] · DMF were reported. The free molecule guaH exhibits one tautomeric form (B) in contrast to the form (A) which was reported in the Merck index. In the copper(II) complex, the anionic form gua⁻ exhibits the third tautomeric form (C). This complex is characterized by a CuN₂O₂ coordination. The EPR spectrum is in agreement with a Cu(II) ion in a square planar configuration.     

Synthesis of (13)C-labeled gamma-hydroxybutyrates for EPR studies with 4-hydroxybutyryl-CoA dehydratase

4-Hydroxybutyryl-CoA dehydratase from Clostridium aminobutyricum catalyses the reversible dehydration of its substrate 4-hydroxybutyryl-CoA (4-HB-CoA) to crotonyl CoA. The enzyme contains one [4Fe-4S](2+) cluster and one flavin adenine dinucleotide (FAD) molecule per homotetramer. Incubation of the enzyme with its substrate under equilibrium conditions followed by freezing at 77K induced the EPR-spectrum of a neutral flavin semiquinone (g=2.005, linewidth 2.1 mT), while at 10K additional signals were detected. In an attempt to characterize these signals, 4-HB-CoA molecules specifically labeled with (13)C have been synthesized. This was achieved via (13)C-labeled gamma-butyrolactones, which were obtained from (13)C-labeled bromoacetic acids by efficient synthetic routes. Incubation of the (13)C-labeled 4-hydroxybutyrate-CoA molecules with 4-hydroxybutyryl-CoA dehydratase did not lead to marked broadening of the signals.     

EPR and H NMR spectroscopy and DFT study of pentaammineruthenium(III)phenylcyanamide complexes

The EPR and ¹H NMR spectroscopy of seven [Ru(NH₃)₅L]²⁺ complexes, where L = 3,5-dimethoxyphenylcyanamide (MeO₂pcyd), 3,4,5-trimethoxyphenylcyanamide (MeO₃pcyd), 4-nitrophenylcyanamide (NO₂pcyd), 2,3-dichlorophenylcyanamide (Cl₂pcyd), 2,4,6-trichlorophenylcyanamide (Cl₃pcyd), 2,3,5,6-tetrachlorophenylcyanamide (Cl₄pcyd) and pentachlorophenylcyanamide (Cl₅pcyd), was performed. EPR spectra of the complexes showed an axial signal with g_|| and g_⊥ at high and low field, respectively. The g_|| axis is suggested to lie along the Ru-cyanamide bond. Gas-phase DFT calculations of [Ru(NH₃)₅ phenylcyanamide]²⁺ showed spin density localized mostly on the phenylcyanamide ligand, in disagreement with EPR data. DFT/polarizable continuum model (PCM, water solvation) calculations shifted spin density towards ruthenium so that spin density was shared between ruthenium and phenylcyanamide ligand. Proton contact shifts were determined from NMR and EPR data and were used to estimate spin density distributions on phenyl ring carbons. The results showed that the DFT/PCM calculation overestimated spin density on phenyl ring carbons by approximately one order of magnitude. Donor-acceptor interactions between the solute and solvent that are not fully accounted for in the DFT/PCM method are suggested to stabilize the Ru(III) oxidation state.     

Studies on the Circular Dichroism (CD) and Electron Paramaguetic Resonance(EPR) Spe-ctroscopy of the Partially Oxidized FeMo Protein from Azotobacter vinelandii Nitrogenase

After nitrogenase FeMo protein from A. vinelandii was incubated with a large excess (5–6 equivalents) of indigo carmine for 30–60 min., all of P-clusters in the partially oxidized FeMo protein were oxidized, but all of FeMoCo in the protein were still in the reductive state. The oxidized P-clusters in the protein were able to be completely reduced by Na2S2O4(DT) and the reduction was accelerated by methyl viologen (MV). And all of FeMoCo in the protein were firstly oxidized by some oxidants such as methylene blue. The numbers of the redox equivalent of P-cluster and FeMoCo have been obtained by the CD reductive titration of and EPR/ ABS time course on the oxidation of the partially oxidizeA FeMo protein, respectivelly.     

Single-crystal EPR studies of a spin-frustrated imidazolate-bridged trinuclear copper(II) complex

The single-crystal and polycrystalline powder EPR spectra of the trinuclear compound [L₃Cu₃(Im)₃](ClO₄)₃, L = 1,4,7-trimethyl-1,4,7-triazacyclononane, and Im = imidazolate, have been measured in the temperature range 4.2–300 K. The spectra are explained based on the spin-frustration, the low symmetry, and the intercluster exchange interactions.     

Investigations of different carbohydrate anomers in copper(II) complexes with D-glucose, D-fructose, and D-galactose by Raman and EPR spectroscopy

With the aim of verifying different carbohydrate anomers coordinated to copper(II) ions, some copper(II) complexes with D-glucose (Glc), D-fructose (Fru), and D-galactose (Gal) were prepared and investigated by spectroscopic techniques. Their compositions were verified by elemental, ICP-AES and thermal analyses, in addition to conductivity measurements. The compounds isolated were consistent with the formula Na2[Cu2(carbohydrate)3].8H2O and Na[Cu2(carbohydrate)3].6H2O for the aldoses Glc and Gal, respectively, and Na2[Cu3(carbohydrate)4].8H2O in the case of the ketose, Fru. EPR spectra of these solids showed a rhombic environment around the metal center and suggested the presence of different anomers of the carbohydrates in each case. By Raman spectroscopy, it was possible to verify the predominance of the beta anomer of d-glucose in the corresponding copper complex, while in the free ligand the alpha anomer is predominant. In the case of the analogous complex with d-galactose, the spectrum of the complex shows bands of both anomers (alpha and beta) in approximately the same relative intensities as those observed in the isolated free ligand spectrum. On the other hand, for the complex with d-fructose a mixture of both furanose (five-membered ring) and pyranose (six-membered ring) structures was detected with prevalence of the furanose structure. Based on variations in the relative intensities of characteristic Raman bands, the binding site for copper in the fructose ligand was identified as most likely the 1-CH2OH and the anomeric 1-OH, while in beta-D-glucose it is presumably the anomeric 1-OH and the O-5 atom. These results indicated that EPR and Raman spectroscopy are suitable supporting techniques for the characterization of carbohydrate anomers coordinated to paramagnetic ions.     

重组人组织型纤溶酶原激活剂(rht-PA)及其突变体的纯化

稳定高效表达重组人组织型纤溶酶原激活剂 (rht PA)的CHO细胞株和表达组合突变体的细胞株进行了 3L转瓶培养 .将培养上清分别进行了Lys Sepharose 4B亲和层析和Zn2 + Sepharose 4B层析两步纯化 ,rht PA纯度提高了 5 34倍 ,比活达 2 5× 10 5IU mg ,产率为 73% ;突变体纯度提高了1119倍 ,比活达 5 9× 10 5IU mg ,产率为 6 9% .纯化产物SDS PAGE分析显示 ,rht PA和突变体基本都呈单一条带 ,扫描分析均达到 98%以上纯度 .rht PA和突变体在纯化系统中的行为作对照分析发现 ,突变体的构建思想在Lys Sepharose 4B亲和层析过程中有充分体现 .这两步层析组合是很好的纯化t PA及其突变体的方法 ,尤其是Lys Sepharose 4B纯化突变体效果更好     

High-field (94-GHz) EPR spectroscopy on the S(2) multiline signal of Photosystem II

The multiline signal of the S(2) state in Photosystem II was measured both in frozen-solution and single-crystal preparations from the cyanobacterium Thermosynechococcus elongatus. The frozen-solution EPR spectrum shows a gaussian-like line shape without any resolution of Mn hyperfine couplings. This line shape can be understood on the basis of the single-crystal spectra, where a strong orientation dependence of partially resolved hyperfine structures appears. Simulation of the frozen-solution spectrum on the basis of Mn hyperfine couplings taken from published pulse-ENDOR data yields a fully rhombic g-matrix for the multiline signal with principal components 1.997, 1.970, and 1.965. The resulting isotropic g-value g(iso)=1.977 is surprisingly small compared to other manganese complexes containing manganese ions in the formal oxidation states three and four.     

大肠杆菌亮氨酰－tRNA合成酶LeuRS67R的纯化及其动力学研究

本实验室已得到的亮氨酰－ｔＲＮＡ合成酶（ＬｅｕＲＳ）基因,与文献相比,６７位氨基酸残基由Ｈｉｓ变为Ａｒｇ,此酶被定名为ＬｅｕＲＳ６７Ｒ。我们从该基因与ｐＵＣ１９重组质粒的大肠杆菌ＴＧ１转化子ＴＧ１－９１中得到ＬｅｕＲＳ的高表达,粗抽液中ＬｅｕＲＳ的表达量在转化子中比在宿主菌ＴＧ１中高２０倍以上。用三步拉层析得到电泳一条带的酶,其比活为１７８９单位／毫克。测定其动力学常数,氨酰化活力对Ｌｅｕ、ＡＴＰ的Ｋｍ值分别为０．０２７ｍｍｏｌ／Ｌ、０．４７ｍｍｏｌ／Ｌ,Ｋｃａｔ值分别为３．５～５．１ｓ－１。ＡＴＰ－ＰＰｉ交换活力对Ｌｅｕ、ＡＴＰ的Ｋｍ值分别为０．０３ｍｍｏｌ／Ｌ、１．０ｍｍｏｌ／Ｌ,Ｌｃａｔ值分别为１４０～１５５ｓ－１。此结果与从野生型大肠杆菌Ｋ－１２中提纯的ＬｅｕＲＳ的动力学常数差别很小,６７位氨基酸残基在与活性中心无直接关系的域可能是大肠杆菌的种间差异。     

Synthesis of two persistent fluorinated tetrathiatriarylmethyl (TAM) radicals for biomedical EPR applications

Tetrathiatriarylmethyl radicals are attractive spin probes extensively used in biomedical magnetic resonance applications. We report a straightforward synthesis of two original tetrathiatriarylmethyl radicals incorporating, respectively, 15 and 45 fluorine atoms, and thus possessing a high affinity to fluorous media. F15T-03 and F45T-03 exhibit a single sharp EPR spectrum and their EPR line broadening is highly sensitive to molecular oxygen. These spin probes are specially designed for assessment of tumor oxygenation using perfluorocarbon formulations.     

重组人睫状神经营养因子突变体的构建、表达及生物活性分析

目的:通过对原始CNTF的突变改造,降低表达蛋白的免疫原性,同时增加其稳定性,以期能用于临床治疗肥胖症。方法:以原始CNTF为模板,去掉N端的12个氨基酸、C端的14个氨基酸,并将C端末2个氨基酸点突变为极性较强的赖氨酸,在大肠杆菌中表达,获得CNTF-T突变体。结果:基因序列分析与原设计吻合,目的蛋白表达量占全菌体的35%左右,表达蛋白以包涵体形式存在,经变性、复性、DEAE-FF纯化,纯度可达95%以上,小鼠体内生物活性测定,能明显抑制小鼠体重增长,且CNTF-T的生物活性比原始序列CNTF的活性高。结论:突变的CNTF-T有望成为新一代的生物减肥制剂应用于临床。     

Optical and EPR spectra of the thionitrosyl complex [Cr(OH2)5(NS)]

The green thionitrosyl complex [Cr(OH₂)₅(NS)]²⁺ was isolated in solution by the hydrolysis of [Cr(NCCH₃)₅(NS)]²⁺. The optical absorption spectra of both compounds are dominated by a band with vibrational progression around 600 nm assigned as a {d_yz,zx, π^∗(NS)} → {π^∗(NS), d_yz,zx}^∗ transition. The optical data indicate that the NS ligand is a weaker π-acceptor than the NO ligand. The EPR parameters of [Cr(OH₂)₅(NS)]²⁺ were determined: g_iso, g_∥ and g_⊥: 1.96515, 1.92686(5) and 1.986860(8); A_iso(⁵³Cr), A_∥(⁵³Cr) and A_⊥(⁵³Cr): 25.3 × 10⁻⁴, 38 × 10⁻⁴ and 18.5 × 10⁻⁴ cm⁻¹; A_iso(¹⁴N), A_∥(¹⁴N) and A_⊥(¹⁴N): 6.5 × 10⁻⁴, 2.81 × 10⁻⁴ and 8.346(12) × 10⁻⁴ cm⁻¹.     

DNA-fiber EPR spectroscopy as a tool to study DNA-metal complex interactions: DNA binding of hydrated Cu(II) ions and Cu(II) complexes of amino acids and peptides

DNA-fiber EPR spectroscopy and its application to studies of the DNA binding orientation and dynamic properties of Cu(II) ions and their complexes with amino acids and peptides are reviewed. Cu(II) ions bind in at least two different binding modes; one mode was mobile while the other mode fixed the orientation of the coordination plane. The hydroxyl groups of L-Ser and L-Thr fixed the coordination plane of their respective Cu(II) complexes parallel to the DNA base pair plane, whereas Cu(II) complexes of Lys and Arg induced several binding modes, depending on the tertiary structure of the DNA and the chirality of the amino acids. Unusually broadened signals observed for the His complex were assigned to a mono-L-His complex stacked stereospecifically along the DNA double helix. In comparison, Cu(II). Xaa-Xaa' -His type complexes oriented in the minor groove with different affinities and extents of randomness depending on the Xaa-Xaa' sequence and the chirality of Xaa or Xaa' while the C-terminal Xaa residues in Cu(II).Arg-Gly-His-Xaa (Xaa=L-Leu or L-Glu) decreased the stereospecificity and the stability of the complexes bound to DNA. In contrast to Xaa-Xaa'- His complexes, the coordination planes of Cu(II).Gly-L-His-Gly and Cu(II).Gly-L-His-L-Lys complexes were found to lie parallel to the DNA-fiber axis. Dinuclear Cu(II).carnosine complexes were also shown to bind to DNA stereospecifically.     

Accurate predictions of the EPR parameters in planar cobalt(II) complexes by hybrid density functional theory

The hybrid density functional theory is applied to calculate the electron paramagnetic resonance parameters, i.e, the g- and A-tensors of some planar Cobalt(II) complexes with a C_2v symmetry. Calculations were done for four systems: Co(acacen), Co(tacacen), Co(seacacen) and Co(sacsac)₂. The following hybrid functionals were employed: B3LYP, B3PW91, mPW1PW91 and PBE0. The expected large deviation of the g- and A-tensors is well reproduced, and is in very good agreement with the experimental observations. Comparative study shows that the PBE0 hybrid model yields the best agreement with experimental data.     

EPR detection of paramagnetic chromium in liver of fish (Anguilla anguilla) treated with dichromate(VI) and associated oxidative stress responses—Contribution to elucidation of toxicity mechanisms

The impact of chromium (Cr) on fish health has been the subject of numerous investigations, establishing a wide spectrum of toxicity, attributed particularly to the hexavalent form [Cr(VI)]. However, reports on the simultaneous assessment of Cr toxicity in fish and its toxico-kinetics, namely involving metal speciation, are scarce. Therefore, keeping in view the understanding of the mechanisms of Cr(VI) toxicity, this work intended to detect the formation of paramagnetic Cr species in liver of Anguilla anguilla following short-term dichromate(VI) intraperitoneal treatment (up to 180 min), assessing simultaneously the pro-oxidant properties. The formation of Cr(V) and Cr(III) was examined by electron paramagnetic resonance (EPR), as an innovative approach in the context of fish toxicology, and related with the levels of total Cr. Cr(V) was successfully detected and quantified by EPR spectrometry, showing a transient occurrence, mostly between 15 and 90 min post-injection, with a peak at 30 min. The limitations of EPR methodology towards the detection and quantification of Cr(III) were confirmed. Although Cr(VI) exposure induced the antioxidant system in the eel's liver, the oxidative deterioration of lipids was not prevented. Overall, the results suggested that Cr(V), as a short-lived species, did not appear to be directly and primarily responsible for the cellular damaging effects observed, since stress responses persisted up to the end of exposure regardless Cr(V) drastic decay. Though further research is needed, ROS mediated pathways (suggested by superoxide dismutase and catalase activity induction) and formation of Cr(III) complexes emerged as the most plausible mechanisms involved in Cr(VI) toxicity.