Investigation of the Gas-Phase Structure of Electrosprayed Proteins Using Ion-Molecule Reactions

Proton transfer reactions of ammonia, dimemylamine, diethylamine, and trimethylarnine with multiply protonated proteins generated by electrospray ionization (ESI) were examined to probe the relationship between solution and gas-phase protein structure and the relationship with ion-molecule reactivity. The ion-molecule reactions were carried out in an atmospheric pressure capillary inlet/reactor based upon an ESI interface to a quadrupole mass spectrometer. Two types of systems were explored: (1) proteins possessing cysteine-cysteine disulfide bonds and the analogous disulfide-reduced proteins, and (2) proteins sprayed from solution compositions where the protein has different conformations. While the cysteine-cysteine disulfide-bound proteins were more reactive than equally charged disulfide-reduced proteins under these conditions, no significant reactivity differences were noted for ions arising from different solution conformations. The effect of inlet/reactor temperature on charge distributions with and without amine reagent was also explored, demonstrating that thermal denaturation of proteins can occur in heated capillary inlets. The results are discussed in the context of recent results indicating the persistence of at least some higher order protein structure in the gas phase.     

Mössbauer spectra of mixed-valence dimeric iron clusters in charge-ordered crystals

The theory of charge ordering and phase transitions in molecular crystals consisting of dimeric mixed-valence iron clusters is developed. It is supposed that the cluster contains high-spin Fe²⁺ and Fe³⁺ ions. The model employed involves intracluster Heisenberg-type exchange interaction between iron ions, “extra” electron transfer (double exchange), as well as dipole-dipole intercluster interaction. It is shown that depending on the parameters of the abovementioned interactions three types of order parameter (the mean value of the crystal dipole moment) temperature dependence are possible. These types of dependences lead to one-, two- and three-phase transitions. The mechanism of temperature transitions of the type of “localization-delocalization” in the Mössbauer spectra of mixed valence crystals is discussed. It is shown that at low temperatures spectra with averaged parameters may exist.     

Rheological properties of deionized Chinese ink

Rheological properties for Chinese ink in exhaustively deionized aqueous media were carefully examined. In the steady shear measurement, the shear viscosities of the ink could be well explained by considering the “effective” volume fraction of the particles in the ink including the electrical double layers and by using Einstein's equation for dilute suspension viscosity, when the particle volume fraction was substantially low. In the case that the volume fraction was higher, the shear viscosities showed extremely higher than those from Einstein's prediction, though the ink remained a Newtonian liquid. In the stress-strain measurement, the shear moduli were observed at strain smaller than 0.2. The “weak” aggregation among the particles in the ink under no shear or low shear rates was supported. It should be noted that the glue in the suspension plays an important role for the good liquidity of the ink and for the “weak” bridges among the particles resulting its good dispersion stability.     

Electrochemical impedance spectroscopy analysis of chalcopyrite CuFeS2 electrodes

A chalcopyrite CuFeS₂ electrode obtained from the “El Teniente” mine has been studied by Electrochemical Impedance Spectroscopy (EIS) in an alkaline solution for different oxidation potentials. The experimental results can be interpreted from a Randles equivalent circuit, V_dc<0.4 V vs. saturated calomel electrode (SCE), and a surface layer model for V_dc>0.4 V vs. SCE. From these results, the variation with the d.c. applied potentials of charge transfer electrical resistance of the redox reaction, the double layer capacitance and other characteristic parameters are considered.     

The influence of H2SO4 electrolyte concentration on proton transfer resistance of membrane/solution interface

The proton transfer resistance of membrane/solution interface is investigated in this paper by employing H₂SO₄ aqueous solution with different concentration. Two commercial cation exchange membranes, Nafion1135 and PE01 membranes with different ion exchange capacity were selected as test membranes; Proton transfer resistance measurements were made by A.C impedance techniques. The proton transfer resistance of membrane/solution interface increases quickly from 0.059 to 2.22 Ω with the decrease of H₂SO₄ concentration from 2.0 to 0.05 mol/L. The ion exchange capacity of the membrane, or more exactly, the surface charge of the membrane has obviously effect on the membrane/solution resistance due to the formation of electrical double layer (EDL). The effect of electrolyte concentration on membrane/solution interface resistance can be explained by the electrical interactions between ions and charged groups of the membrane: high concentration of ions in the medium can compress the EDL and reduce the electrical interactions between ions and charged groups of the membrane.     

Protein-coated β-ferric hydrous oxide particles: An electrokinetic and electrooptic study

Using microelectrophoresis and electric light scattering techniques, we investigated the adsorption characteristics, surface coverage and surface electric parameters of superstructures from two isoforms of plastocyanin, PCa and PCb, in an oxidized state adsorbed on β-ferric hydrous oxide particles. The surface electric charge and electric dipole moments of the composite particles and the thickness of the protein adsorption layer are determined in a wide pH range, at different ionic strengths and concentration ratios of PC to β-FeOOH. The adsorption of the two proteins was found to shift the particles’ isoelectric point and to alter the total electric charge and the electric dipole moments of the oxide particles to different extent. A “reversal” in the direction of the permanent dipole moment is observed at lower pH for PCb- than for PCa-coated oxide particles. Strict correlation is found between the changes in the electrokinetic charge of the composite particles and the variation in their “permanent” dipole moments. Data suggest that the adsorption of the proteins is driven by electrostatic and/or hydrophobic interactions with the oxide surfaces dependent on pH. The adsorption behaviour is consistent with the involvement of the “eastern” and “northern” patches of the plastocyanin molecules in their adsorption on the oxide surfaces that are differently charged depending on pH.     

On the maximum charge state and proton transfer reactivity of peptide and protein ions formed by electrospray ionization

A relatively simple model for calculation of the energetics of gas-phase proton transfer reactions and the maximum charge state of multiply protonated ions formed by electrospray ionization is presented. This model is based on estimates of the intrinsic proton transfer reactivity of sites of protonation and point charge Coulomb interactions. From this model, apparent gas-phase basicities (GB^app) of multiply protonated ions are calculated. Comparison of this value to the gas-phase basicity of the solvent from which an ion is formed enables a maximum charge state to be calculated. For 13 commonly electrosprayed proteins, our calculated maximum charge states are within an average of 6% of the experimental values reported in the literature. This indicates that the maximum charge state for proteins is determined by their gas-phase reactivity. Similar results are observed for peptides with many basic residues. For peptides with few basic residues, we find that the maximum charge state is better correlated to the charge state in solution. For low charge state ions, we find that the most basic sites Arg, Lys, and His are preferentially protonated. A significant fraction of the less basic residues Pro, Trp, and Gln are protonated in high charge state ions. The calculated GB^app of individual protonation sites varies dramatically in the high charge state ions. From these values, we calculate a reduced cross section for proton transfer reactivity that is significantly lower than the Langevin collision frequency when the GB^app of the ion is approximately equal to the GB of the neutral base.     

Synthesis of multi-unit protein hetero-complexes in the gas phase via ion-ion chemistry

The synthesis of protein hetero-complex ions via ion-ion reactions in the gas phase is demonstrated in a quadrupole ion trap. Bovine cytochrome c cations and bovine ubiquitin anions are used as reactant species in the stepwise construction of complexes containing as many as six protein sub-units. For any set of reactants, a series of competitive and consecutive reactions is possible. The yield of complex ions for any given sequence of reactions is primarily limited by the presence of competitive reactions. Proton transfer represents the most important competitive reaction that adversely affects protein complex synthesis. In the present data, proton transfer takes place most extensively in the first step of complex synthesis, when single protein sub-units are subjected to reaction with one another. Proton transfer is found to be less extensive when one of the reactants is a protein complex. The generation of hexameric hetero-complexes containing two cytochrome c molecules and four ubiquitin molecules is demonstrated with two different synthesis approaches. The first involved the initial reaction of several charge states of cytochrome c and several charges states of ubiquitin. The sequence of reactions in this example illustrates the array of possible competitive and consecutive reactions associated with even a relatively simple set of multiply charged reactants. The second approach involved the initial reaction of the 9(+) charge state of cytochrome c and the 5(-) charge state of ubiquitin. The latter approach highlights the utility of the multi-stage mass spectrometric (MS(n)) capabilities of the ion trap in defining reactant ion identities (i.e. charge states and polarities) so that synthesis reactions can be directed along a particular set of pathways.     

Quantum-classical Liouville dynamics of proton and deuteron transfer rates in a solvated hydrogen-bonded complex

Proton and deuteron transfer reactions in a hydrogen-bonded complex dissolved in a polar solution are studied using quantum-classical Liouville dynamics. Reactive-flux correlation functions that involve quantum-classical Liouville dynamics for species operators and quantum equilibrium sampling are used to calculate the rate constants. Adiabatic and nonadiabatic reaction rates are computed, compared, and analyzed. Large variations of the kinetic isotope effect (KIE) for this reaction have been observed in the literature, which depend on the nature of the approximate calculation used to estimate the proton and deuteron transfer rates. Our estimate of the KIE lies at the low end of the range of previously observed values, suggesting a rather small KIE for this reaction.     

Spectrophotometric and voltammetric study of the interaction between iodine and poly[bis(p-tolylamino)]phosphazene in methylene chloride

The reaction between iodine and poly[bis(p-tolylamino)]phosphazene (PTAP) in methylene chloride was investigated and attributed to a charge-transfer interaction between the imine donor sites of the polymer and the halogen molecules. The mechanism of this process seems to be similar to that already proposed for the charge transfer between p-toluidine and iodine. In particular, the formation of a polar “inner” complex is strongly favoured when a polar substance, such as tetrabutylammonium perchlorate (TBAP), is added to the polymer-iodine system. Even if experimental evidence was not obtained, iodination of the aromatic ring, following the “inner” complex formation, cannot be excluded.     

Gas-phase proton transfer reactions involving multiply charged cytochrome c ions and water under thermal conditions

Investigations of gas-phase proton transfer reactions have been performed on protein molecular ions generated by electrospray ionization (ESI). Their reactions were studied in a heated capillary inlet/reactor prior to expansion into a quadrupole mass spectrometer. Results from investigations involving protonated horse heart cytochrome c and H, O suggest that Coulombit effects can lower reaction barriers as well as aid in entropically driven reactions. For example, the charge state distribution observed by a quadrupole mass spectrometer for multiply protonated cytochrome c without the addition of any reactive gas ranges from 9+ to 19+ , with the [M + 15H]¹⁵⁺ ion being the most intense peak. With the addition of H₂O (proton affinity approximately 170.3±2 kcal/mol) to the capillary reactor at 120°C, the charge state distribution shifts to a lower charge, ranging from 13+ to less than 9+. Under the same conditions with argon (proton affinity approximately 100 kcal/mol) as the reactive gas, no shift in the charge state distribution is observed. The results demonstrate that proton transfer to water can occur for highly protonated molecular ions, a process that would be expected to be highly endothermic for singly protonated molecules (for which Coulombic destabilization is not significant). The results imply that the charge state distribution from ESI is somewhat dependent upon the mechanism and speed of the droplet evaporation/ion desolvation process, which may vary substantially with the ESI/mass spectrometry interface design.     

Further studies on the origins of asymmetric charge partitioning in protein homodimers

Dissociation of gas-phase protonated protein dimers into their constituent monomers can result in either symmetric or asymmetric charge partitioning. Dissociation of alpha-lactalbumin homodimers with 15+ charges results in a symmetric, but broad, distribution of protein monomers with charge states centered around 8+/7+. In contrast, dissociation of the 15+ heterodimer consisting of one molecule in the oxidized form and one in the reduced form results in highly asymmetric charge partitioning in which the reduced species carries away predominantly 11+ charges, and the oxidized molecule carries away 4+ charges. This result cannot be adequately explained by differential charging occurring either in solution or in the electrospray process, but appears to be best explained by the reduced species unfolding upon activation in the gas phase with subsequent separation and proton transfer to the unfolding species in the dissociation complex to minimize Coulomb repulsion. For dimers of cytochrome c formed directly from solution, the 17+ charge state undergoes symmetric charge partitioning whereas dissociation of the 13+ is asymmetric. Reduction of the charge state of dimers with 17+ charges to 13+ via gas-phase proton transfer and subsequent dissociation of the mass selected 13+ ions results in a symmetric charge partitioning. This result clearly shows that the structure of the dimer ions with 13+ charges depends on the method of ion formation and that the structural difference is responsible for the symmetric versus asymmetric charge partitioning observed. This indicates that the asymmetry observed when these ions are formed directly from solution must come about due either to differences in the monomer conformations in the dimer that exist in solution or that occur during the electrospray ionization process. These results provide additional evidence for the origin of charge asymmetry that occurs in the dissociation of multiply charged protein complexes and indicate that some solution-phase information can be obtained from these gas-phase dissociation experiments.     

Origin of asymmetric charge partitioning in the dissociation of gas-phase protein homodimers

The origin of asymmetric charge and mass partitioning observed for gas-phase dissociation of multiply charged macromolecular complexes has been hotly debated. These experiments hold the potential to provide detailed information about the interactions between the macromolecules within the complex. Here, this unusual phenomenon of asymmetric charge partitioning is investigated for several protein homodimers. Asymmetric charge partitioning in these ions depends on a number of factors, including the internal energy, charge state, and gas-phase conformation of the complex, as well as the conformational flexibility of the protein monomer in the complex. High charge states of both cytochrome c and disulfide-reduced alpha-lactalbumin homodimers dissociate by a symmetrical charge partitioning process in which both fragment monomers carry away roughly an equal number of charges. In contrast, highly asymmetric charge partitioning dominates for the lower charge states. Cytochrome c dimer ions with eleven charges formed by electrospray ionization from two solutions in which the solution-phase conformation differs dissociate with dramatically different charge partitioning. These results demonstrate that these gas-phase complexes retain a clear "memory" of the solution from which they are formed, and that information about their solution-phase conformation can be obtained from these gas-phase dissociation experiments. Cytochrome c dimer ions formed from solutions in which the conformation of the protein is native show greater asymmetric charge partitioning with increasing ion internal energy. Cytochrome c dimers that are conformationally constrained with intramolecular cross-linkers undergo predominantly symmetric charge partitioning under conditions where asymmetric charge partitioning is observed for cytochrome c dimers without cross-links. Similar results are observed for alpha-lactalbumin homodimers. These results provide convincing evidence that the origin of asymmetric charge partitioning in these homodimers is the result of one of the protein monomers unfolding in the dissociation transition state. A mechanism that accounts for these observations is proposed.     

The role of organic acids in mineral weathering

Organic acids and their anions (for brevity we shall use the term “acids” to include both) may affect mineral weathering rates by at least three mechanisms: by changing the dissolution rate far from equilibrium through decreasing solution pH or forming complexes with cations at the mineral surface; by affecting the saturation state of the solution with respect to the mineral; and by affecting the speciation in solution of ions such as Al³⁺ that themselves affect mineral dissolution rate. In this paper we review the effects of organic acids on the dissolution rates of silicate minerals, particularly feldspars, under conditions approximating the natural weathering environment −25°C, pH 4–7 and with concentrations of organic acids comparable to those measured in soil solutions.

Feldspar dissolution rates far from equilibrium increase with decreasing pH below pH 4–5. They appear to be independent of pH between pH 4–5 and about 8, and above pH 8 feldspar dissolution rates increase with increasing pH.

Small chelating ligands such as oxalate appear to accelerate feldspar dissolution through complexation of Al at the surface of the mineral. Feldspar dissolution rates in the presence of 1 mM oxalic acid show effects ranging from no enhancement to enhancements of a factor of 15, depending upon the data set, pH, and aluminum content of the mineral: there is a great deal of scatter in the available data. In general, concentrations of oxalate of the order of 1 mM are necessary to cause a significant effect. Humic acids do not appear to increase feldspar dissolution rates significantly.

Dissolution rates must decrease as the solution approaches saturation with respect to the primary phase (the chemical affinity effect). Organic acids will influence chemical affinity by complexing Al (and possibly other elements) in solution and hence decreasing the chemical activity of Al³⁺. There are essentially no data on the effect of chemical affinity on feldspar dissolution rate at 25°C and mildly acid pH, so it is hard to evaluate the importance of organic acids in accelerating silicate dissolution through the chemical affinity effect. The effect of complexation of dissolved Al does not appear to be an important determinant of silicate dissolution rates in nature.

Observed rates of silicate weathering in the field are typically much slower than predicted from laboratory experiments far from equilibrium, suggesting control by transport of solutes between “micropores” and “macropores” (“micropores” include fractures and crystal defects within grains). If such transport is rate-controlling, analysis of the effect of organic acids on weathering rates in nature in terms of dissolution rates far from equilibrium may be misleading.     

Protein structural effects in gas phase ion/molecule reactions with diethylamine.

The relationship between gas-phase protein structure and ion/molecule reactivity is explored in comparisons between native and disulfide-reduced aprotinin, lysozyme, and albumin. Reactions are performed in the atmospheric-pressure inlet to a quadrupole mass spectrometer employing a novel capillary interface-reactor. In reactions with equal concentrations of diethylamine, multiply protonated molecules generated by electrospray ionization (ESI) of 'native' proteins shifted to lower charge states than did multiply protonated molecules from ESI of the disulfide-reduced counterparts, suggesting that the disulfide-reduced protein ions are less reactive than native protein ions of the same charge state. Differences in reactivity may arise from protonation of different amino acid residues and/or differences in the proximities of charge sites in the two molecules. These results suggest that the reactivity of multiply charged proteins can be significantly affected by their gas-phase structure.     

Peculiarities of the excitation energy transfer in europium and terbium aromatic carboxylates and nitrate complexes with sulfoxides: Blocking effect

The influence of modification of the aromatic ligands on the excitation energy transfer to Ln³⁺ ions in europium and terbium carboxylates and nitrates was examined. The luminescence excitation spectra of three groups of the europium and terbium compounds: phenyl-, diphenyl-, triphenylacetates, phenoxyacetates and triphenylpropionates; 1- and 2-naphthylcarboxylates and 2-naphthoxyacetates; lanthanide nitrates with diarylsulfoxides (diphenyl- and dibenzylsulfoxides) and dialkylsulfoxides were investigated. The spectra of adducts of terbium phenylcarboxylates with 1,10-phenanthroline were also analyzed. The effect of the aliphatic bridges, which decouple the π–π- or p–π-conjugation in the ligand, on the energy transfer to Ln³⁺ ions (so-called blocking effect) was investigated. It was shown, that this decoupling leads to significant lowering of the energy of “ligand–metal ion” charge transfer states (LM CTS) in the europium carboxylate salts, just down to 27,800 cm⁻¹ in europium 2-naphthoxyacetate. As a consequence, the probability of the LM CTS participation in the excitation energy dissipation processes increases. A channel of the excitation energy dissipation in the region of 31,750 cm⁻¹ related to ligand electronic transitions was found in the europium and terbium nitrates with sulfoxides. It was demonstrated that a part of the energy absorbed by the aromatic ligand having aliphatic bridge can be emitted as the ligand fluorescence.     

Role of structural flexibility in the fluorescence and photochromism of salicylideneaniline: the general scheme of the phototransformations

The mechanism of light-induced transformation in the salicylideneaniline molecule was studied by semiempirical PM3 calculations. The structures and energies of the minima and saddle points (transition states) on the S₀, S₁ and T₁ potential energy hypersurfaces (PESs) were obtained, together with the gradient lines on the PESs. The structure-energy scheme was compared with the experimental findings. According to the results obtained, the following principle processes are observed: fast S₁ excited state intramolecular proton transfer (ESIPT), followed by typical ESIPT fluorescence; the formation of two S₁ twisted intramolecular charge transfer (TICT) structures which quench the ESIPT fluorescence; the diabatic formation of two ground state metastable coloured “post-TICT” structures responsible for photochromism.     

喹唑啉酮互变异构的理论计算

采用密度泛函理论B3LYP/6-311+G(d,p)方法,计算并考察了喹唑啉酮进行结构互变的质子迁移过程的两种可能途径:(a)分子内质子迁移,(b)水助质子迁移.结果表明,途经b所需要的能垒小,氢键在降低反应能垒方面起重要作用.     

Zur nomenklatur der phane—II

The new term “Phanes” has been clearly defined and a nomenclature system tentatively developed. This system is comprehensible and of general application and at the same time relatively simple. The notations “nucleus”, “bridge”, “number of bridge members” and “number of ring members” are defined. In order to get a definite characterisation of the phanes which contain a carbocyclic nucleus, a carbocyclic and heterocyclic bridge the following terms: “carbophanes”, “carba-phanes” and “hetera-phanes” have been newly introduced. The prefix “hetera-” has been proposed as a general expression and as a representative term for the syllables “aza-”, “oxa-”, “thia-” and so on. The so called “a-nomenclature” is clearly called “hetera-nomenclature”. The new expressions “heteralogous” and “substitulogous” are explained. As the various examples will show, the “Phane-Nomenclature” can also be applied to the naming of complicated metallocenophanes.     

Colloidal forces between bitumen surfaces in aqueous solutions measured with atomic force microscope

Colloidal forces between bitumen surfaces in aqueous solutions were measured with an atomic force microscope (AFM). The results showed a significant impact of solution pH, salinity, calcium and montmorillonite clay addition on both long-range (non-contact) and adhesion (pull-off) forces. Weaker long-range repulsive forces were observed under conditions of lower solution pH, higher salinity and higher calcium concentration. Lower solution pH, salinity and calcium concentration resulted in a stronger adhesion forces. The addition of montmorillonite clays increased long-range repulsive forces and decreased adhesion forces, particularly when co-added with calcium ions. The measured force profiles were fitted with extended DLVO theory to show the repulsive electrostatic double layer and attractive hydrophobic forces being the dominant components in the long-range forces between the bitumen surfaces. At a very short separation distance (less than 4–6 nm), a strong repulsion of steric origin was observed. The findings provide a fundamental understanding of bitumen emulsion stability and a mechanism of bitumen “aeration” in bitumen recovery processes from oil sands.