Letter: collision-induced dissociation of [metal(L)(2)](2+) complexes (metal = Cu, Ca and Mg) of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine allows distinction of the acyl groups at the sn1 and sn2 positions

The collision induced dissociation (CID) spectra of the divalent metal complexes of 1-palmitoyl-2-oleoyl-sn- glycero-3-phosphocholine, [Metal(lI)(L)(2)](2+) (where metal = Cu(2+), Mg(2+) and Ca(2+), L = [16:0/18:1GPCho]), formed by electrospray ionization, reveal interesting metal dependant fragmentation chemistry. Six main classes of reaction are observed corresponding to: two competing carboxylate abstraction pathways (from the sn1 and sn2 positions); phosphate abstraction; competing losses of the two different carboxylic acids from the sn1 and sn2 positions; loss of a protonated ligand, [L + H](+). The relative ratios of the competing carboxylate abstraction reactions are dependant on the metal, with the Cu and Ca complexes favouring the abstraction of the larger carboxylate (18:1) and the Mg complex favoring the abstraction of the smaller carboxylate (16:0).     

Interaction of apo cytochrome c with sulfonated polystyrene nanoparticles

Stable nanoparticle dispersion in aqueous solutions was obtained with partially sulfonated polystyrene. The hydrophobic association of the backbone chains and phenyl groups is balanced by the electrostatic repulsion of the sulfonate groups on the particle surface. The size distribution of the sulfonated polystyrene particles in relation to concentration, degree of sulfonation and chain length, and pH was characterized by dynamic laser light-scattering. The structure and morphology of the particles were characterized with fluorescence and atom force microscopy. Highly sulfonated polystyrene particles can form large complex particles with positively charged protein, apo cytochrome c. Dynamic laser light-scattering and atom force microscopy studies show that the size and distribution of the complex particles depend on the relative amount of apo cytochrome c and sulfonated polystyrene. When sulfonated polystyrene is in excess, apo cytochrome c interacts with sulfonated polystyrene particles forming stable complexes and excessive sulfonated polystyrene particles bind to the periphery of the complexes preventing them from further aggregation. When apo cytochrome c is in excess, apo cytochrome c links the complexes forming much larger particles. Fluorescence study demonstrates that the hydrophobicity/hydrophility of the complex particles is relative to the ratio of apo cytochrome c and sulfonated polystyrene, degree of sulfonation, and pH. Apo cytochrome c not only can neutralize the negative charges on the surface of sulfonated polystyrene particles, but may also insert into the cores disrupting the original structure of sulfonated polystyrene particles.     

Phase changes in supported planar bilayers of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine

We studied the thermal response of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE) by comparing the differential scanning calorimetry (DSC) data of liposomes with atomic force microscopy (AFM) observations on supported planar bilayers. Planar bilayers were obtained by using the Langmuir-Blodgett (LB) technique: the first leaflet transferred at 30 mN m(-1) and the second at 25 mN m(-1). The topographic evaluation of supported POPE bilayers above room temperature showed changes between 43.8 and 59.8 degrees C. These observations are discussed in relation to the main roughness (Ra) variations and are interpreted as the result of the lamellar liquid crystalline (Lalpha) to inverted hexagonal (HII) phase transition. High-magnification images obtained at 45 degrees C revealed intermediate structures in the transformation. Force spectroscopy (FS) was subsequently applied to gain further structural and nanomechanical insight into the POPE planar bilayers as a function of temperature. These measurements show that the threshold force (Fy), which is the maximum force, that the sample can withstand before breaking, increases from 1.91+/-0.11 nN at 21 degrees C up to 3.08+/-0.17 nN at 43.8 degrees C. This behavior is interpreted as a consequence of the formation of intermediate structures or stalks in the transition from the L alpha to H II phase.     

Hopping in the electron-transfer photocycle of the 1:1 complex of Zn-cytochrome c peroxidase with cytochrome c

The physiological electron-transfer (ET) partners, cytochrome c peroxidase (CcP) and cytochrome c (Cc)1, can be modified to exhibit photoinitiated ET through substitution of Zn (or Mg) for Fe in either partner. Laser excitation of the Zn-porphyrin (ZnP) to its triplet excited state (3ZnP) initiates direct heme-heme ET to the ferriheme center of its partner across the protein-protein interface. This photoinitiated ET produces the charge-separated intermediate, I = [ZnP+CcP, Fe2+Cc], with a metalloporphyrin pi-cation radical (ZnP+) in the donor protein and a ferroheme acceptor protein. I, in general, is thought to return to the ground state by a thermal ET process that involves direct heme-heme back-ET to complete a simple photocycle. We here contrast intracomplex ET between yeast iso-1 Cc and ZnCcP(WT) (wild-type) with that for two ZnCcP(X) variants: X = W191F, with redox-active W191 replaced by Phe; WYM4, a W191F mutant with further replacement of four other potentially redox-active sites (W51F, Y187F, Y229F, and Y236F). The results show that W191 acts as an ET mediator, which "short-circuits" the direct heme-heme back-ET through a two-step, hopping process in which the ZnP+ cation radical formed by photoinitiated ET rapidly oxidizes W191, and the resultant W191+, in turn, rapidly oxidizes Fe2+Cc.     

An improved NMR study of liposomes using 1-palmitoyl-2-oleoyl-sn-glycero-3-phospatidylcholine as model

In this paper we report a comparative characterization of Small Unilamellar Vesicles (SUVs), Large Unilamellar Vesicles (LUVs) and Multilamellar Vesicles (MLVs) prepared from 1-palmitoyl-2-oleoyl-sn-glycero-3-phospatidylcholine (POPC), carried out using two NMR techniques, namely High Resolution NMR in solution and High Resolution-Magic Angle Spinning (HR-MAS). The size and size distributions of these vesicles were investigated using the dynamic light scattering technique. An improved assignment of the (1)H-NMR spectrum of MLVs is also reported.     

Studies on the constituents of asclepiadaceae plant—LVII: The structures of six glycosides,wilfoside c1n,c2n,c3n,c1g,c2g and c3g,with novel sugar chain containing a pair of optically isomeric sugars

Six new glycosides named wilfoside C3N (l), C1N (2), C2N (3), C3G (4), C1G (5) and C2G (6) were isolated from Cynanchum wilfordi hemsley (Asclepiadaceae) and their structures were deduced on the basis of the chemical and spectral evidence. It is quite unusual that 2, 3, 5 and 6 include both D-cymarose and L-cymarose in each sugar chain.     

Interaction of N-nitrosodiethylamine/bovine serum albumin complexes with 1,2-dipalmitoyl-sn-glycero-3-phosphocholine monolayers at the air-water interface

We report the effect of N-nitrosodiethylamine (NDA) on the interaction between bovine serum albumin (BSA) and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine monolayers (DPPC) at the air-water interface. We prepared aqueous solutions of NDA/BSA complexes maintaining a constant concentration of BSA of 1.49 x 10(-9) M and using NDA concentrations to obtain 2000, 4000, 6000, 12,500, and 25,000 NDA/BSA molar ratios. The hysteresis area and the compressional modulus of the compression-expansion cycles performed at different times were dependent on the NDA concentration. The cycles performed demonstrate the stability of the new phase of DPPC/BSA and DPPC/NDA/BSA monolayers. This was achieved probably because the BSA concentration used was lower than the one needed for BSA to inhibit the return of DPPC molecules to the interface. Results of the compressional modulus at the onset of the new phase, obtained around 17 mN/m, 15 min and 1, 3, 5, and 12 h after DPPC deposition, indicated that the 3.0 x 10(-6) M NDA concentration produced a more rigid film, probably due to the higher alpha-helix content of BSA. AFM images were obtained for DPPC/BSA and two DPPC/NDA/BSA complexes. Our images show that 12,500 NDA/BSA molecules were mostly adsorbed in the liquid condensed phase. However, BSA molecules were distributed more homogeneously.     

Interaction of azide ion with hemin and cytochrome c immobilized on Au and Ag nanoparticles

This paper presents a set of investigations on the binding of a metabolic inhibitor, azide with prosthetic heme group of biomolecules, hemin chloride (Hem) and cytochrome c (Cyt c) immobilized on Au and Ag nanoparticles. A variety of spectroscopic tools have been used to understand the chemistry occurring on the nanoparticle surface. While the nature of binding of the model system, hemin has been investigated by UV-visible, fluorescence, FTIR, and Raman spectroscopies, the azide binding has been studied in detail by MALDI-TOF MS. Hemin binding on the nanoparticle surface occurs through the carboxylic acid groups. The hemin-N(3) adduct on the nanoparticle surface has been detected by mass spectrometry and its fragments have been studied by post source decay analysis. The chemistry of hemin on the nanoparticle surface has been compared with that of the protein, Cyt c. Azide binding of Cyt c requires thermal activation due to reduced accessibility of the heme center, unlike in the case of hemin. The binding chemistry is similar for free Cyt c and Cyt c bound to the nanoparticles.     

1H and 13C NMR spectra of 1-palmitoyl-2-oleyl-sn-glycero-3-phosphorylcholine in CDCl3 solution and in sonicated dispersions in 2H2O

A mixed-acid monounsaturated lecithin, 1-palmitoyl-2-oleyl-sn-glycero-3-phosphorylcholine (POL), has been synthesized by phospholipase A₂ digestion of 1,2 dipalmitoyl-sn-glycero-3-phosphorylcholine followed by reacylation of the lysolecithin with oleic anhydride. ¹H (90 MHz) and ¹³C (25.2 MHz) NMR spectra of POL in CDCl₃ solution and in sonicated dispersions in ²H₂O have been obtained, and spin-lattice relaxation times measured. The relaxation times were characteristic of the type of structure formed and reflect molecular motion within the lecithin molecule in each structure. In both systems the spin-lattice relaxation times increase along the alkyl chains towards the terminal methyl group, showing a corresponding increase in the chain molecular motion, although there are significant differences in the gradation of the changes.     

Role of type II thioesterases: evidence for removal of short acyl chains produced by aberrant decarboxylation of chain extender units

BACKGROUND: Modular polyketide synthases (PKSs) function as molecular assembly lines in which polyketide chains are assembled by successive addition of chain extension units. At the end of the assembly line, there is usually a covalently linked type I thioesterase domain (TE I), which is responsible for release of the completed acyl chain from its covalent link to the synthase. Additionally, some PKS clusters contain a second thioesterase gene (TE II) for which there is no established role. Disruption of the TE II genes from several PKS clusters has shown that the TE II plays an important role in maintaining normal levels of antibiotic production. It has been suggested that the TE II fulfils this role by removing aberrant intermediates that might otherwise block the PKS complex. RESULS: We show that recombinant tylosin TE II behaves in vitro as a TE towards a variety of N-acetylcysteamine and p-nitrophenyl esters. The trends of hydrolytic activity determined by the kinetic parameter k(cat)/K(M) for the analogues tested indicates that simple fatty acyl chains are effective substrates. Analogues that modelled aberrant forms of putative tylosin biosynthetic intermediates were hydrolysed at low rates. CONCLUSIONS: The behaviour of tylosin TE II in vitro is consistent with its proposed role as an editing enzyme. Aberrant decarboxylation of a malonate-derived moiety attached to an acyl carrier protein (ACP) domain may generate an acetate, propionate or butyrate residue on the ACP thiol. Our results suggest that removal of such groups is a significant role of TE II.     

FTIR-ATR characterization of langmuir-blodgett assemblies of 1-palmitoyl-2-pyrenedecanoylphosphatidylcholine and dipalmitoylphosphatidylcholine

Langmuir-Blodgett assemblies of 1-palmitoyl-2-pyrenedecanoylphosphatidylcholine and dipalmitoylphosphatidylcholine were transferred from an air/water interface onto germanium or silicon attenuated total reflection (ATR) crystals and were investigated by Fourier transform infrared spectroscopy. A detailed attention has been paid to optimize the deposition conditions and to study the organization of transferred mono- and multimolecular layers.     

细胞色素c在Fe_2O_3修饰电极上的直接电化学和电催化特性

采用全氟磺酸树脂Nafion将金属氧化物Fe2O3颗粒细胞色素c(Cyt c)固定玻碳电极(GCE)表面,制备了Nafion-Cyt c-Fe2O3修饰的玻碳电极,构建了基于直接电子传递的过氧化氢生物传感器。在0.10mol/L pH7.0的磷酸盐缓冲溶液中,修饰电极的循环伏安曲线上显示出一对准可逆的氧化还原峰,式量电位为22mV。Cyt c在修饰电极表面的异相电子转移速率常数为1.21s-1。修饰后的电极对过氧化氢有良好响应,响应时间小于10s,电极的安培响应与过氧化氢浓度在2.0×10-6～3.0×10-3mol/L范围内成线性关系,检出限为1.0×10-6mol/L,米氏常数为1.35mmol/L,显示出较好的亲和力。     

Interaction of decacarbonyldimanganese and decacarbonyldirhenium with 1-aryl-3-cyanoprop-2-en-1-ones

The reactions of M₂(CO)₁₀ (M = Mn, Re) withtrans-1-aryl-3-cyanoprop-2-en-1-ones in the presence of Me₃NO proceed with replacement of the CO ligand and lead to complexeseq-(1-aryl-3-cyanoprop-2-en-1-on)nonacarbonyldimanganese andeq-(1-aryl-3-cyanoprop-2-en-1-on)nonacarbonyldirhenium complexes where the metals are bonded with the ligand through the nitrogen atom of the cyanogroup. The treatment of (1-phenyl-3-cyanoprop-2-en-1-on)nonacarbonyldirhenium with a second equivalent of the ligand resulted in the disubstituted complex,eq,eq-bis(1-phenyl-3-cyanoprop-2-en-1-on)octacarbonyldirhenium in a good yield. The structures of the obtained complexes are discussed on the basis of¹³C,¹H NMR, and IR-spectroscopy.Translated fromIzvestiya Akademii Nauk. Seriya Khimicheskaya, No. 8, pp. 1485–1487, August, 1994.This work was supported in part by the Russian Foundation for Basic Research (grant No 93-03-4028).     

Identification by electrospray tandem mass spectrometry of spin-trapped free radicals from oxidized 2-oleoyl-1-palmitoyl-sn-glycero-3-phosphocholine

GPC radical species formed during oxidation of a glycerophosphocholine (16:0/18:1) under the Fenton reaction conditions were detected using a spin trap, 5,5-dimethyl-1-pyrrolidine N-oxide (DMPO). The stable spin-trapped radical adducts were identified by mass spectrometry (MS) using electrospray (ES) as ionization method and characterized by tandem mass spectrometry (MS/MS). Radical adducts of oxidized free sn-2 fatty acid and of oxidized intact GPC, containing one, two and three additional oxygen atoms, were assigned. DMPO adducts of oxidized intact GPC were observed as singly and doubly charged ions in ES-MS, while adducts of oxidized free fatty acids were observed as singly charged ions. Oxidized free sn-2 fatty acids and intact GPC-DMPO adducts correspond to carbon- and oxygen-centered radicals that were identified by MS/MS as alkyl, hydroxy-alkyl, alkoxyl, hydroxy-alkoxyl, peroxyl and hydroperoxide-alkoxyl spin adducts. The DMPO molecule was attached predominantly at C(9) of the oleic chain. The fragmentation pathway of spin adducts with two DMPO molecules strongly suggests the presence of species that were simultaneously carbon- and oxygen-centered radicals. Several fragments identified are consistent with the presence of isomeric structures contributing to the same ions.     

Structural evidence for Pπ-Pπ conjugation in aminosubstituted phosphaalkynes

The molecular structure of 2,2,6,6-tetramethylpiperidinophosphaalkyne was determined by the X-ray structural method. The main geometrical parameters are as follows: PC 1.559(2), N C(sp) 1.316(2) Å, PC N 178.9(1)°, with an almost planar trigonal bond configuration for the N atom and the chair conformation of the piperidine ring. Structural evidence for the nitrogen lone pair conjugation with the π-system of the triple bond was found to be different in phosphaalkynes PC-NR₂ and nitriles NC NR₂. Quantum-chemical ab initio calculations (HF/631G*) showed that this is caused by a different character of polarization of the PC and NC triple bonds.     

Supramolecular complex of cytochrome c with lariat ether: solubilization, redox behavior and catalytic activity of cytochrome c in methanol

A variety of lariat ethers were employed to solubilize water-soluble cytochrome c in methanol, in which alcohol, ether, ester, amine, and amide functionalities were attached as cation-ligating side arms to 18-crown-6, 15-crown-5, and 12-crown-4 rings. Among these lariat ethers, the alcohol-armed 18-crown-6 derivative offered the highest solubilization efficiency for cytochrome c via supramolecular complexation. The resulting cytochrome c-lariat ether complexes were electrochemically and spectroscopically characterized and confirmed to have redox-active heme structures of 6-coordinate low-spin population in methanol. Some of them catalyzed the oxidation of pinacyanol chloride with hydrogen peroxide in methanol and exhibited higher activities than unmodified cytochrome c and its poly(ethylene glycolated) derivative. Since the supramolecular complexation between lariat ether and cytochrome c includes extremely simple procedures, it provides a facile preparation method of effective biocatalysts working in organic solvents from metalloproteins.     

Characteristics of excimer formation in langmuir-blodgett assemblies of 1-palmitoyl-2-pyrenedecanoylphosphatidylcholine and dipalmitoylphosphatidylcholine

Langmuir-Blodgett film alloys of PPDPC (1-palmitoyl-2-[10-(pyren-1-yl)]decanoyl-sn-glycero-3-phosphocholine) and DPPC (1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine) were assembled in varying molar proportions on quartz glass slides. The transferred layers were then characterized according to their pyrene excimer to monomer fluorescence intensity ratio i_E/i_M and fluorescence quantum yields as a function of film composition. The observed deviation from non-ideal mixing is considered to be due to the formation of regular distribution patterns of PPDPC in a DPPC lattice. The observed critical mole fractions of PPDPC evident as steps in I_E/I_M versus X_PPDPC plots can be accounted for by a model involving a trigonal distribution of pyrenedecanoyl chains in the phospholipid acyl chain lattice. The implications of the distribution of PPDPC as superlattices to the excimer formation mechanism are discussed.