Thermodynamic and kinetic aspects of metal binding to the histidine-rich protein, Hpn

The histidine-rich protein, Hpn, binds to essential metals Ni2+, Cu2+, Zn2+ and a therapeutic metal Bi3+ with the in vitro affinities in the order of Cu2+ > Ni2+ > Bi3+ > Zn2+. In contrast, the in vivo (in E. coli) protection by the protein is in the order of Ni2+ > Bi3+ > Cu2+ approximately Zn2+. The release of Ni2+ from the protein follows a two-step process consisting of a rapidly established equilibrium and subsequently a rate-determining step (dissociation of Hpn-Ni...EDTA to Ni-EDTA). Our work suggests the nickel storage and homeostasis in H. pylori as the primary role of Hpn.     

A third mode of DNA binding: Phosphate clamps by a polynuclear platinum complex

We describe a 1.2 A X-ray structure of a double-stranded B-DNA dodecamer (the Dickerson Dodecamer, DDD, [d(CGCGAATTCGCG)]2) associated with a cytotoxic platinum(II) complex, [{trans-Pt(NH3)2(NH2(CH2)6(NH3+)}2-mu-{trans-Pt(NH3)2(NH2(CH2)6NH2)2}] (TriplatinNC). TriplatinNC is a multifunctional DNA ligand, with three cationic Pt(II) centers, and directional hydrogen bonding functionalities, linked by flexible hydrophobic segments, but without the potential for covalent interaction. TriplatinNC does not intercalate nor does it bind in either groove. Instead, it binds to phosphate oxygen atoms and thus associates with the backbone. The three square-planar tetra-am(m)ine Pt(II) coordination units form bidentate N...O...N complexes with OP atoms, in a motif we call the Phosphate Clamp. The geometry is conserved among the 8 observed phosphate clamps in this structure. The interaction appears to prefer O2P over O1P atoms (frequency of interaction is O2P > O1P, base and sugar oxygens > N). The high repetition and geometric regularity of the motif suggests that this type of Pt(II) center can be developed as a modular nucleic acid binding device with general utility. TriplatinNC extends along the phosphate backbone, in a mode of binding we call "Backbone Tracking" and spans the minor groove in a mode of binding we call "Groove Spanning". Electrostatic forces appear to induce modest DNA bending into the major groove. This bending may be related to the direct coordination of a sodium cation by a DNA base, with unprecedented inner-shell (direct) coordination of penta-hydrated sodium at the O6 atom of a guanine.     

Metal-binding thermodynamics of the histidine-rich sequence from the metal-transport protein IRT1 of Arabidopsis thaliana

The widespread ZIP family of transmembrane metal-transporting proteins is characterized by a large intracellular loop that contains a histidine-rich sequence whose biological role is unknown. To provide a chemical basis for this role, we prepared and studied a peptide corresponding to this sequence from the first iron-regulated transporter (IRT1) of Arabidopsis thaliana, which transports Fe2+ as well as Mn2+, Co2+, Zn2+, and Cd2+. Isothermal titration calorimetry (ITC) measurements, which required novel experiments and data analysis, and supporting spectroscopic methods were used to quantify IRT1's metal-binding affinity and associated thermodynamics. The peptide, PHGHGHGHGP, binds metal ions with 1:1 stoichiometry and stabilities that are consistent with the Irving-Williams series. Comparison of the metal-binding thermodynamics of the peptide with those of trien provides new insight about enthalpic and entropic contributions to the stability of the metal-peptide complex. Although Fe2+ and other IRT1-transported metal ions do not bind very tightly, this His-rich sequence has a very high entropy-driven affinity for Fe3+, which may have biological significance.     

Thickness dependence of microstructure in semiconducting films of an oligofluorene derivative

The measurement and optimization of microstructure development in organic semiconductor films is valuable because microstructure in many cases critically impacts electronic performance. We demonstrate a general method to measure microstructure thickness dependence in thin films using surface-sensitive near edge X-ray absorbance fine structure (NEXAFS) spectroscopy. The method is applied to an oligofluorene derivative DDFTTF, which consists of a fluorene-bithiophene-fluorene core that is end-substituted with linear dodecyl groups. The substrate-relative orientations of the aromatic core and the aliphatic end chains are independently determined, and comparing these orientations to terrace heights from atomic force micrographs proves that the end chains are interdigitated or folded. By measuring microstructure development from 6 to 150 nm, we find that DDFTTF exhibits two different preferential microstructures: one with large terraces within which molecules exhibit a strongly vertical orientation, and one with much smaller domains within which molecules exhibit a mildly horizontal orientation. The relative distribution of these two preferential microstructures depends on the distance of the domains from the substrate and the substrate temperature during deposition. The utility of this method is tested using a lamination technique to measure the saturation hole mobility at the top and bottom interface of DDFTTF films. We find that local microstructures with greater pi orbital alignment in the source-drain plane correlate directly to better local saturation hole mobilities.     

Titanium(II) and titanium(III) tetrahydroborates. Crystal structures of [Li(Et2O)2][Ti2(BH4)5(PMe2Ph)4], Ti(BH4)3(PMe2Ph)2, and Ti(BH4)3(PEt3)2

The molecular structures of the titanium(III) borohydride complexes Ti(BH4)3(PEt3)2 and Ti(BH4)3(PMe2Ph)2 have been determined. If the BH4 groups are considered to occupy one coordination site, both complexes adopt distorted trigonal bipyramidal structures with the phosphines in the axial sites; the P-Ti-P angles deviate significantly from linearity and are near 156 degrees. In both compounds, two of the three BH4 groups are bidentate and one is tridentate. The deduced structures differ from the one previously described for the PMe3 analogue Ti(BH4)3(PMe3)2, in which two of the tetrahydroborate groups were thought to be bound to the metal in an unusual "side-on" (eta(2)-B,H) fashion. Because the PMe3, PEt3, and PMe2Ph complexes have nearly identical IR spectra, they most likely have similar structures. The current evidence strongly suggests that the earlier crystal structure of Ti(BH4)3(PMe3)2 was incorrectly interpreted and that these complexes all adopt structures in which two of the BH4 groups are bidentate and one is tridentate. The synthesis of the titanium(III) complex Ti(BH4)3(PMe2Ph)2 affords small amounts of a second product: the titanium(II) complex [Li(Et2O)2][Ti2(BH4)5(PMe2Ph)4]. The [Ti2(BH4)5(PMe2Ph)4]- anion consists of two Ti(eta(2)-BH4)2(PMe2Ph)2 centers linked by a bridging eta(2),eta(2)-BH4 group that forms a Ti...(mu-B)...Ti angle of 169.9(3) degrees. Unlike the distorted trigonal bipyramidal geometries seen for the titanium(III) complexes, the metal centers in this titanium(II) species each adopt nearly ideal tbp geometries with P-Ti-P angles of 172-176 degrees. All three BH4 groups around each Ti atom are bidentate. One of the BH4 groups on each Ti center bridges between Ti and an ether-coordinated Li cation, again in an eta(2),eta(2) fashion. The relationships between the electronic structures and the molecular structures of all these titanium complexes are briefly discussed.     

Method for determining intracapillary solution temperatures: application to sample zone heating for enhanced fluorescent labeling of proteins

A fundamental premise in CE relies heavily on the assumption that temperature within the capillary is accurately known and controlled. Theoretical calculations for sample zone and BGE temperature during voltage application are presented. We propose that transient elevation of the sample zone temperature allowed for denaturing and renaturing of proteins in the presence of a fluorescent dynamic labeling reagent. Comparison with the extent of labeling possible with standard on-column dynamic labeling in the absence of elevated temperatures showed order-of-magnitude increases in the fluorescence detection sensitivity of proteins with low surface hydrophobicity. As a result, this represents an example where excess heating in the sample zone during electrophoresis can be exploited advantageously.     

Determination of paeoniflorin in rat plasma by a liquid chromatography-tandem mass spectrometry method coupled with solid-phase extraction

A rapid, sensitive and selective liquid chromatography-tandem spectrometry method was developed and validated for determination of paeoniflorin in rat plasma using geniposide as the internal standard. The samples were pretreated with solid-phase extraction using Extract-Clean cartridges. Separation of paeoniflorin and IS was achieved on a reversed-phase C18 column (50x4.6 mm i.d.) with a mobile phase made up of acetonitrile and 0.05% formic acid (25:75, v/v) at a flow rate of 0.5 mL/min. Detection was carried out on a triple quadrupole tandem mass spectrometer by multiple-reaction monitoring and an electrospray ionization source was employed as the ionization source. The lower limit of quantification obtained was 4 ng/mL (n=6) using 200 microL plasma with an accuracy of -3.67% (relative error) and a precision of 4.13% (relative standard deviation). A good linearity was found in the range of 4-1000 ng/mL. The intra- and inter-day relative standard deviations in the measurement of quality control samples 10, 150 and 800 ng/mL ranged from 3.73 to 4.94% and from 4.31 to 6.56%, respectively. The accuracy was from -3.93 to -1.11% in terms of relative error. The analyte and IS were stable in the battery of stability studies. This method was successfully applied to a pharmacokinetic study of paeoniflorin after a single oral administration of 53.36 mg/kg paeoniflorin to rats.     

Characterization of tanshinones in the roots of Salvia miltiorrhiza (Dan-shen) by high-performance liquid chromatography with electrospray ionization tandem mass spectrometry

The qualitative analysis of tanshinones in the roots of Salvia miltiorrhiza (Dan-shen in Chinese) was performed using high-performance liquid chromatography with electrospray ionization tandem mass spectrometry (ESI-MS(n)). Tanshinones are the major bioactive constituents of Dan-shen, which is used in China for the treatment of haematological abnormalities and cardiovascular diseases. The ESI-MS(n) fragmentation behavior of tanshinones was investigated. For tanshinones with the tanshinone I nucleus, the fragmentation was triggered by loss of a molecule of CO except bearing a substituent at C17 or C18, followed by sequential eliminations of CO. If C(15-16) was a saturated bond, the fragmentation was triggered by elimination of a molecule of H2O. For tanshinones with the tanshinone IIA nucleus, the fragmentation was triggered by loss of a molecule of H2O, followed by successive eliminations of CO. Ions corresponding to loss of a molecule of propylene (Delta m = 42) were also observed. Moreover, when C(15-16) was a saturated bond, ions corresponding to losses of CH3, H2O and propylene were more abundant. If no D-ring existed, the presence of isopropyl resulted in an elimination of a molecule of H2O with an adjacent CO or OH. In addition, the extension of the pi-conjugation in the A-ring (especially at C(1-2)) induced the fragmentation by loss of a molecule of CO. These fragmentation rules were applied to the identification of tanshinones in a chloroform/methanol (3:7) extract of Dan-shen, which was separated on a C18 column with gradient elution. A total of 27 tanshinones were identified, including five new constituents. The established method could be used for the sensitive and rapid identification of tanshinones in the Dan-shen drug and its pharmaceutical preparations.     

Cutting boards in Salmonella cross-contamination

Cutting boards are commonly perceived as important fomites in cross-contamination of foods with agents such as Salmonella spp., despite the lack of supporting epidemiological data. A variety of woods and plastics have been used to make work surfaces for cutting. In general, wood is said to dull knives less than plastic, and plastic is seen as less porous than wood. Research to model the hypothetical cross-contamination has been done in a variety of ways and has yielded a variety of results. At least some of the work with knife-scarred plastic indicates that the surface is very difficult to clean and disinfect, although this may vary among the polymers used. High-density polyethylene, which is most used in commercial applications, has been shown to delaminate in response to knife scarring. Wood is intrinsically porous, which allows food juices and bacteria to enter the body of the wood unless a highly hydrophobic residue covers the surface. The moisture is drawn in by capillary action until there is no more free fluid on the surface, at which point immigration ceases. Bacteria in the wood pores are not killed instantly, but neither do they return to the surface. Destructive sampling reveals infectious bacteria for hours, but resurrection of these bacteria via knife edges has not been demonstrated. Small plastic cutting boards can be cleaned in a dishwasher (as can some specially treated wooden boards), but the dishwasher may distribute the bacteria onto other food-contact surfaces. Most small wooden boards (i.e., those with no metal joiners in them) can be sterilized in a microwave oven, but this should be unnecessary if accumulation of food residues is prevented. However, 2 epidemiological studies seem to show that cutting board cleaning habits have little influence on the incidence of sporadic salmonellosis. Further, one of these studies indicated that use of plastic cutting boards in home kitchens is hazardous, whereas use of wooden cutting boards is not.     

Nonclassical carbocations as C-H hydrogen bond donors

Computed [B3LYP/6-31+G(d,p) and MP2/6-31+G(d,p)] structures and binding energies for complexes of nonclassical cations (carbonium ions) with ammonia, in the gas phase and several solvents, are described. Overall, nonclassical cations are found to be competent C-H hydrogen bond donors. The potential relevance of the C-H...N interactions holding the carbocation.amine complexes together for enzyme-catalyzed terpenoid synthesis is discussed.