Main Binding Site Research Articles

Determination of unbound fraction of pazopanib in vitro and in cancer patients reveals albumin as the main binding site

Pazopanib exhibits wide inter-patient pharmacokinetic variability which may contribute to differences in treatment outcome. Unbound drug concentrations are believed to be more relevant to pharmacological responses than total concentrations. Thus it is desirable to evaluate pazopanib binding on plasma proteins and different factors potentially affecting this process. An equilibrium dialysis method coupled with UPLC-MS/MS assay has been optimized and validated for the determination of pazopanib unbound fraction (fu%) in human plasma. Pazopanib binding in the plasma of healthy volunteers and in isolated protein solutions was investigated. The unbound fraction was determined for 24 cancer patients treated daily with pazopanib. We found that pazopanib was extensively bound in human plasma (>99.9 %) with a mean fu% value of 0.0106 ± 0.0013 % at 40 μg/mL. Protein binding was concentration independent over a clinically relevant range of concentrations. In isolated protein solutions, pazopanib at 40 μg/mL was mainly bound to albumin (40 g/L) and to a lesser extent to α1-acid glycoprotein (1 g/L) and low density lipoproteins (1.2 g/L), with a mean fu% of 0.0073 ± 0.0022 %, 0.992 ± 0.44 % and 7.4 ± 1.7 % respectively. Inter-patient variability (CV%) of fu% in cancer patients was limited (27.2 %). A correlation was observed between individual unbound fraction values and albuminemia. Pazopanib exhibits extensive binding to plasma proteins in human plasma. Variable albumin concentrations, frequently observed in cancer patients, may affect pazopanib unbound fraction with implications for inter-patient variability in drug efficacy and toxicity.

Abstract 15853: Identification of Novel Sites Required for PCSK9 Autocatalytic Cleavage

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a secreted protein that promotes degradation of cell surface LDL receptors (LDLRs). Decrease in circulating PCSK9 levels by monoclonal antibodies is correlated with improved lipid profiles in humans. In contrast to therapeutic antibodies, strategies to inhibit PCSK9 with a small molecule have proven to be challenging. Here we explored a potential strategy to decrease PCSK9 level by inhibiting its autocatalytic cleavage and subsequent secretion. We discovered a tool compound, which represents a highly reactive lactone series irreversible inhibitors, that can non-specifically bind to multiple sites on PCSK9 and prevent its autocatalytic cleavage and secretion. Mass spectrometry studies confirmed that serine residues 326 (S326) and 329 (S329) are the main binding sites. Structurally, S326 and S329 are far away from the PCSK9 autocatalytic cleavage site serine 386 (S386). Mutagenesis of S326/S329 reduced PCSK9 autocatalytic cleavage intracellularly. Through structural modeling, we identified additional residues serine 294 (S294) and asparagine 298 (N298) that interact with S326 and S329. Mutagenesis of S294 and N298 also reduced PCSK9 autocatalytic cleavage and secretion. In addition, by using the in-trans expression system of PCSK9, we determined that the effect of S326/S329 on PCSK9 secretion is not through their direct impact on PCSK9 autocatalytic cleavage. In conclusion, we have discovered a novel mechanism that regulates PCSK9 secretion, and we have determined the critical residues that are involved in this regulatory mechanism.

Neutral bis(benzimidazole) Λ-shaped anion receptor

A neutral receptor, integrating a series of –N(H) and –C(H) donor groups organized in a Λ-shape motif was designed and prepared. This compound can form stable 1:1 complexes in acetonitrile solution with halides (F−, Cl−, Br−, and I−) and nitrate (NO3−) anions. The main binding site in the receptor is on the cleft and recognition occurs through a series of co-operative hydrogen bonding interactions including intramolecular hydrogen bonds which stabilizes the co-planar conformation of the host–guest complex. For halides, complex stability is determined by anion basicity, but for anions having other geometries, size and shape complementarity play a major role.

Combined spectroscopy and molecular modeling studies on the binding of galbanic acid and MMP9

The molecular mechanism of galbanic acid (GBA) binding to matrix metalloproteinase 9 (MMP9) was investigated by fluorescence quenching, absorption spectroscopy, FT-IR, molecular docking and molecular dynamics (MD) simulation procedures. The fluorescence emission of MMP9 was quenched by GBA. The titration of MMP9 by various amount of GBA was also followed by UV–Vis absorption spectroscopy. The results revealed that GBA, as a biologically active sesquiterpene coumarin derivative, has an ability to bind strongly to MMP9.Molecular docking results indicated that the main active binding site for GBA has been located in a hydrophobic cavity in the vicinity of Zn atom. Moreover, MD simulation results suggested that GBA as a coumarin derivative can interact with MMP9, without affecting the secondary structure of MMP9. MD simulations, molecular docking as computational methods from one hand and experimental data from other hand reciprocally supported each other.

Structural evidence for asymmetric ligand binding to transthyretin.

Human transthyretin (TTR) represents a notable example of an amyloidogenic protein, and several compounds that are able to stabilize its native state have been proposed as effective drugs in the therapy of TTR amyloidosis. The two thyroxine (T4) binding sites present in the TTR tetramer display negative binding cooperativity. Here, structures of TTR in complex with three natural polyphenols (pterostilbene, quercetin and apigenin) have been determined, in which this asymmetry manifests itself as the presence of a main binding site with clear ligand occupancy and related electron density and a second minor site with a much lower ligand occupancy. The results of an analysis of the structural differences between the two binding sites are consistent with such a binding asymmetry. The different ability of TTR ligands to saturate the two T4 binding sites of the tetrameric protein can be ascribed to the different affinity of ligands for the weaker binding site. In comparison, the high-affinity ligand tafamidis, co-crystallized under the same experimental conditions, was able to fully saturate the two T4 binding sites. This asymmetry is characterized by the presence of small but significant differences in the conformation of the cavity of the two binding sites. Molecular-dynamics simulations suggest the presence of even larger differences in solution. Competition binding assays carried out in solution revealed the presence of a preferential binding site in TTR for the polyphenols pterostilbene and quercetin that was different from the preferential binding site for T4. The TTR binding asymmetry could possibly be exploited for the therapy of TTR amyloidosis by using a cocktail of two drugs, each of which exhibits preferential binding for a distinct binding site, thus favouring saturation of the tetrameric protein and consequently its stabilization.

Synthesis, structure, protein binding of Cu(II) complexes with a tridentate NNO Schiff-base ligand

Four new Cu(II) complexes (1, 2, 3 and 4) in the presence of different anions (Cl−, Br−, I− and ClO4−) have been prepared by tridentate NNN Schiff-base ligand (N,N-dimethyl-N′-[phenyl(2-pyridyl)methylene]ethane-1,2-diamine) and well characterized by single-crystal X-ray diffraction, elemental analysis, IR and UV–Vis spectroscopy. The interactions of complexes 1–4 with human serum albumin (HSA) have been investigated in Tris–HCl buffer solution at pH 7.4 by spectroscopic methods and a molecular docking technique. Experimental results proved that the four complexes quench the fluorescence of HSA through a static quenching mechanism. Thermodynamic parameters were calculated from Van’t Hoff equation. The distance r between the donor (HSA) and acceptor (complexes 1–4) has been obtained by means of Förester resonance energy transfer (FRET). Molecular docking results indicated that the main active binding sites for complexes 1, 2 and 4 are site III in subdomain IB and for complex 3 is site II in subdomain III A. The combination of molecular docking results and fluorescence experimental results indicate that the interaction between 1–4 and HSA are dominated by hydrophobic forces as well as hydrogen bonds.

Isolation of High Level Macrolide Resistant Bordetella pertussis Without Transition Mutation at Domain V in Iran.

Background:Bordetella pertussis, as a causative agent of whooping cough, due to the annual rise y of infection cases, failure of prophylaxis and treatment by macrolides, is considered as the new concern in the health care system.Objectives:The main objective of this study was the determination of single nucleotide polymorphisms (SNPs) at domain V, as the main binding site for macrolides, following the identification of high level macrolides resistant B. pertussis.Materials and Methods:Following the identification of 11 recovered B. pertussis isolates, from a total of 1084 nasopharyngeal swabs, by using the biochemical and molecular methods, the activities of erythromycin, azithromycin and clarithromycin antibiotics against the recovered isolates were examined. Subsequently, A-G transition mutations in domain V were analyzed by molecular techniques, such as Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP) and sequencing.Results:After susceptibility testing, one strain was detected as a high level macrolide resistant B. pertussis (Erythromycin = 128 μg/mL, Clarithromycin > 256 μg/mL). After sequencing and PCR-RFLP methods, transition mutations in positions 2047 and 2058 of the mentioned domain were not observed.Conclusions:Although previous studies have shown that A-G transition mutations in 23 SrRNA gene (domain V) are the main reason for the occurrence of high level macrolides resistance in B. pertussis, however, the mentioned single nucleotide polymorphisms (SNPs) have not been detected in our resistant strain. This is the first report of high level macrolide resistant B. pertussis, without SNPs in domain V, in Iran.

Groove binding interaction between daphnetin and calf thymus DNA

The binding characteristics of daphnetin with calf thymus DNA (ctDNA) were investigated by multispectroscopic and chemometric approaches coupled with DNA viscosity measurements, melting studies and molecular docking technique. The expanded UV–vis spectral data matrix was processed by multivariate curve resolution-alternating least-squares method to obtain the concentration profiles of the components (daphnetin, ctDNA and daphnetin–ctDNA complex) to quantitatively monitor the daphnetin–ctDNA interaction. The groove mode of daphnetin binding to ctDNA was concluded by little change in melting temperature, viscosity of ctDNA and iodide quenching effect as well as increase in single-stranded DNA quenching effect. Moreover, the quantitative data for the competitive binding between daphnetin and Hoechst 33258 for ctDNA obtained by resolving the three-way synchronous fluorescence spectra data using parallel factor analysis modeling further supported the groove binding. The molecular docking visualized the results of the Fourier transform infrared analysis that the adenine and thymine bases in the minor groove of ctDNA were the main binding sites for daphnetin, and the circular dichroism spectra showed that the groove binding of daphnetin to ctDNA led to the conformational change in ctDNA from B-form to A-form. This study revealed the interaction mechanism of daphnetin with ctDNA.

New pyrimido-indole compound CD-160130 preferentially inhibits the KV11.1B isoform and produces antileukemic effects without cardiotoxicity.

KV11.1 (hERG1) channels are often overexpressed in human cancers. In leukemias, KV11.1 regulates pro-survival signals that promote resistance to chemotherapy, raising the possibility that inhibitors of KV11.1 could be therapeutically beneficial. However, because of the role of KV11.1 in cardiac repolarization, blocking these channels may cause cardiac arrhythmias. We show that CD-160130, a novel pyrimido-indole compound, blocks KV11.1 channels with a higher efficacy for the KV11.1 isoform B, in which the IC50 (1.8 μM) was approximately 10-fold lower than observed in KV11.1 isoform A. At this concentration, CD-160130 also had minor effects on Kir2.1, KV 1.3, Kv1.5, and KCa3.1. In vitro, CD-160130 induced leukemia cell apoptosis, and could overcome bone marrow mesenchymal stromal cell (MSC)-induced chemoresistance. This effect was caused by interference with the survival signaling pathways triggered by MSCs. In vivo, CD-160130 produced an antileukemic activity, stronger than that caused by cytarabine. Consistent with its atypical target specificity, CD-160130 did not bind to the main binding site of the arrhythmogenic KV11.1 blockers (the Phe656 pore residue). Importantly, in guinea pigs CD-160130 produced neither alteration of the cardiac action potential shape in dissociated cardiomyocytes nor any lengthening of the QT interval in vivo. Moreover, CD-160130 had no myelotoxicity on human bone marrow-derived cells. Therefore, CD-160130 is a promising first-in-class compound to attempt oncologic therapy without cardiotoxicity, based on targeting KV11.1. Because leukemia and cardiac cells tend to express different ratios of the A and B KV11.1 isoforms, the pharmacological properties of CD-160130 may depend, at least in part, on isoform specificity.

Intercalation of herbicide propyzamide into DNA using acridine orange as a fluorescence probe

The binding action of PRO associated with calf thymus DNA (ctDNA) was determined using multispectroscopic methods combined with parallel factor analysis (PARAFAC) and molecular docking. The fluorescence titration suggested a static fluorescence quenching of PRO was induced by ctDNA. The positive values of enthalpy changes and entropy changes suggested that the binding reaction was predominantly driven by hydrophobic interactions. Increases in melting temperature and viscosity of ctDNA and decrease in iodide-quenching effect indicated that an intercalative binding of PRO to ctDNA occurred. Moreover, the three-way synchronous fluorescence spectra data from the competitive interaction between PRO and fluorescence probe acridine orange (AO) with ctDNA were resolved by PARAFAC modeling to provide the concentration information and spectra of the three components (PRO, AO and ctDNA–AO complex) for the reaction system, which further supported the intercalation of PRO molecule into ctDNA double-helix by replacing bound AO probe. The molecular docking predicted that the A–T base pairs of ctDNA were the main binding sites for PRO, which was confirmed by Fourier transform infrared analysis, and the conformational change of ctDNA from B-form to A-form induced by PRO was inferred from the circular dichroism spectra studies.

Krüppel-like factor 8 involved in hypoxia promotes the invasion and metastasis of gastric cancer via epithelial to mesenchymal transition.

Previously, we reported that hypoxia was able to induce invasion and metastasis in gastric cancer and that hypoxia-inducible factor-1 (HIF-1) is a key factor involved in this tumor type. Krüppel-like factor 8 (KLF8) as a transcriptional repressor has been suggested as a promoter of tumor metastasis in breast cancer and an inducer of the epithelial‑mesenchymal transition (EMT). KLF8 is also highly expressed in gastric cancer tissues, contributing to poor prognosis. However, the association between KLF8 and HIF-1 in regulating the progression of human gastric cancer in hypoxia is unclear. In the present study, we found that KLF8 was overexpressed in gastric cancer metastatic tissues and cells. Additionally, KLF8 siRNA significantly inhibited SGC7901 cell invasion and migration compared with SGC7901, SGC7901/Scr-si cells. Hypoxia is thus able to induce KLF8 expression and EMT in SGC7901 cells. However, following the examination of changes in cell morphology and epithelial and mesenchymal markers, it was found that KLF8 siRNA and HIF-1 siRNA strongly reversed EMT in cells undergoing hypoxia. Furthermore, hypoxia-induced KLF8 overexpression was attenuated by HIF-1 siRNA. Experiments using luciferase promoter constructs resulted in a marked increase in the activity of cells exposed to hypoxia and decreased activity in cells co-transfected with HIF-1 siRNA. The chromatin immunoprecipitation assay revealed proximal HRE at -133 is the main HIF-1 binding site in the KLF8 promoter. In conclusion, the results demonstrated that KLF8 is actively enhanced by hypoxia and is a novel HIF-1 target. KLF8 is a novel EMT regulating transcription factor that involved in the progression of gastric cancer. The specific anti-EMT drugs in combination with anti-hypoxia are new promising cancer therapies.

Abstract 307: Structural Characterization of Calcineurin A-Calsarcin 1 Assembly

Signaling by the calcium-dependent phosphatase calcineurin (Cn) plays key roles in regulating cardiac development, hypertrophy, and pathological remodeling. Cn binds to and is negatively regulated by calsarcins (CS), a family of muscle-specific proteins. However, the molecular mechanisms involved in the inhibition of Cn by CS remain unclear. Understanding the architecture and structure of Cn-CS complex is critical to unravel the regulation of Cn by CS. Here we combined biochemical assays, chemical cross-linking coupled to mass spectrometry experiments (MS/MS), mutational analysis and a modeling strategy for structural characterization of CnA-CS1 assembly. The MS/MS data obtained from the cross-linked peptides of both proteins were used to guide an in silico docking of their polypeptide models. The protein complex models with the smallest estimated binding energy were clustered according to structural similarity and submitted to molecular dynamics simulation. The interacting surface of CnA was mapped in a pocket between the 1st and 3rd α-helixes and surrounding loops, while the corresponding surface of CS1 was mapped to the carboxyterminal loops within the Leu179-Phe185, Phe195-Ser199 and Thr250-Leu264 regions. Notably, the region of CnA that interacts with CS1 was found to be located in close proximity, but not coincident, to the β-sheet 14, the main binding site for the PxIxIT sequence of NFAT. Experiments performed with several CnA (FLAG-CnA) and CS1 (myc-CS1) mutants were used to validate the structural model of the CnA-CS1 assembly. The Lys40 (CnA) and Glu254 (CS1) residues were identified as critical for the complex stability. The model that emerges from this study supports the notion that CS1 interacts with an allosteric site to inhibit the activity of CnA. Alternatively, the close proximity of the CS1 to NFAT interacting site supports an interference of CS1 on the ability of CnA to bind and activate NFAT.

Characteristics of the adsorbed heavy metals onto aerobic granules: isotherms and distributions

Biosorption experiments were performed using aerobic granules as bioadsorbent for Cd2+, Cu2+, Mn2+, Zn2+, Fe2+, and Co2+. Aerobic granule shows high adsorption capacity [Qe (mg/g) = 189 − 232]. Both Langmuir and Freundlich isotherm fitted the adsorption data well. Especially, the R2 value exceeds 0.96 in most cases when Langmuir model was adopted. In order to look into the distribution of the adsorbed heavy metal in aerobic granules and to verify metal uptake in surface layer, in middle zone and center of aerobic granule, energy dispersive X-ray (EDX) spectra were obtained. According to field emission scanning electron microscope-EDX observation, it was found that whole granule body can act as an active site for heavy metals uptake because of a lot of pore existed in the inside of aerobic granule. It is clear that the penetration of dissolved metal solution to the center of aerobic granule has occurred, which confirms that metal uptake can occur even in the center of aerobic granule. The analyses by Fourier transform infrared spectroscopy and X-ray photoelectron spectroscopy showed that functional groups on aerobic granules, such as alcoholic, carboxylate and ether, would be the main binding sites for biosorption of heavy metals. The area distributions further reveal that ether and alcoholic would be the dominant carbon forms in aerobic granules, and the interaction and coordination affinity between the functional groups and metals would be more complicated than expected.

Interactions that influence the binding of synthetic heparan sulfate based disaccharides to fibroblast growth factor-2.

Heparan sulfate (HS) is a linear sulfated polysaccharide that mediates protein activities at the cell-extracellular interface. Its interactions with proteins depend on the complex patterns of sulfonations and sugar residues. Previously, we synthesized all 48 potential disaccharides found in HS and used them for affinity screening and X-ray structural analysis with fibroblast growth factor-1 (FGF1). Herein, we evaluated the affinities of the same sugars against FGF2 and determined the crystal structures of FGF2 in complex with three disaccharides carrying N-sulfonated glucosamine and 2-O-sulfonated iduronic acid as basic backbones. The crystal structures show that water molecules mediate different interactions between the 3-O-sulfonate group and Lys125. Moreover, the 6-O-sulfonate group forms intermolecular interactions with another FGF2 unit apart from the main binding site. These findings suggest that the water-mediated interactions and the intermolecular interactions influence the binding affinity of different disaccharides with FGF2, correlating with their respective dissociation constants in solution.

Kinetic simulating of Cr(VI) removal by the waste Chlorella vulgaris biomass

Waste microalgae biomass, one of the most abundant residues from biodiesel production, can be used as a low-cost biosorbent for heavy metal removal. The ability and mechanism of Cr(VI) removal by lipid-extraction residue of Chlorella vulgaris were investigated in this study. The removal of Cr(VI) increased as the pH decreased from 4.0 to 0.5, or temperature increased from 15°C to 45°C. The Sips isotherm well-fitted the experiment data, and the maximal biosorption capacity of waste C. vulgaris biomass for total chromium was 43.3mg/g at pH 1.5 and a temperature of 25°C. X-ray photoelectron spectroscopy revealed that the majority of Cr(VI) bound on the biomass was reduced to Cr(III) with its subsequent partial biosorption. The results of Fourier Transform Infrared Spectrometer study indicated that both carboxyl and amino groups on the biomass were the main binding sites for Cr(VI) biosorption, while carbohydrate was mainly responsible for reduction of Cr(VI). Finally, the proposed kinetic model based on the indirect reduction mechanism well described the Cr(VI) removal behavior at various pHs (0.5–2) and temperatures (15–45°C).

β-Lactoglobulin–naringenin complexes: Nano-vehicles for the delivery of a hydrophobic nutraceutical

Naringin, a glycosylated polyphenol of the flavanones class and its aglycone, naringenin, have been shown to possess many health benefits, therefore they show promise for supplementation of staple foods and beverages, as a mean of preventive medicine. Major obstacles to the successful implementation of these flavanones as nutraceuticals are their low water-solubility and the related low oral bioavailability. Naringin is more soluble but is also very bitter. To allow the enrichment of clear beverages with these important health promoters, we have used native and preheated β-Lactoglobulin (β-Lg) based nanovehicles. Using UV spectrophotometry and intrinsic fluorescence methods, we have found that naringenin forms complexes with preheated and non-preheated β-Lg, with Ka in the order of 104 M−1. No binding was found between naringin and β-Lg, probably due to the lower hydrophobicity of naringin and the steric interference of its sugar. Naringenin: β-Lg binding stoichiometry was 2:1, suggesting naringenin was probably bound at the two main hydrophobic binding sites of the protein, the calyx and the cleft near the alpha-helix. The complex formation with both native and preheated protein solubilized naringenin up to 3 times its solubility limit and prevented crystal formation in the aqueous medium. Such crystallization is responsible for both reduced bioavailability and hampered visual attractiveness of the product. Dynamic light scattering revealed the existence of nanoparticles of ∼10 nm, marginally larger than those of the pure protein (∼7 nm), therefore the solution was clear, hence suitable for clear beverages. Reconstitution after freeze drying was good, but only after quench freezing.

Metal counterion modulated single proton transfer process in guanine base

The influence of metal cations (Mn+=Na+, K+, Mg2+, Ca2+, Zn2+) on the single proton transfer process in guanine base has been studied at the MP2/6-31G* and B3LYP/6-311++G** levels of theory. Two main basic binding sites, N3 and N7 of guanine, are considered for Mn+ coordination. It is observed that the tautomerism behavior of guanine, induced by intramolecular single proton transfer, can be modulated by surrounding metal counterions. Calculated results show that the single proton transfer process in guanine is favored and even becomes thermodynamically spontaneous due to the presence of Mn+ interacting at the N3 position of guanine. On the contrary, if Mn+ coordinated to N7 of guanine, the single proton transfer process will become unfavorable than that in the neutral system. Moreover, the effects are more pronounced for the divalent cations than for the monovalent ones. All of them can be understood from the electrostatic and oxidative effects of metal cations on the guanine base obtained from further natural bond orbital (NBO) analyses.

Species-dependent binding of tocainide analogues to albumin: Affinity chromatography and circular dichroism study

A series of novel tocainide analogues were characterized for their HSA and RSA binding, by using high-performance liquid affinity chromatography (HPLAC) and circular dichroism (CD). In this HPLAC study, HSA and RSA were covalently immobilized to the silica matrix of HPLC columns, with a procedure that maintained unaltered the binding properties of the proteins. The tocainide analogues were ranked for their affinity to HSA and RSA on the basis of their bound fractions measured by the two albumin-based columns. This technique was also applied to characterize the high affinity binding sites of these tocainide analogues to the protein. For this purpose displacement experiments were carried out by means of increasing concentrations in the mobile phase of competitors known to bind selectively to the main binding sites of HSA. The results obtained with the immobilized proteins were confirmed by investigating the same drug-protein systems in solution by circular dichroism. The comparison of the data collected with both methodologies highlighted the dramatic effect of small differences in the amino acidic sequences of the two proteins. In fact, despite their similar primary and secondary structures, a small difference in the amino acidic sequence leads to significant differences in their three-dimensional structure reflecting their different binding capacity and their stereoselectivity. Therefore, this study confirms how it is crucial to consider the significant differences among the animal models when performing pharmacokinetic studies. It is also clear that the knowledge of serum carrier binding parameters at an early stage of drug discovery represents a great advantage that may help to save time and efforts.

Removal of Cu(II), Pb(II) and Zn(II) Ions from Aqueous Solutions Using Selected Agricultural Wastes: Adsorption and Characterisation Studies

The feasibility of durian tree sawdust (DTS), coconut coir (CC) and oil palm empty fruit bunch (EFB) as low-cost biosorbents for the removal of Cu(II), Pb(II) and Zn(II) ions from aqueous solutions was investigated. The effects of solution pH and initial metal concentration on adsorption capacity were examined in batch experiments. The affinity and the adsorption capacity of DTS, CC and EFB were evaluated. The adsorption behaviour of Cu(II), Pb(II) and Zn(II) ions onto DTS, CC and EFB was described using Freundlich and Langmuir isotherm models. The separation factor (RL) analysis suggests that the removal of metal ions onto three agricultural wastes studied was favourable. The maximum adsorption capacities (Q) estimated from the Langmuir isotherm model for Cu(II), Pb(II) and Zn(II) were 18.42, 20.37 and 22.78 mg/g for DTS, 18.38, 37.04 and 24.39 mg/g for CC, and 26.95, 37.59 and 21.19 mg/g for EFB, respectively. The characterisation studies were carried out using Scanning Electron Microscope (SEM), Energy Dispersive X-ray Spectrometer (EDX) and Fourier Transform Infrared Spectrometer (FTIR). The surface morphology of the biosorbents changed significantly following interaction with metal ions. The primary adsorption mechanism was complexation between metal ions and binding sites of biosorbents. Both hydroxyl and amine groups are the main binding sites in DTS, CC and EFB.

Structure of the Pb²⁺-deprotonated dGMP complex in the gas phase: a combined MS-MS/IRMPD spectroscopy/ion mobility study.

The structure of the Pb(2+)-deprotonated 2'-deoxyguanosine-5'-monophosphate (dGMP) complex, generated in the gas phase by electrospray ionization, was examined by combining tandem mass spectrometry, mid-infrared multiple-photon dissociation (IRMPD) spectroscopy and ion mobility. In the gas phase, the main binding site of Pb(2+) onto deprotonated dGMP is the deprotonated phosphate group, but the question is whether an additional stabilization of the metallic complex can occur via participation of the carbonyl group of guanine. Such macrochelates indeed correspond to the most stable structures according to theoretical calculations. A multiplexed experimental approach was used to characterize the gas-phase conformation of the metallic complex and hence determine the binding mode of Pb(2+) with [dGMP](-). MS/MS analysis, observation of characteristic bands by IRMPD spectroscopy, and measurement of the ion mobility collision cross section suggest that gaseous [Pb(dGMP)-H](+) complexes adopt a macrochelate folded structure, which consequently differs strongly from the zwitterionic forms postulated in solution from potentiometric studies.