Direct detection of oxygen intermediates in the non-heme Fe enzyme taurine/alpha-ketoglutarate dioxygenase

The reaction of substrate-bound taurine/alpha-ketoglutarate dioxygenase with O2 has been studied using cryogenic continuous-flow spectroscopy. Transient absorption spectra acquired at -38 degrees C show an exponential decay of a 318-nm chromophore with an apparent rate of 1.3 s-1. The observed optical changes and their kinetics are consistent with the profile of an Fe(IV) species detected recently by M?ssbauer spectroscopy (Price et al., Biochemistry 2003, 42, 7497-7508). Resonance Raman measurement upon excitation at 363.7 nm reveal at least two oxygen isotope-sensitive vibrations at 821/787 cm-1 and 583/555 cm-1 for 16O and 18O derivatives, respectively. An additional mode is likely to be obscured by an ethylene glycol vibration at 865 cm-1 and/or 1089 cm-1. The 821 cm-1 vibration is assigned to the stretching mode of Fe(IV)=O species on the basis of its frequency and isotopic shift amplitude. The 583 cm-1 band is likely to originate from an Fe-O2 precursor of the Fe(IV)=O species, although its structural details are unclear at present.     

The dependence of collision induced fragmentation pattern on the internal energy of the precursor ion

A Method is presented whereby product organic ions formed as the result of fragmentation of metastable ions can be selected on the basis of their internal energies. The method requires requires angular collimation of the beam of reactant ions issuing from the ion source and that the fragmentation of the metastable ions is studied in the first field free region of a reversed geometry double focusing mass spectrometer. The product ions that make up the metastable peak are allowed to fall on the intermediate resolving slit and, by adjusting the magnet current over a small range, ions contained in different regions of the peak can be allowed into the second field free region. It is shown that the position of an ion within the metastable peak correlates with its internal energy, ions near the edges of the peak being the least excited. The ions entering the second field free region can be investigated by collisional activation. This has been done for molecular ions of p-chlorophenol, methylbenzoate, benzaldehyde, m-chlorotoluene and n-butane. The in which the collision induced fragmentation pattern varies with internal energy of the ions is illustrated.     

Effects of electric fields and electric field-gradients on X-ray scattering factors and the compton profile of hydrogenic atoms

Analytic expressions for the modification of the hydrogenic atom X-ray atomic scattering factor and Compton profile due to a proton at distance R are derived. It is shown that electric field-gradient effects (order R^?3) are comparable in importance to electric field effects (order R^?2).     

Investigating bidentate and tridentate carbamoylmethylphosphine oxide ligand interactions with rare-Earth elements using electrospray ionization quadrupole ion trap mass spectrometry

Electrospray ionization (ESI) quadrupole ion trap mass spectrometry (QIT-MS) and collisionally activated dissociation (CAD) were used to evaluate the rare-earth binding properties of two hydrophobic carbamoylmethylphosphine oxide (CMPO) ligands, the normal bidentate variety, (t-BuC6H4)2P(O)CH2C(O)N(i-Bu)2 (A), a new potentially tridentate extractant, (t-BuC6H4)2P(O)CH[CH2C(O)N(i-Bu)2]C(O)N(i-Bu)2 (B), and tributyl phosphate. The mass spectral results obtained from analysis of 1% HNO3/methanol solution containing the ligands and dissolved lanthanide salts reveal that the favorable stoichiometries of the ligand/metal/nitrate complexes are 2:1:2 for the bidentate ligand A, 1:1:2 for the tridentate ligand B, and 3:1:2 for the monodentate tributyl phosphate. These observed stoichiometries correlate with the number of available binding sites on each ligand as well as with potential steric effects. Energy-variable collisionally activated dissociation experiments showed that for the 2:1:2 complexes involving ligand A or B, as the ionic radius of the bound metal decreased, the removal of nitric acid required less energy and resulted in less extensive spontaneous solvent coordination. This experimental trend suggests that, as the ionic radius of the lanthanide ion decreases, a pair of the carbamoylmethylphosphine ligands is able to more completely solvate the bound metal ion thereby weakening the nitrate-metal interaction.     

Label-free chiral detection of melittin binding to a membrane

The study presented here describes an innovative approach for the detection of surface-confined proteins using chiral second harmonic generation (C-SHG). A unique optical geometry has been employed which allows for the separation of the chiral and achiral nonlinear response. By utilizing this optical arrangement, the detection of chirality originating from melittin adsorbed to a planar supported lipid bilayer has been performed for the first time by C-SHG. Melittin binding to the membrane was monitored as a function of bulk concentration through detection of the C-SHG signal. Analysis of the C-SHG adsorption isotherms reveals Frumkin adsorption behavior with a positive interaction energy. The binding constant (Ka) obtained was determined to be (8.3 +/- 1.0) x 105 M-1. The results of these studies have far-reaching implication in the use of C-SHG for the label-free detection of protein association to surfaces and in the analysis of protein interfacial phenomena.     

Carbon dioxide emulsion assisted loading of polymer microspheres toward sustained release materials

An organic solvent-free method for encapsulating progesterone at high loadings within micron-sized inert latex polymer beads is reported. This approach makes use of a polymeric surfactant to emulsify carbon dioxide into an aqueous latex suspension. In this way, preformed approximately 4 microm polystyrene (PS) microparticles surface-grafted with poly(N-vinylpyrrolidone) (PVP) were plasticized and swollen followed by rapid partitioning of progesterone into the polymer matrix. The as-prepared polystyrene beads incorporated over 10% progesterone by weight while maintaining their initial size and morphological uniformity. Dissolution experiments were also carried out to obtain the release profile of progesterone entrapped within the PVP/PS particles.     

Flexible sorption and transformation behavior in a microporous metal-organic framework

Crystals of the metal-organic framework material Ni(2)(4,4'-bipyridine)(3)(NO(3))(4) (A) have been grown by reaction of Ni(NO(3))(2).6H(2)O and 4,4'-bipyridine in methanol solution. Single-crystal X-ray diffraction experiments show that the ladder structure of the framework is maintained after desolvation of the material, resulting in the production of a porous solid stable to 215(4) degrees C. Powder X-ray diffraction has been employed to confirm the bulk purity and temperature stability of this material. The crystal structure indicates that the pore window has an area of 12.3 A(2). However, sorption experiments show these windows will admit toluene, which has a minimum cross-sectional area of 26.6 A(2), with no significant change in the structure. Monte Carlo docking calculations show that toluene can be accommodated within the large pores of the structure. Exposure of the related microporous material Ni(2)(4,4'-bipyridine)(3)(NO(3))(4).2C(2)H(5)OH (B) to methanol vapor causes a guest-driven solid-state transformation to A which is observed using powder X-ray diffraction. This structural rearrangement proceeds directly from crystalline B to crystalline A and is complete in less than 1 day. Mechanisms for the transformation are proposed which require breaking of at least one in six of the covalent bonds that confer rigidity on the framework.     

An investigation of mononuclear and binuclear palladium(II) complexes of egta (egta = glycine,N,N′-(1,2-ethanediylbis(oxy-2,1-ethanediyl)bis[N-carboxymethyl]) by 1H-, 13C- and 15N-n.m.r. methods

1:1 and 2:1 palladium(II) complexes of egta^4– (egta^4– = glycine, N,N-(1,2-ethanediylbis)(oxy-2,1-ethanediyl)bis[N-carboxymethyl]) were prepared by 1:1 and 2:1 addition of K₂PdCl₄ to K₄egta, and examined by ¹H-, ¹³C- and ¹⁵N-n.m.r. methods. The 1:1 complex, [Pd(egta)]^2– in solution, utilizes a square-planar coordination comprised of two nitrogen and two glycinato carboxylate donors of egta^4–, leaving two glycinato carboxylates pendant. The complex has a cis-(R,S) stereochemistry which places both pendant carboxylates below the PdN₂O₂ square plane and the tether backbone of egta^4– in the up, up sense above the same plane. The cis-(R,S) assignment was assisted by computer simulations of the ¹³C-n.m.r. spectrum for four possible isomers. Only cis-(R,S) and trans-(R,R) calculated ¹³C-spectra were compatible with the observed ¹³C-n.m.r. pattern. The HH NOESY spectrum of [Pd(egta)]^2– detects long range coupling of the backbone –OCH₂CH₂O– linkage with both coordinated and pendant glycinato CH₂ moieties. The cis-(R,S) isomer's tortional movements allow such contacts whereas a trans-(R,R) isomer does not. The 2:1 complex, [Pd₂(egta)(H₂O)₂] in solution has an extended-chain structure with each palladium(II) center coordinated in the mer-iminodiacetate-like coordination with two bound glycinato-functionalities.     

Photochemical and Photophysical Studies of 3-Amino-6-lodoacridine and the Inactivation of λ Phage

Abstract— The photochemistry and photophysics of 3-amino-6-io-doacridine (Acr-I) was studied. Photolysis (350 nm) of Acr-I (free base) generates products consistent with a free radical intermediate in methanol, benzene and carbon tetrachloride. The Acr-I hydrochloride is shown to bind to calf thymus DNA and to the self-complementary dinucleotide cytidylyl-(3′-5′)-guanosine (CpG) minidu-plex in a manner similar to that of proflavine (Acr-NH₂), a known DNA intercalator. The Acr-I is shown to more efficiently nick supercoiled plasmid DNA pBR322 upon 350 nm or 420 nm photolysis than Acr-NH₂. The efficiency of Acr-I-sensitized DNA nicking is not oxygen dependent. Photolysis of the Acr-I/(CpG)₂ complex leads to cleavage of the dinucleotide and to cytidine base release by selective damage to a specific ribose moiety. Dinucleotide cleavage occurs equally well in the presence or absence of oxygen, thereby eliminating a singlet oxygen- or peroxyl radical-mediated process. Photolysis of Acr-I in the presence of a mononucleotide (GMP) or a non-self-complementary dinucleotide (uridylyl-[3′-5′]-cytidine– UpC) does not lead to fragmentation and base release. Similarly, photolysis of the Acr-NH₂/(CpG)₂ complex does not lead to fragmentation and base release. The data indicate that photolysis of an iodinated intercalator bound to CpG or plasmid DNA generates an intercalated aryl radical and that the reactive intermediate initiates a sequence of reactions that efficiently nick nucleic acids. The inactivation of Λ phage sensitized by Acr-I with UV (350 nm) light is oxygen independent but with visible (420 nm) light is strongly oxygen dependent. The Acr-I fluoresces more intensely when excited at 446 than at 376 nm. Thus, UV photolysis may lead to C-I bond homolysis and free radical formation, a process that is not energetically feasible with visible light. The results demonstrate the difficulty of extrapolating model studies involving simple molecules and DNA to understanding the mechanism of viral inactivation with a particular sensitizer.