Qualitative and quantitative characterization of the arsenic‐binding behaviour of sulfur‐containing peptides and proteins by the coupling of reversed phase liquid chromatography to electrospray ionization mass spectrometry

Phenylarsenic‐substituted cysteine‐containing peptides and proteins were completely differentiated from their unbound original forms by the coupling of reversed phase liquid chromatography with electrospray ionization mass spectrometry. The analysis of biomolecules possessing structure‐stabilizing disulfide bridges after reduction provides new insights into requirements concerning the accessibility of cysteine residues for reducing agents as well as for arsenic compounds in a spatial protein structure. Complementary binding studies performed using direct ESI‐MS without chromatographic coupling in different solvent systems demonstrated that more than one binding site were activated for aprotinin and lysozyme in denaturing solvents because of a stronger defolding. From the intensities of the different charge states occurring in the mass spectra as well as from the LC elution behaviour, it can be deduced that the folding state of the arsenic‐bound protein species resembles the native, oxidized conformation. In contrast, although the milk protein α‐lactalbumin has several disulfide bridges, only one phenylarsenic moiety was bound under strongly denaturing conditions. Because of the charge state distribution in the ESI mass spectra, a conformational change to a molten globule structure is assumed. For the second considered milk protein ß‐lactoglobulin, a noncovalent interaction with phenylarsine oxide was detected. In general, smaller apparent binding constants for the condensation reactions of the biomolecules with phenylarsine oxide leading to covalent arsenic–sulfur bindings were determined from direct injection ESI‐MS measurements than from LC‐ESI‐MS coupling. The following order of binding affinities for one phenylarsenic group can be assumed from both ESI‐MS and LC‐ESI‐MS: nonapeptide vasopressin > nonapeptide vasotocin > lysozyme > aprotinin > α‐lactalbumin > thioredoxin. Kinetic investigations by LC‐ESI‐MS yielded a partial reaction order of 2 for vasopressin, Lys and α‐lactalbumin and corresponding half‐lives of 0.93, 2.56 and 123.5 min, respectively. Copyright © 2012 John Wiley & Sons, Ltd.     

Characterization of a peptide affinity support that binds selectively to staphylococcal enterotoxin B

The influences of mass transfer and adsorption-desorption kinetics on the binding of staphylococcal enterotoxin B (SEB) to an affinity resin with the peptide ligand, Tyr-Tyr-Trp-Leu-His-His (YYWLHH) have been studied. The bed and particle porosities, the axial dispersion coefficient and the pore diffusivity were measured using pulse experiments under unretained conditions. Adsorption isotherms for SEB on YYWLHH resins with peptide densities in the range from 6 to 220 micromol/g were measured and fitted to a bi-Langmuir equation. At peptide densities below 9 micromol/g and above 50 micromol/g, dissociation constants were lower (2 x 10(-3) to 7 x 10(-3) mol/m3), and binding capacities were larger (43-47 mg SEB/g). In the range from 9 to 50 micromol/g dissociation constants were larger (13 x 10(-3) to 24 x 10(-3) mol/m3) and capacities were lower (33-37 mg SEB/g). These observations are consistent with a transition from single point attachment of the protein to the ligand at low peptide densities to multipoint attachment at high peptide densities. The general rate (GR) model of chromatography was used to fit experimental breakthrough curves under retained conditions to determine the intrinsic rate constants for adsorption, which varied from 0.13 to 0.50 m3 mol(-1) s(-1), and exhibited no clear trend with increasing peptide density. An analysis of the number of transfer units for the various mass transfer steps in the column indicated that film mass transfer, pore diffusion (POR) and the kinetics of adsorption can all play an important role in the overall rate of adsorption, with the intrinsic adsorption step apparently being the rate determining step at peptide densities below 50 micromol/g.     

Some critical aspects in the determination of binding constants by electrospray ionisation mass spectrometry at the example of arsenic bindings to sulphur‐containing biomolecules

The influences of reactant concentrations, solvent type, acid strength, pH conditions and ionic strength on the determination of apparent gas‐phase equilibrium constants K using electrospray ionisation mass spectrometry (ESI‐MS) were elucidated. As example serves the interaction of the tripeptide glutathione (GSH) with phenylarsine oxide (PAO). It was shown that rising initial concentrations of both reactants were not adequately compensated by increasing signal intensities of the reaction products in the mass spectra. The equilibrium constant for the formation of the phenylarsenic‐substituted peptide species decreased from 1.42 × 10⁵ ± 1.81 × 10⁴ l µmol^?1 to 1.54 × 10⁴ ± 1.5 × 10³ l µmol^?1 with rising initial GSH concentrations from 1 to 10 µM at fixed PAO molarity of 50 µM . K values resulting from a series with a fixed GSH molarity of 5 µM and a PAO molarity varied from 10 to 100 µM remained in a narrower range between 4.59 × 10⁴ ± 2.15 × 10⁴ l µmol^?1 and 1.07 × 10⁴ ± 4.0 × 10³ l µmol^?1. In contrast, consumption numbers calculated from the ion intensity ratios of reaction products to the unreacted peptide were not influenced by the initial reactant concentrations. In a water–acetonitrile–acetic acid mixture (48:50:2, v:v), the consumption of 5 µ M GSH increased from 8.3 ± 1.4% to 39.6 ± 1.6% with increased molar excess of PAO from 2 to 20, respectively. The GSH consumption was considerably enhanced in a changed solvent system consisting of 25% acetonitrile and 75% 10 mM ammonium formate, pH 5.0 (v:v) up to 80% of the original peptide amount at an only threefold molar arsenic excess. Copyright © 2010 John Wiley & Sons, Ltd.     

电喷雾质谱法研究谷胱甘肽与L型芳香性氨基酸非共价复合物

为探索谷胱甘肽和L型芳香性氨基酸的非共价相互作用, 将一定化学剂量比的还原型γ-谷胱甘肽分别与L型芳香性氨基酸(包括苯丙氨酸、酪氨酸和色氨酸)在室温和生理pH条件下混合后, 温育1 h, 生成非共价复合物, 并使反应完全. 电喷雾质谱测量结果揭示谷胱甘肽和L型芳香性氨基酸反应可以生成非共价复合物. 在二级串级质谱MS2测得的复合物碎片离子峰中, 除芳香性氨基酸离子峰外, 还包括谷胱甘肽及其它再次碎裂产生的b2和y2碎片离子, 进一步确认了非共价复合物的形成. 紫外光谱也证实了电喷雾质谱的实验结果. 为避免严重的离子化效率差异和质谱信号的相互抑制作用, 定量评估了谷胱甘肽和酪氨酸的相互作用, 结果显示反应物的初始浓度应该选择在5×10-5~3.00×10-4 mol/L范围内. 用质谱滴定法测定了谷胱甘肽与3个芳香性氨基酸非共价复合物的解离常数, 结果表明, 谷胱甘肽复合物的稳定性按Tyr, Trp和Phe次序依次增大.     

Studying aminoglycoside antibiotic binding to HIV-1 TAR RNA by electrospray ionization mass spectrometry

The recognition of the aminoglycosides neomycin and streptomycin by HIV-1 TAR RNA was studied by electrospray ionization mass spectrometry (ESI-MS). Members of the aminoglycoside family of antibiotics are known to target a wide variety of RNA molecules. Neomycin and streptomycin inhibit the formation of the Tat protein–TAR RNA complex, an assembly that is believed to be necessary for HIV replication. The noncovalent complexes formed by the binding of aminoglycosides to TAR RNA and the Tat–TAR complex were detected by ESI-MS. Neomycin has a maximum binding stoichiometry of three and two to TAR RNA and to the Tat–TAR complex, respectively. Data from the ESI-MS experiments suggest that a high affinity binding site of neomycin is located near the three-nucleotide bulge region of TAR RNA. This is consistent with previous solution phase footprinting measurements [H.-Y. Mei et al., Biochemistry 37 (1998) 14204]. Neomycin has a higher affinity toward TAR RNA than streptomycin, as measured by ESI-MS competition binding experiments. A noncovalent complex formed between a small molecule inhibitor of TAR RNA, which has a similar solution binding affinity as the aminoglycosides, and TAR RNA is much less stable than the RNA–aminoglycoside complexes to collisional dissociation in the gas phase. It is believed that the small molecule inhibitor interacts with TAR RNA via hydrophobic interactions, whereas the aminoglycosides bind to RNAs through electrostatic forces. This difference in gas phase stabilities may prove useful for discerning the types of noncovalent forces holding complexes together.     

Gas-Phase Binding of Noncovalent Complexes Between α-cyclodextrin and Amino Acids Investigated by Mass Spectrometry

Noncovalent complexes between cyclodextrins and small molecules have been extensively studied recently because of their widespread application in the pharmaceutical industry for chiral and molecular recognition. To date, gas phase noncovalent binding affinities between α-cyclodextrin and amino acids have not been widely investigated. In this study, gas-phase binding of noncovalent complexes between α-CD and amino acids was investigated by electrospray ionization mass spectrometry (ESI-MS), demonstrating the formation of 1:1 stoichiometric noncovalent complexes. The binding of the complexes were further confirmed by collision-induced dissociation by tandem mass spectrometry. Mass spectrometric titrations between α-cyclodextrin and phenylalanine, glutamic acid, and arginine were performed to provide binding constants (lgK_a) as references for competitive ESI-MS. Calibration curves for the complexes of α-cyclodextrin with phenylalanine, glutamic acid, and arginine were plotted. Through competitive ESI-MS, the lgK_a for the complexes of α-CD with aspartic acid, lysine, proline, glycine, alanine, asparagine, cystine, glutamine, histidine, leucine, isoleucine, methionine, serine, threonine, and valine were measured directly. By comparison, it is seen that the measured binding constants for the complexes of α-cyclodextrin with basic amino acids such as arginine and lysine are lower than those for most complexes of neutral amino acids. The chiral selectivity of α-cyclodextrin for L- and D-isomers of methionine, threonine, asparagine, and phenylalanine determined by ESI-MS revealed its application as a chiral selector.     

谷胱甘肽与D型氨基酸非共价复合物的质谱

为了研究谷胱甘肽和D型氨基酸的非共价相互作用, 将一定化学剂量比的还原型谷胱甘肽与D-苯丙氨酸、D-组氨酸或D-谷氨酰胺在室温下混合后, 温育1 h, 使反应达到平衡. 电喷雾质谱测量结果表明, 在生理pH条件下, 谷胱甘肽可以和D 型氨基酸经反应生成非共价复合物. 串级质谱中的碰撞诱导解离(CID)以及紫外光谱进一步确认了非共价复合物的生成. 为了避免严重的离子化效率和质谱信号相互抑制作用, 对谷胱甘肽和D-谷氨酰胺的相互作用作了定量的评估. 配制一系列不同初始浓度的谷胱甘肽和D型氨基酸的混合溶液, 并用电喷雾质谱测定混合溶液中不同质点的质谱峰强度, 计算了谷胱甘肽与三个D型氨基酸结合形成的复合物的解离常数. 计算结果表明, 谷胱甘肽可以和D型氨基酸结合形成不同键合强度的非共价复合物, 其稳定性按照D-谷氨酰胺、D-苯丙氨酸、D-组氨酸的次序逐渐增大.     

Complexation of phenols and thiophenol by phosphine oxides and phosphates. Extraction, isothermal titration calorimetry, and ab initio calculations

To develop a new solvent-impregnated resin system for the removal of phenols from water the complex formation of triisobutylphosphine sulfide (TIBPS), tributylphosphate (TBP), and tri-n-octylphosphine oxide (TOPO) with a series of phenols (phenol, thiophenol, 3-chlorophenol, 3,5-dichlorophenol, 4-cyanophenol, and pentachlorophenol) was studied. The investigation of complex formation between the extractants and the phenols in the solvent toluene was carried out using liquid-liquid extraction, isothermal titration calorimetry (ITC), and quantum chemical modeling (B3LYP/6-311+G(d,p)//B3LYP/6-311G(d,p) and MP2/6-311++G(2d,2p)//B3LYP/6-311G(d,p)). The equilibrium constant (binding affinity, Kchem), enthalpy of complex formation (DeltaH), and stoichiometry (N) were directly measured with ITC, and the entropy of complexation (DeltaS) was derived from these results. A first screening of K chem toward phenol revealed a very high binding affinity for TOPO, and very low binding affinities for the other extractants. Modeling results showed that although 1:1 complexes were formed, the TIBPS and TBP do not form strong hydrogen bonds. Therefore, in the remainder of the research only TOPO was considered. Kchem of TOPO for the phenols in toluene increased from 1,000 to 10,000 M(-1) in the order phenol < pentachlorophenol < 3-chlorophenol < 4-cyanophenol approximately 3,5-dichlorophenol (in line with their pKa values, except for pentachlorophenol) in the absence of water, while the stoichiometric ratio remained 1:1. In water-saturated toluene, the binding affinities are lower due to co-complexation of water with the active site of the extractant. The increase in binding affinity for TOPO in the phenol series was confirmed by a detailed ab initio study, in which Delta H was calculated to range from -10.7 kcal/mol for phenol to -13.4 kcal/mol for 4-cyanophenol. Pentachlorophenol was found to behave quite differently, showing a DeltaH value of -10.5 kcal/mol. In addition, these calculations confirm the formation of 1:1 H-bonded complexes.     

Evaluation of binding affinity of protein-mutant dna complexes in solution by laser spray mass spectrometry

We have applied laser spray mass spectrometry developed by Hiraoka et al. to investigate the binding affinity of protein-mutant DNA complexes. The results were compared with our previous data of collision-induced dissociation (CID) experiments using electrospray ionization mass spectrometry (ESI-MS). Systematic experiments were carried out on the complexes of the c-Myb DNA binding domain (c-Myb DBD) bound to eight kinds of 16- or 22-mer point mutant double-stranded DNA (dsDNA), whose solution K(d) values are different in the range from 10(-9) M to 10(-7) M. The dissociation curve as a function of laser power was plotted for each complex, and the laser power where 50% of complex was dissociated (E(50%)) in population was obtained. The correlation coefficient between E(50%) and the relative binding free-energy change (DeltaDeltaG) of each complex formation in solutions was 0.9808, which is much better than the coefficient obtained by the previous ESI-CID experiments that was 0.859. In addition, complexes of the c-Myb DBD with five other mutant dsDNA were also examined to confirm that laser spray can be used to estimate the K(d) values of a DNA-protein complex in solutions if an appropriate calibration curve is available. In the process of laser spray, dissociations of these noncovalent complexes occur in solutions, but not in the gas phase. This differs greatly from ESI-CID. Laser spray mass spectrometry has been found to be better than ESI-CID in evaluating binding affinity of a protein to various mutant DNA.     

Protonation thermochemistry of aminoacetonitrile

The gas-phase basicity (GB) of aminoacetonitrile (NH2CH2CN, 1) has been determined from measurement of proton transfer equilibrium constants in an ion cyclotron resonance mass spectrometer (GB(1) = 789.3 +/- 1.0 kJ x mol(-1)). Molecular orbital calculations up to the G2 level demonstrate that protonation occurs preferentially on the nitrogen atom of the NH2 group, and provide a theoretical proton affinity (PA(1)) of 824.0 kJ x mol(-1). Exact calculation of the entropy associated with hindered rotations and consideration of Boltzman distribution of conformers allow a theoretical estimate of the molar protonation entropy S degrees (1H+) - S degrees (1) = 8.6 J x mol(-1) x K(-1). Combining this value with experimental GB(1) leads to an 'experimental' proton affinity of 819.2 kJ x mol(-1), in close agreement with the G2 expectation.     

High-relaxivity magnetic resonance imaging (MRI) contrast agent based on supramolecular assembly between a gadolinium chelate, a modified dextran, and poly-beta-cyclodextrin

Nanosized contrast agents have great potential in magnetic resonance molecular imaging applications for clinical diagnosis. This study proposes new nanoparticles spontaneously formed under mild conditions and composed of a noncovalent adduct between a gadolinium complex, a polymer of beta-cyclodextrin (pbetaCD: MW 1.5 x 10(6) g mol(-1)) and a dextran grafted with alkyl chains (MD). The formation of this supramolecular nanoassembly is based upon a "lock-and-key" recognition process in which the hydrophobic alkyl chains of MD and the adamantyl moieties of macrocyclic Gd(III) chelates are included in the cavities of pbetaCD. The large number of betaCDs contained in the pbetaCD resulted in the formation of 200 nm diameter nanoparticles, each entrapping 1.8 x 10(5) molecules of a low-molecular-weight Gd complex. This system, which exhibits a great relaxivity enhancement (48.4 mM(-1) s(-1), at 20 MHz and 37 degrees C) compared to the Gd(III) chelate itself (5.2 mM(-1) s(-1)), appears to be a promising strategy for the in vivo targeted delivery of Gd(III) complexes. The mechanisms of particle formation, conjugation strategies, and relaxometric characterizations in the field of contrast-enhanced magnetic resonance imaging are discussed.     

Fluorimetric and mass spectrometric study of the interaction of beta-cyclodextrin and osthole

The inclusion complex of beta-cyclodextrin (beta-CD) and osthole was studied by the electrospray ionization mass spectrometry (ESI-MS) and fluorescence spectrometry. From the mass spectrum, the 1:1 stoichiometric inclusion complex of beta-CD and osthole was observed. The tandem mass spectrum was performed. The fluorescence intensity of osthole increased in the present of beta-CD. According to the 1:1 beta-CD-osthole mode, the dissociation constant (KD) was obtained by ESI-MS and fluorescence spectrometry. The KD of beta-CD-osthole inclusion complex is 6.96 x 10(-3) mol L(-1) obtained by mass spectrometry and that is 8.14 x10 (-3) mol L(-1) obtained by fluorescence spectrometry, which is consistent with each other.     

Biomimetic interactions of proteins with functionalized nanoparticles: a thermodynamic study

Gold nanoparticles (NPs) functionalized with L-amino acid-terminated monolayers provide an effective platform for the recognition of protein surfaces. Isothermal titration calorimetry (ITC) was used to quantify the binding thermodynamics of these functional NPs with alpha-chymotrypsin (ChT), histone, and cytochrome c (CytC). The enthalpy and entropy changes for the complex formation depend upon the nanoparticle structure and the surface characteristics of the proteins, e.g., distributions of charged and hydrophobic residues on the surface. Enthalpy-entropy compensation studies on these NP-protein systems indicate an excellent linear correlation between DeltaH and TDeltaS with a slope (alpha) of 1.07 and an intercept (TDeltaS0) of 35.2 kJ mol(-1). This behavior is closer to those of native protein-protein systems (alpha = 0.92 and TDeltaS0 = 41.1 kJ mol(-1)) than other protein-ligand and synthetic host-guest systems.     

Characterization of non-covalent complexes of rutin with cyclodextrins by electrospray ionization tandem mass spectrometry

Electrospray ionization tandem mass spectrometry (ESI-MS(n)) and the phase solubility method were used to characterize the gas-phase and solution-phase non-covalent complexes between rutin (R) and alpha-, beta- and gamma-cyclodextrins (CDs). The direct correlation between mass spectrometric results and solution-phase behavior is thus revealed. The order of the 1 : 1 association constants (K(c)) of the complexes between R and the three CDs in solution calculated from solubility diagrams is in good agreement with the order of their relative peak intensities and relative collision-induced dissociation (CID) energies of the complexes under the same ESI-MS(n) condition in both the positive and negative ion modes. Not only the binding stoichiometry but also the relative stabilities and even binding sites of the CD-R complexes can be elucidated by ESI-MS(n). The diagnostic fragmentation of CD-R complexes, with a significant contribution of covalent fragmentation of rutin leaving the quercetin (Q) moiety attached to the CDs, provides convincing evidence for the formation of inclusion complexes between R and CDs. The diagnostic fragment ions can be partly confirmed by the complexes between Q and CDs. The gas-phase stability order of the deprotonated CD-R complexes is beta-CD-R > alpha-CD-R > gamma-CD/R; beta-CD seems to bind R more strongly than the other CDs.     

Determination of dissociation constants of cyclodextrin-ligand inclusion complexes by electrospray ionization mass spectrometry

The inclusion complexes of four ligands binding to cyclodextrins (CDs) were studied by electrospray ionization mass spectrometry (ESI-MS) and the dissociation constants of the complexes were obtained. The 1:1 stoichiometric inclusion complex was found in the system of CD and fenbufen or aspirin. The obtained KD values of the inclusion complexes of fenbufen binding to alpha-CD and to beta-CD are 4.38x10(-4) mol L(-1) and 2.12x10(-4) mol L(-1), respectively. The KD values of the inclusion complexes of alpha-CD-aspirin and beta-CD-aspirin are 3.33x10(-4) mol L(-1) and 1.83x10(-4) mol L(-1), respectively. A non-linear least squares regression method was applied to validate the results which were consistent with each other. For the system of tetracycline hydrochloride and CD, the 1:1 and 1:2 stoichiometric inclusion complexes were found in the mass spectra. The KD,1 and KD,2 values of the 1:1 and 1:2 stoichiometric inclusion complexes of alpha-CD and tetracycline hydrochloride are 4.47x10(-4) mol L(-1) and 6.51x10(-4) mol L(-1), respectively, and those of beta-CD and tetracycline hydrochloride are 2.26x10(-4) mol L(-1) and 8.57x10(-4) mol L(-1), respectively. For the system of norfloxacin and CD, besides the 1:1 and 1:2 inclusion complexes, the 1:3 stoichiometric inclusion complex was also found. The KD,1, KD,2 and KD,3 of alpha-CD and norfloxacin inclusion complexes are 4.61x10(-4) mol L(-1), 6.05x10(-4) mol L(-1) and 1.45x10(-3) mol L(-1), respectively. The three KD values of beta-CD and norfloxacin are 1.96x10(-4) mol L(-1), 4.93x10(-4) mol L(-1) and 1.15x10(-3) mol L(-1), respectively.     

A study of Src SH2 domain protein-phosphopeptide binding interactions by electrospray ionization mass spectrometry

The noncovalent binding of various peptide ligands to pp60^src (Src) SH2 (Src homology 2) domain protein (12.9 ku) has been used as a model system for development of electrospray ionization mass spectrometry (ESI-MS) as a tool to study noncovalently bound complexes. SH2 motifs in proteins are critical in the signal transduction pathways of the tyrosine kinase growth factor receptors and recognize phosphotyrosine-containing proteins and peptides. ESI-MS with a magnetic sector instrument and array detection has been used to detect the protein-peptide complex with low-picomole sensitivity. The relative abundances of the multiply charged ions for the complex formed between Src SH2 protein and several nonphosphorylated and phosphorylated peptides have been compared. The mass spectrometry data correlate well to the measured binding constants derived from solution-based methods, indicating that the mass spectrometry-based method can be used to assess the affinity of such interactions. Solution-phase equilibrium constants may be determined by measuring the amount of bound and unbound species as a function of concentration for construction of a Scatchard graph. ESI-MS of a solution containing Src SH2 with a mixture of phosphopeptides showed the expected protein-phosphopeptide complex as the dominant species in the mass spectrum, demonstrating the method’s potential for screening mixtures from peptide libraries.     

The role of phosphorylated residues in peptide-peptide noncovalent complexes formation

Electrospray mass spectrometry (ESI-MS) has become the tool of choice for the study of noncovalent complexes. Our previous work has highlighted the role of phosphorylated amino acid residues in the formation of noncovalent complexes through electrostatic interaction with arginine residues’ guanidinium groups. In this study, we employ tandem mass spectrometry to investigate the gas-phase stability and dissociation pathways of these noncovalent complexes. The only difference in the three phosphopeptides tested is the nature of the phosphorylated amino acid residue. In addition the absence of acidic residues and an amidated carboxyl terminus insured that the only negative charge came from the phosphate, which allowed for the comparison of the noncovalent bond between arginine residues and each of the different phosphorylated residues. Dissociation curves were generated by plotting noncovalent complex ion intensities as a function of the nominal energy given to the noncovalent complex ion before entering the collision cell. These results showed that noncovalent complexes formed with phosphorylated tyrosine were the most stable, followed by serine and threonine, which had similar stability.     

New ligand, N-(2-pyridylmethyl)aminoacetate, for use in the immobilised metal ion affinity chromatographic separation of proteins.

A new chelating compound has been developed for use in the immobilised metal ion affinity chromatographic separation of proteins. The tridentate ligand, sodium N-(2-pyridylmethyl)aminoacetate (carbpyr), 1, was prepared via a one-step synthesis from 2-picolylamine, 3 and then immobilised onto Sepharose CL-4B through the epoxide coupling procedure. The binding behaviour of the resulting IMAC sorbent, following chelation with Cu2+ ions to a density of 152 micromol Cu2+ ions/g gel was characterised by frontal analysis experiments using horse heart myoglobin (HMYO) at pH 7.0 and pH 9.0. From the derived isotherms, the adsorption capacity, q(m), for the binding of HMYO to immobilised Cu2+-N-(2-pyridylmethyl)aminoacetate (im-Cu2+-carbpyr)-Sepharose CL-4B at these pH values was found to be 1.92 and 1.91 micromol/g sorbent, respectively, whilst the dissociation constants K(D) were 0.0092 x 10(-6) M and 0.0062 x 10(-6) M at pH 7.0 and pH 9.0, respectively, indicating that the HMYO-im-Cu2+-N-(2-pyridylmethyl)aminoacetate complex was more stable under alkaline conditions, although the binding capacity in terms of micromol protein/g gel remained essentially unchanged. The selectivity features of the im-Cu2+-carbpyr-Sepharose CL-4B sorbent were further characterised in terms of the binding properties with several human serum proteins at pH 5.0, pH 7.0 and pH 9.0.     

Guest encapsulation and self-assembly of molecular capsules in polar solvents via multiple ionic interactions

Herein we report the formation and characterization of a novel type of capsules resulting from the self-association between oppositely charged complementary building blocks in MeOH/H2O. The assembly is based on the interaction between tetraamidinium calix[4]arenes 1a-d and tetrasulfonato calix[4]arene 2. Evidence for the formation of the expected 1:1 assemblies is provided by proton NMR, ESI-MS, and ITC. The association process is fast on the NMR time scale and strongly entropy driven, with association constants in the range of 10(6) M-1. The system 1a.2 shows binding affinity toward acetylcholine, tetramethylammonium, and N-methylquinuclidinium cations.     

Efficient determination and evaluation of model cyclodextrin complex binding constants by electrospray mass spectrometry

Electrospray ionization mass spectrometry (ESI-MS) is now routinely used for the detection of cyclodextrin noncovalent complexes, complementing previously established methods. Host-guest complexes formed in solution are also stable for characterization by ESI in the gas phase. This paper reports the first investigations to characterize the stability of three inclusion complexes between beta-cyclodextrin (beta-CD) and three model "guest" molecules, by determining the cyclodextrin compound complex stability constant (K(st)) with the use of mass spectrometric studies. The relative signal intensity of the complexes were monitored in the positive ion mode by mixing each "guest" molecule with an up to 50-fold molar excess of betaCD. A novel linear equation, similar to Benesi-Hildebrand, was derived allowing the determination of K(st) for 1:1 stoichiometry in all complexes. These values were compared with the K(st) obtained by spectrophotometric experiments and they were evaluated to be slightly different, indicating the validity of the described method.