Primary Dissociation Research Articles

Base-Pairing Energies of Protonated Nucleoside Base Pairs of dCyd and m(5)dCyd: Implications for the Stability of DNA i-Motif Conformations.

Hypermethylation of cytosine in expanded (CCG)n•(CGG)n trinucleotide repeats results in Fragile X syndrome, the most common cause of inherited mental retardation. The (CCG)n•(CGG)n repeats adopt i-motif conformations that are preferentially stabilized by base-pairing interactions of protonated base pairs of cytosine. Here we investigate the effects of 5-methylation and the sugar moiety on the base-pairing energies (BPEs) of protonated cytosine base pairs by examining protonated nucleoside base pairs of 2'-deoxycytidine (dCyd) and 5-methyl-2'-deoxycytidine (m(5)dCyd) using threshold collision-induced dissociation techniques. 5-Methylation of a single or both cytosine residues leads to very small change in the BPE. However, the accumulated effect may be dramatic in diseased state trinucleotide repeats where many methylated base pairs may be present. The BPEs of the protonated nucleoside base pairs examined here significantly exceed those of Watson-Crick dGuo•dCyd and neutral dCyd•dCyd base pairs, such that these base-pairing interactions provide the major forces responsible for stabilization of DNA i-motif conformations. Compared with isolated protonated nucleobase pairs of cytosine and 1-methylcytosine, the 2'-deoxyribose sugar produces an effect similar to the 1-methyl substituent, and leads to a slight decrease in the BPE. These results suggest that the base-pairing interactions may be slightly weaker in nucleic acids, but that the extended backbone is likely to exert a relatively small effect on the total BPE. The proton affinity (PA) of m(5)dCyd is also determined by competitive analysis of the primary dissociation pathways that occur in parallel for the protonated (m(5)dCyd)H(+)(dCyd) nucleoside base pair and the absolute PA of dCyd previously reported.

Quantum chemical characterization of the CF2(OH)CF2OONO2 and CF3CF2OONO2 peroxynitrates and related radicals

We present a detailed study of the molecular conformations, vibrational spectra and thermochemistry of the CF2(OH)CF2OONO2 and CF3CF2OONO2 peroxynitrates. In addition, we studied the CF2(OH)CF2OO, CF2(OH)CF2O, CF3CF2OO and CF3CF2O radicals, formed by the rupture of ON and OO bonds of the above peroxynitrates. The geometric structures of the most stable conformations were determined by density functional theory calculations. At the B3LYP/6-311++G(3df,3pd) level of theory, both peroxynitrates present dihedral angle COON values of about 104°. At the best levels of theory employed, G3(MP2)B3 and G4(MP2), the standard enthalpies of formation at 298K derived from isodesmic reactions, are −265.6, −248.6 and −248.5kcalmol−1 for CF2(OH)CF2OONO2, CF2(OH)CF2OO and CF2(OH)CF2O respectively; while for CF3CF2OONO2, CF3CF2OO and CF3CF2O the values of −268.2, −252.6 and −251.9kcalmol−1 were obtained. Bond dissociation enthalpies of 25.1 and 35.0kcalmol−1 have been predicted for the first time for CF2(OH)CF2OONO2 and CF2(OH)CF2OONO2 and of 23.7 and 34.2kcalmol−1 have been estimated for CF3CF2OONO2 and CF3CF2OONO2. The obtained result of the dissociation enthalpy of CF3CF2OONO2 is in excellent agreement with the available experimental determination. Therefore, the ON bond fission is the primary thermal dissociation pathway for both studied peroxynitrates.

Influence of organelle geometry on the apparent binding kinetics of peripheral membrane proteins.

Information processing in living cells frequently involves an exchange of peripheral membrane proteins between the cytosol and organelle membranes. The typical time scale τ of these association-dissociation cycles is commonly quantified invivo via fluorescence recovery after photobleaching (FRAP). Contrary to common assumptions, we show here that τ values determined by FRAP depend on the size and number of target structures. Hence, FRAP times alone are insufficient to draw conclusions about the proteins' binding kinetics. In contrast, extracting primary molecular association and dissociation rates from FRAP approaches provides a size-independent and therefore robust measure for the proteins' binding kinetics. We support our theoretical considerations with experiments on the small GTPase Arf-1 that transiently associates with Golgi membranes: While Arf-1 recovery times in untreated cells and in cells with disrupted microtubules are significantly different, the molecular kinetic rates are shown to be the same in both cases.

Base-Pairing Energies of Protonated Nucleobase Pairs and Proton Affinities of 1-Methylated Cytosines: Model Systems for the Effects of the Sugar Moiety on the Stability of DNA i-Motif Conformations

Expansion of (CCG)n·(CGG)n trinucleotide repeats leads to hypermethylation of cytosine residues and results in Fragile X syndrome, the most common cause of inherited intellectual disability in humans. The (CCG)n·(CGG)n repeats adopt i-motif conformations that are preferentially stabilized by base-pairing interactions of noncanonical protonated nucleobase pairs of cytosine (C(+)·C). Previously, we investigated the effects of 5-methylation of cytosine on the base-pairing energies (BPEs) using threshold collision-induced dissociation (TCID) techniques. In the present work, we extend our investigations to include protonated homo- and heteronucleobase pairs of cytosine, 1-methylcytosine, 5-methylcytosine, and 1,5-dimethylcytosine. The 1-methyl substituent prevents most tautomerization processes of cytosine and serves as a mimic for the sugar moiety of DNA nucleotides. In contrast to permethylation of cytosine at the 5-position, 1-methylation is found to exert very little influence on the BPE. All modifications to both nucleobases lead to a small increase in the BPEs, with 5-methylation producing a larger enhancement than either 1-methyl or 1,5-dimethylation. In contrast, modifications to a single nucleobase are found to produce a small decrease in the BPEs, again with 5-methylation producing a larger effect than 1-methylation. However, the BPEs of all of the protonated nucleobase pairs examined here significantly exceed those of canonical G·C and neutral C·C base pairs, and thus should still provide the driving force stabilizing DNA i-motif conformations even in the presence of such modifications. The proton affinities of the methylated cytosines are also obtained from the TCID experiments by competitive analyses of the primary dissociation pathways that occur in parallel for the protonated heteronucleobase pairs.

Shock wave study and theoretical modeling of the thermal decomposition of c-C4F8.

The thermal dissociation of octafluorocyclobutane, c-C4F8, was studied in shock waves over the range 1150-2300 K by recording UV absorption signals of CF2. It was found that the primary reaction nearly exclusively produces 2 C2F4 which afterwards decomposes to 4 CF2. A primary reaction leading to CF2 + C3F6 is not detected (an upper limit to the yield of the latter channel was found to be about 10 percent). The temperature range of earlier single pulse shock wave experiments was extended. The reaction was shown to be close to its high pressure limit. Combining high and low temperature results leads to a rate constant for the primary dissociation of k1 = 10(15.97) exp(-310.5 kJ mol(-1)/RT) s(-1) in the range 630-1330 K, over which k1 varies over nearly 14 orders of magnitude. Calculations of the energetics of the reaction pathway and the rate constants support the conclusions from the experiments. Also they shed light on the role of the 1,4-biradical CF2CF2CF2CF2 as an intermediate of the reaction.

Guided ion beam studies of the collision-induced dissociation of CuOH+(H2O)n (n = 1-4): comprehensive thermodynamic data for copper ion hydration.

Threshold collision-induced dissociation (TCID) using a guided ion beam tandem mass spectrometer is performed on CuOH(+)(H2O)n where n = 1-4. The primary dissociation pathway for the n = 2-4 reactants consists of loss of a single water molecule followed by the sequential loss of additional water molecules at higher collision energies. The n = 1 reactant departs from this trend by losing the OH ligand and the H2O ligand competitively. Loss of the OH ligand is thermodynamically favored, whereas H2O loss is the kinetically favored process, consistent with heterolytic cleavage of the dative bond. The data are analyzed using a statistical model after accounting for internal and kinetic energy distributions, multiple collisions, and kinetic shifts to obtain 0 K bond dissociation energies (BDEs). These are also converted using a rigid rotor/harmonic oscillator approximation to yield thermodynamic values at room temperature. Experimental BDEs are compared to theoretical BDEs determined at the B3LYP, cam-B3LYP, B3P86, M06, CCSD(T), and MP2(full) levels of theory with a 6-311+G(2d,2p) basis set using geometries and vibrational frequencies determined at the B3LYP/6-311+G(d,p) level. In addition, BDEs for the loss of OH from CuOH(+)(H2O)n where n = 0, 2-4 are derived using the experimental BDEs for dissociation of CuOH(+)(H2O)n and literature values for Cu(+)(H2O)n.

High-energy collision-activated and electron-transfer dissociation of gas-phase complexes of tryptophan with Na+, K+, and Ca2+

The structure and reactivity of gas-phase complexes of tryptophan (Trp) with Na+, K+, and Ca2+ were examined by high-energy collision-activated dissociation (CAD) and electron transfer dissociation (ETD) using alkali metal targets. In the CAD spectra of M+Trp (M = Na and K), neutral Trp loss was the primary dissociation pathway, and the product ion of collision-induced intracomplex electron transfer from the indole π ring of Trp to the alkali metal ion was observed, indicating a charge-solvated structure in which Trp is non-zwitterionic. The NH3 loss observed in the CAD spectrum of Ca2+Trp2 is ascribed to a CZ (mixed charge-solvated/zwitterionic)-type structure, in which one Trp is non-zwitterionic and the other Trp adopts a zwitterionic structure with an NH 3 + moiety. The H atom and NH3 losses observed in the ETD spectrum of Ca2+Trp2 indicate the formation of a hypervalent radical in the complex, R–NH3, via electron transfer from the alkali metal target to the NH 3 + group of the CZ-type structure. Ca2+ attachment to Trp cluster induces the zwitterionic structure of Trp in the gas phase, and an electron transfer to the zwitterionic Trp forms the hypervalent radical as a reaction intermediate.

CO2dissociation using the Versatile atmospheric dielectric barrier discharge experiment (VADER)

Dissociation of CO2 is investigated in an atmospheric pressure dielectric barrier discharge (DBD) with a simple, zero dimensional (0-D) chemical model and through experiment. The model predicts that the primary CO2 dissociation pathway within a DBD is electron impact dissociation and electron-vibrational excitation. The relaxation kinetics following dissociation are dominated by atomic oxygen chemistry. The experiments included investigating the energy efficiencies and dissociation rates of CO2 within a planar DBD, while the gas flow rate, voltage, gas composition, driving frequency, catalyst, and pulse modes were varied. Some of the VADER results include a maximum CO2 dissociation energy efficiency of 2.5 +/- 0.5%, a maximum CO$_2$ dissociation rate of 4 +/- 0.4*10^-6 mol CO2/s (5 +/- 0.5% percent dissociation), discovering that a resonant driving frequency of ~30 kHz, dependent on both applied voltage and breakdown voltage, is best for efficient CO2 dissociation and that TiO2, a photocatalyst, improved dissociation efficiencies by an average of 18% at driving frequencies above 5 kHz.

Synthesis and characterization of novel tetra cyclo[4]pyrrole ether as an anion recognition element for nanocomposite nitrate ion selective carbon paste electrode

Novel tetra cyclo[4]pyrrole ether cation (C4PE) was synthesized and characterized by different spectroscopy methods. Cyclo[4]pyrrole ether was synthesized by oxidation reaction of pyrrole with Cu(II) nitrate in acetonitrile. Conductometric data in acetonitrile shows C4PE behaves as a divalent cation salt with two nitrate ions undergoing primary and secondary dissociations. C4PE was used as a recognition element in the form of a composite with carbon paste as a selective electrode for the potentiometry sensing of nitrate in water samples. The electrode has a linear response to nitrate with a detection limit of 1×10−5molL−1 and exhibit a Nernstian slope (57.5±0.6mV/decade) between pH 3.6 and 9.6 with a fast response time less than 10s. The selectivity coefficient values indicate high selectivity for nitrate ions over various anions (NO3>NO2−>Cl−>Br−>I−>F−>CH3COO−>SO42−>IO3−>ClO4−). The electrodes were used over a period of 60 days with good reproducibility. The analytical usefulness of the proposed electrode has been evaluated by its application in the determination of nitrate ions in mineral and drinking water samples.

Control of growth process for obtaining high-quality a-SiO:H

Film-growth process of hydrogenated amorphous silicon–oxygen alloys (a-SiO:H) from CO2/(CO2 + SiH4) plasma has been investigated to control the optoelectronic properties in the resulting materials. Optical emission spectroscopy results and simple simulation results for steady-state density of chemical species in the plasma indicate that main film-growth precursors for a-SiO:H are SiH3, OH, and O. Si dangling-bond defect density is drastically increased in a-SiO:H when increasing the CO2 gas ratio in CO2/(CO2 + SiH4) plasma, being caused by the increase in the contribution ratio of Si-related short-lifetime species (SiHx, x < 2) to film growth owing to a severe SiH4-molecule depletion because of high-rate consumption reaction of SiH4 with O produced from CO2 in the plasma. Considering the primary electron impact dissociation reactions of source gas molecules and several secondary chemical reactions in the plasma, the guiding principle for obtaining high quality a-SiO:H has been proposed.

Shock wave study of the thermal dissociations of C3F6 and c-C3F6. I. dissociation of hexafluoropropene.

The thermal dissociation of C3F6 was studied between 1330 and 2210 K in shock waves monitoring the UV absorption of CF2. CF2 yields of about 2.6 per parent C3F6 were obtained at reactant concentrations of 500-1000 ppm in the bath gas Ar. These yields dropped to about 1.8 when reactant concentrations were lowered to 60 ppm. The increase of the CF2 yield with increasing concentration was attributed to bimolecular reactions between primary and secondary dissociation products. Quantum-chemical and kinetic modeling calculations helped to estimate the contributions from the various primary dissociation steps. It was shown that the measurements correspond to unimolecular reactions in their falloff range. Falloff representations of the rate constants are given, leading to an overall high pressure rate constant k∞ = 2.0 × 10(17)(-104 kcal mol(-1)/RT) s(-1) and a relative rate of about 2/3:1/3 for the reactions C3F6 → CF3CF + CF2 versus C3F6 → C2F3 + CF3.

Study on thermal decomposition and oxidation kinetics of cation exchange resins using non-isothermal TG analysis

Kinetics of two successive thermal decomposition reaction steps of cationic ion exchange resins and oxidation of the first thermal decomposition residue were investigated using a non-isothermal thermogravimetric analysis. Reaction mechanisms and kinetic parameters for three different reaction steps, which were identified from a FTIR gas analysis, were established from an analysis of TG analysis data using an isoconversional method and a master-plot method. Primary thermal dissociation of SO3H+ from divinylbenzene copolymer was well described by an Avrami–Erofeev type reaction (n = 2, g(α) = [−ln(1 − α)]1/2]), and its activation energy was determined to be 46.8 ± 2.8 kJ mol−1. Thermal decomposition of remaining polymeric materials at temperatures above 400 °C was described by one-dimensional diffusion (g(α) = α 2), and its activation energy was determined to be 49.1 ± 3.1 kJ mol−1. The oxidation of remaining polymeric materials after thermal dissociation of SO3H+ was described by a phase boundary reaction (contracting volume, g(α) = 1−(1 − α)1/3). The activation energy and the order of oxygen power dependency were determined to be 101.3 ± 13.4 and 1.05 ± 0.17 kJ mol−1, respectively.

Dimetal hydride generated from dimetalated glycine: Formation mechanism and electronic structure

Collision-induced dissociation (CID) of [MaMb(Gly-H)]+ precursors (where Ma=Ag, Cu; Mb=Ag, Cu) results in formation of novel dimetal hydrides, [MaHMb]+ and elimination of MaH in the gas phase. Deuterium labeling studies reveal that the hydride only originates from the CH2 group. Energy-resolved CID of [Ag2(Gly-H)]+ shows that the formation of [Ag2H]+ and the elimination of AgH are two primary dissociation channels, and the latter is always more prevalent than the former. H2 loss, only a minor channel, was also observed. This was confirmed by the correlation of the relative abundance of dimetal hydride with the difference of silver ion affinity between the neutral (Gly-2H) and AgH. Dimetal hydrides are of particular interest due to the three-center two-electron (3c–2e) bond. DFT calculations predict that [Cu2H]+ and [CuAgH]+ have bent structures, while [Ag2H]+ has linear structures. However, bending deformation energies are only 2.6kcal/mol for [Cu2H]+. Population analysis and atom in molecule (AIM) calculations show that in [Cu2H]+ the metal–metal bond is present, while for [Ag2H]+ there is no metal–metal bond. Energies from high-level theory calculations provide direct evidence to explain why the dimetal radical cation cannot be observed in the CID of the dimetal hydride.

Thermodynamics and Mechanism of Protonated Cysteine Decomposition: A Guided Ion Beam and Computational Study

A quantitative molecular description of the decomposition of protonated cysteine, H(+)Cys, is provided by studying the kinetic energy dependence of threshold collision-induced dissociation (CID) with Xe using a guided ion beam tandem mass spectrometer (GIBMS). Primary dissociation channels are deamidation (yielding both NH3 loss and NH4(+) formation) and (H2O + CO) loss reactions, followed by an additional six subsequent decompositions. Analysis of the kinetic energy-dependent CID cross sections provides the 0 K barriers for six different reactions after accounting for unimolecular decay rates, internal energy of reactant ions, multiple ion-molecule collisions, and competition among the decay channels. To identify the mechanisms associated with these reactions, quantum chemical calculations performed at the B3LYP/6-311 + G(d,p) level were used to locate the transition states (TSs) and intermediates for these processes. Single point energies of the reactants, products, and key optimized TSs and intermediates are calculated at B3LYP, B3P86, and MP2(full) levels using a 6-311 + G(2d,2p) basis set. The computational characterization of the elementary steps of these reactions, including the structures of the final products, is validated by quantitative agreement with the experimental energetics. In agreement with previous work, deamidation is facilitated by anchimeric assistance of the thio group, which also leads to an interesting rearrangement of the intact amino acid identified computationally.

A theoretical study of the ozonolysis of C60: primary ozonide formation, dissociation, and multiple ozone additions

We present an investigation of the reaction of ozone with C60 fullerene using electronic structure methods. Motivated by recent experiments of ozone exposure to a C60 film, we have characterized stationary points in the potential energy surface for the reactions of O3 with C60 that include both the formation of primary ozonide and subsequent dissociation reactions of this intermediate that lead to C-C bond cleavage. We have also investigated the addition of multiple O3 molecules to the C60 cage to explore potential reaction pathways under the high ozone flux conditions used in recent experiments. The lowest-energy product of the reaction of a single ozone molecule with C60 that results in C-C bond breakage corresponds to an open-cage C60O3 structure that contains ester and ketone moieties at the seam. This open-cage product is of much lower energy than the C60O + O2 products identified in prior work, and it is consistent with IR experimental spectra. Subsequent reaction of the open-cage C60O3 product with a second ozone molecule opens a low-energy reaction pathway that results in cage degradation via the loss of a CO2 molecule. Our calculations also reveal that, while full ozonation of all bonds between hexagons in C60 is unlikely even under high ozone concentration, the addition of a few ozone molecules to the C60 cage is favorable at room temperature.

Molecular halogen elimination from halogen-containing compounds in the atmosphere

Atmospheric halogen chemistry has drawn much attention, because the halogen atom (X) playing a catalytic role may cause severe stratospheric ozone depletion. Atomic X elimination from X-containing hydrocarbons is recognized as the major primary dissociation process upon UV-light irradiation, whereas direct elimination of the X2 product has been seldom discussed or remained a controversial issue. This account is intended to review the detection of X2 primary products using cavity ring-down absorption spectroscopy in the photolysis at 248 nm of a variety of X-containing compounds, focusing on bromomethanes (CH2Br2, CF2Br2, CHBr2Cl, and CHBr3), dibromoethanes (1,1-C2H4Br2 and 1,2-C2H4Br2) and dibromoethylenes (1,1-C2H2Br2 and 1,2-C2H2Br2), diiodomethane (CH2I2), thionyl chloride (SOCl2), and sulfuryl chloride (SO2Cl2), along with a brief discussion on acyl bromides (BrCOCOBr and CH2BrCOBr). The optical spectra, quantum yields, and vibrational population distributions of the X2 fragments have been characterized, especially for Br2 and I2. With the aid of ab initio calculations of potential energies and rate constants, the detailed photodissociation mechanisms may be comprehended. Such studies are fundamentally important to gain insight into the dissociation dynamics and may also practically help to assess the halogen-related environmental variation.

Strong Photon Energy Dependence of the Photocatalytic Dissociation Rate of Methanol on TiO2(110)

Photocatalytic dissociation of methanol (CH3OH) on a TiO2(110) surface has been studied by temperature programmed desorption (TPD) at 355 and 266 nm. Primary dissociation products, CH2O and H atoms, have been detected. The dependence of the reactant and product TPD signals on irradiation time has been measured, allowing the photocatalytic reaction rate of CH3OH at both wavelengths to be directly determined. The initial dissociation rate of CH3OH at 266 nm is nearly 2 orders of magnitude faster than that at 355 nm, suggesting that CH3OH photocatalysis is strongly dependent on photon energy. This experimental result raises doubt about the widely accepted photocatalysis model on TiO2, which assumes that the excess potential energy of charge carriers is lost to the lattice via strong coupling with phonon modes by very fast thermalization and the reaction of the adsorbate is thus only dependent on the number of electron-hole pairs created by photoexcitation.

Unimolecular dissociation characteristics of cationic complexes between nicotinic acid and Cu(II) and Ni(II)

Cu2+ and Ni2+ form dimeric ML(L−H)+ complexes with nicotinic acid (M=Cu, Ni; L=nicotinic acid) upon electrospray ionization. Quantum chemical calculations indicate thermochemical preference for coordination of the carboxylate groups rather than the ring nitrogens to the central metal ion in both cases. In analogy to the dimeric metal complexes of amino acids the primary dissociation reaction upon collisional activation of ML(L−H)+ is the loss of CO2 in both cases. Further dissociation of the decarboxylated species show preference for loss of a 3-pyridinyl radical for M=Cu and NiCO2 for M=Ni. This can be understood in light of the redox properties of the two metals and from previous studies of similar complexes with amino acids. Loss of the pyridinyl radical bonded to the carboxylate group in these cationic entities does not lead to M(η2-O2C) structures previously observed for similar anionic metal species.

Sialylated ligands interact in cis to modulate PILRα inhibitory receptor in myeloid cells (P1358)

Abstract Paired Ig-like type 2 receptor alpha (PILRα) is an inhibitory receptor expressed in myeloid cells. We show that an evolutionary conserved arginine-containing domain is critical for PILRα interaction with its sialylated ligands. In addition, PILRα is pre-engaged with its ligands in cis on the surface of primary cells and dissociation of PILRa-ligand interactions by sialidase-A treatment unmasks PILRα to bind soluble ligands. At the cellular level, the engagement of PILRα with monoclonal antibody suppresses the production of proinflammatory cytokines. Our data provides novel insights into nature of PILRα interactions and means to modulate the immune response by agonizing this inhibitory pathway.

The Effect of a Covalent and a Noncovalent Small-Molecule Inhibitor on the Structure of Abg β-Glucosidase in the Gas-Phase

The effects of binding two small-molecule inhibitors to Agrobacterium sp. strain ATCC 21400 (Abg) β-glucosidase on the conformations and stability of gas-phase ions of Abg have been investigated. Biotin-iminosugar conjugate (BIC) binds noncovalently to Abg while 2,4-dinitro-2-deoxy-2-fluoro-β-D-glucopyranoside (2FG-DNP) binds covalently with loss of DNP. In solution, Abg is a dimer. Mass spectra show predominantly dimer ions, provided care is taken to avoid dissociation of dimers in solution and dimer ions in the ion sampling interface. When excess inhibitor, either covalent or noncovalent, is added to solutions of Abg, mass spectra show peaks almost entirely from 2:2 inhibitor-enzyme dimer complexes. Tandem mass spectrometry experiments show similar dissociation channels for the apo-enzyme and 2FG-enzyme dimers. The +21 dimer produces +10 and +11 monomers. The internal energy required to dissociate the +21 2FG-enzyme to its monomers (767 ± 30 eV) is about 36 eV higher than that for the apo-enzyme dimer (731 ± 6 eV), reflecting the stabilization of the free enzyme dimer by the 2FG inhibitor. The primary dissociation channels for the noncovalent BIC-enzyme dimer are loss of neutral and charged BIC. The internal energy required to induce loss of BIC is 482 ± 8 eV, considerably less than that required to dissociate the dimers. For a given charge state, ions of the covalent and noncovalent complexes have about 15 % and 25 % lower cross sections, respectively, compared with the apo-enzyme. Thus, binding the inhibitors causes the gas-phase protein to adopt more compact conformations. Noncovalent binding surprisingly produces the greatest change in protein ion conformation, despite the weaker inhibitor binding. ᅟ