Probe Ligand Research Articles

Targeted glycoproteomic identification of cancer cell glycosylation

GalMBP is a fragment of serum mannose-binding protein that has been modified to create a probe for galactose-containing ligands. Glycan array screening demonstrated that the carbohydrate-recognition domain of GalMBP selectively binds common groups of tumor-associated glycans, including Lewis-type structures and T antigen, suggesting that engineered glycan-binding proteins such as GalMBP represent novel tools for the characterization of glycoproteins bearing tumor-associated glycans. Blotting of cell extracts and membranes from MCF7 breast cancer cells with radiolabeled GalMBP was used to demonstrate that it binds to a selected set of high molecular weight glycoproteins that could be purified from MCF7 cells on an affinity column constructed with GalMBP. Proteomic and glycomic analysis of these glycoproteins by mass spectrometry showed that they are forms of CD98hc that bear glycans displaying heavily fucosylated termini, including Lewisx and Lewisy structures. The pool of ligands was found to include the target ligands for anti-CD15 antibodies, which are commonly used to detect Lewisx antigen on tumors, and for the endothelial scavenger receptor C-type lectin, which may be involved in tumor metastasis through interactions with this antigen. A survey of additional breast cancer cell lines reveals that there is wide variation in the types of glycosylation that lead to binding of GalMBP. Higher levels of binding are associated either with the presence of outer-arm fucosylated structures carried on a variety of different cell surface glycoproteins or with the presence of high levels of the mucin MUC1 bearing T antigen.

Trapping Moving Targets with Small Molecules

Structure-based drug design traditionally uses static protein models as inspirations for focusing on "active" site targets. Allosteric regulation of biological macromolecules, however, is affected by both conformational and dynamic properties of the protein or protein complex and can potentially lead to more avenues for therapeutic development. We discuss the advantages of searching for molecules that conformationally trap a macromolecule in its inactive state. Although multiple methodologies exist to probe protein dynamics and ligand binding, our current discussion highlights the use of nuclear magnetic resonance spectroscopy in the drug discovery and design process.

Flow and image cytometry side by side for the new frontiers in quantitative single‐cell analysis

THE role of regulatory (CD4CD25) T-cells (Tregs) as therapeutics to replace common anti-inflammatory drugs is of increasing interest since cell therapy seems to be within reach (1). Practical aspects of purification and propagation of Tregs is now summarized by Trzonkowski et al. in the present issue (2). The authors demonstrate that novel advances in flow cytometry and also cellular immunology opens the door for the clinical application of Tregs. Affordable diagnostic tools for HIV and AIDS are of imminent importance in resource poor countries (3). To this end, PCR (4) and cell-based analysis (i.e. CD4 counting) by flow cytometry (5,6) or automated microscopic cytometry (7) is often the method of choice. The current PCR assays, however, often fail to detect virus variants that are prevalent in the third world. Thereby, these patients, despite clinical symptoms, may be diagnosed as virus negative and will not receive therapy. Now, Greve et al. present a new microparticle-based PCR assay that enables detection and quantification of HIV-1 viral load for most of the common variants including outlier, nonmajor, and circulating recombinant forms (8, commentary in 9). Malaria is also one of the great killers that needs appropriate diagnostic tools (7). Jimenez-Diaz et al. (10) developed an assay to quantify malaria-infected erythrocytes using the cell-permeable nuclear dye SYTO-16. The authors show the applicability of their assay for infected normal mice and human erythrocyte engrafted NOD mice. Because flow cytometers are now available in many resource poor settings, both assays can be introduced, relatively inexpensively. Finally, the research group of Edwards et al. applied a flow-cytometry-based high-throughput screening system (11) to identify novel formylpeptide receptor ligand probes. The authors screened more than 24,000 small molecules and finally came up with several candidates of potential future therapeutic or diagnostic interest (12,13). The aforementioned scientific articles of this issue illustrate that flow cytometry is still going strong with innovative technologies for versatile applications presented in our journal. However, every scientific journal has to evolve and adjust its focus to present and adjust for future requirements of the scientific community. In view of the increasing technological advances in imaging (14), our goal was to become a front journal for image cytometry and quantitative image analysis. As one can see from the citations in 2008 to Cytometry Part A, the readers find an increasing appreciation in our publications in the technological field of quantitative imaging. As one good example, Abella et al. (15) present an assay for automated quantification of cell infiltrates in histological sections based on microscopic images from allergic murine models. After extraction of texture parameters by a stepwise procedure the authors propose an algorithm that appears to be superior to manual cell counting. Yet another application is automated laser scanning cytometry (LSC) for mast cells tracking in skin as described by Zoog et al. (16). Rapid, accurate evaluation of mast cells present around wounds in a wound-healing model by automated LSC may serve as an important tool for tracking pharmacodynamic effects of MC-directed therapies. Without doubt we enter the era of automated cell detection in tissues (17). New labeling fluorescent tools for life-cell in vivo imaging are introduced by Robers et al. (18). High-affinity and selective ligand–receptor binding of 50-fluorescein-tagged synthetic ligand for FKBP12 (immunophilin) and mutated receptor

Duplex high‐throughput flow cytometry screen identifies two novel formylpeptide receptor family probes

Of recent, clinical interest have been two related human G-protein coupled receptors: formylpeptide receptor (FPR), linked to antibacterial inflammation and malignant glioma cell metastasis; and FPR like-1 (FPRL1), linked to chronic inflammation in systemic amyloidosis, Alzheimer's disease, and prion diseases. In association with the National Institutes of Health (NIH) Molecular Library Screening Network, we implemented a flow-cytometry-based high-throughput screening (HTS) approach for identifying selective small molecule FPR and FPRL1 ligands. The screening assay measured the ability of test compounds to competitively displace a high-affinity, fluorescein- labeled peptide ligand from FPR, FPRL1, or both. U937 cells expressing FPR and rat basophil leukemia (RBL) cells expressing FPRL1 were tested together in a "duplex" format. The U937 cells were color coded with red-fluorescent dye allowing their distinction during analysis. Compounds, cells, and fluorescent ligand were sequentially combined (no wash) in 15 microl assay volumes in 384-well plates. Throughput averaged approximately 11 min per plate to analyze approximately 4,000 cells ( approximately 2,000/receptor) in a 2 microl aspirate from each well. In primary single concentration HTS of 24,304 NIH Small Molecule Repository compounds, 253 resulted in inhibition >30% (181 for FPR, 72 for FPRL1) of which 40 had selective binding inhibition constants (K(i)) < or = 4 microM (34 for FPR and 6 for FPRL1). An additional 1,446 candidate compounds were selected by structure-activity-relationship analysis of the hits and screened to identify novel ligands for FPR (3570-0208, K(i) = 95 +/- 10 nM) and FPRL1 (BB-V-115, K(i) = 270 +/- 51 nM). Each was a selective antagonist in calcium response assays and the most potent small molecule antagonist reported for its respective receptor to date. The duplex assay format reduced assay time, minimized reagent requirements, and provided selectivity information at every screening stage, thus proving to be an efficient means to screen for selective receptor ligand probes.

In vivo Incorporation of Unnatural Amino Acids to Probe Structure, Dynamics, and Ligand Binding in a Large Protein by Nuclear Magnetic Resonance Spectroscopy

In vivo incorporation of isotopically labeled unnatural amino acids into large proteins drastically reduces the complexity of nuclear magnetic resonance (NMR) spectra. Incorporation is accomplished by coexpressing an orthogonal tRNA/aminoacyl-tRNA synthetase pair specific for the unnatural amino acid added to the media and the protein of interest with a TAG amber codon at the desired incorporation site. To demonstrate the utility of this approach for NMR studies, 2-amino-3-(4-(trifluoromethoxy)phenyl)propanoic acid (OCF 3Phe), (13)C/(15)N-labeled p-methoxyphenylalanine (OMePhe), and (15)N-labeled o-nitrobenzyl-tyrosine (oNBTyr) were incorporated individually into 11 positions around the active site of the 33 kDa thioesterase domain of human fatty acid synthase (FAS-TE). In the process, a novel tRNA synthetase was evolved for OCF 3Phe. Incorporation efficiencies and FAS-TE yields were improved by including an inducible copy of the respective aminoacyl-tRNA synthetase gene on each incorporation plasmid. Using only between 8 and 25 mg of unnatural amino acid, typically 2 mg of FAS-TE, sufficient for one 0.1 mM NMR sample, were produced from 50 mL of Escherichia coli culture grown in rich media. Singly labeled protein samples were then used to study the binding of a tool compound. Chemical shift changes in (1)H-(15)N HSQC, (1)H-(13)C HSQC, and (19)F NMR spectra of the different single site mutants consistently identified the binding site and the effect of ligand binding on conformational exchange of some of the residues. OMePhe or OCF 3Phe mutants of an active site tyrosine inhibited binding; incorporating (15)N-Tyr at this site through UV-cleavage of the nitrobenzyl-photocage from oNBTyr re-established binding. These data suggest not only robust methods for using unnatural amino acids to study large proteins by NMR but also establish a new avenue for the site-specific labeling of proteins at individual residues without altering the protein sequence, a feat that can currently not be accomplished with any other method.

Docking to RNA via Root-Mean-Square-Deviation-Driven Energy Minimization with Flexible Ligands and Flexible Targets

Structure-based drug design is now well-established for proteins as a key first step in the lengthy process of developing new drugs. In many ways, RNA may be a better target to treat disease than a protein because it is upstream in the translation pathway, so inhibiting a single mRNA molecule could prevent the production of thousands of protein gene products. Virtual screening is often the starting point for structure-based drug design. However, computational docking of a small molecule to RNA seems to be more challenging than that to protein due to the higher intrinsic flexibility and highly charged structure of RNA. Previous attempts at docking to RNA showed the need for a new approach. We present here a novel algorithm using molecular simulation techniques to account for both nucleic acid and ligand flexibility. In this approach, with both the ligand and the receptor permitted some flexibility, they can bind one another via an induced fit, as the flexible ligand probes the surface of the receptor. A possible ligand can explore a low-energy path at the surface of the receptor by carrying out energy minimization with root-mean-square-distance constraints. Our procedure was tested on 57 RNA complexes (33 crystal and 24 NMR structures); this is the largest data set to date to reproduce experimental RNA binding poses. With our procedure, the lowest-energy conformations reproduced the experimental binding poses within an atomic root-mean-square deviation of 2.5 A for 74% of tested complexes.

Chiral Bifurcation in Aggregating Insulin: An Induced Circular Dichroism Study

The structural unambiguity of folding is lost when disordered protein molecules convert into β-sheet-rich fibrils. The resulting polymorphism of protein aggregates has been studied in the context of its biomedical consequences. Events underlying the conformational variance of amyloid fibrils, as well as physicochemical boundaries between folding and misfolding pathways, remain obscure. Bifurcation and chiral mesoscopic-scale organization of amyloid fibrils are new aspects of protein misfolding. Here we characterize bifurcation events accompanying insulin fibrillation upon intensive vortexing. Upon agitation, two types of insulin fibrils with opposite chiral senses are formed; however, predominance of either species is only stochastically determined. The uncertainty of fibrils’ chiral sense holds only for fibrils grown within the physiological temperature range, while above 50 °C, the bifurcation is no longer observed—fibrils’ chiral moieties become uniformly biased towards ligand probes, as revealed by the extrinsic Cotton effect of thioflavin T, Congo red, and molecular iodine. According to transmission electron microscopy and scanning electron microscopy data, chiral variants of insulin fibrils consist of fibrous superstructures, distinct from spherulites, formed by the protein in nonagitated solutions. Gradual dissociation of the fibrils in the presence of dimethyl sulfoxide is noncooperative and can be resolved into three distinct phases: decay of the higher-order chiral structures, breakdown of fibrils, and unfolding of intermolecular β-sheet. The chiral aggregates are also destabilized by elution of NaCl implying that Debye screening of charged β-sheets provided by chloride counterions is needed for sustaining their kinetic stability. At elevated temperatures, cross-seeding of agitated insulin samples with preformed fibrils revealed a chiral conflict that prevented the passing of structural features of mother seeds to daughter fibrils in a manner typical of amyloid “strains.”

Expression of the tyrosine kinase receptor EphA5 and its ligand ephrin-A5 during mouse spinal cord development

To study the expression patterns of two Eph family molecules, the receptor EphA5, and the ligand ephrin-A5, during spinal cord development. The receptor expression was analyzed using beta-galactosidase knockin mice, and affinity ligand probe binding. The ligand expression was assessed using two different affinity probes, and knockout mouse tissues as controls. EphA5 was expressed in the ventral spinal cord, while ephrin-A5 was located in the dorsolateral regions of the spinal cord throughout development. These results show that EphA5 and ephrin-A5 are expressed over broad developmental stages and may play important roles in establishing the dorsoventral organization of the spinal cord.

Investigating 2,2‘-Bipyridine-3,3‘-diol as a Microenvironment-Sensitive Probe: Its Binding to Cyclodextrins and Human Serum Albumin

The 2,2'-bipyridine-3,3'-diol molecule (BP(OH)2) was investigated as a potential photophysical probe in inclusion and biological studies. Binding of BP(OH)2 to cyclodextrins (CDs) and human serum albumin (HSA) was studied by following the changes in its absorption and fluorescence spectra. The stoichiometric ratios and binding constants of the complexes were deduced by fitting the changes in the spectral intensity to binding isotherms. The stoichiometric ratio in the BP(OH)2/(alpha-CD) complex is dominated by 1:2, whereas in all other CDs and in HSA this ratio is 1:1. The structure of the BP(OH)2:(alpha-CD)2 complex, calculated using ab initio methods, indicates that the inclusion of the BP(OH)2 molecule is axial and centered between the two cavities of alpha-CD with van der Waals and electrostatic interactions dominating the binding. Analysis of these results along with the inclusion results of BP(OH)2 in beta-CD, methyl-beta-CD, 2,6-di-O-methyl-beta-CD, and gamma-CD shows that absorption and fluorescence of BP(OH)2 are very sensitive to the change in the cavity size of CD and its hydrophobicity. This change is reflected in the form of a decrease in the intensity of the absorption peaks of the BP(OH)2/water complex in the region 400-450 nm and a red shift in the fluorescence peak as the cavity size decreases and its hydrophobicity increases. Binding of BP(OH)2 as a probe ligand to HSA, a prototype protein, reflects the hydrophobic interior of HSA in a similar manner. The spectral changes indicate that BP(OH)2 binds in the hydrophobic cavity of HSA's subdomain IIA. The results presented here show that BP(OH)2 can be used in binding sites and biological systems as a microenvironment-sensitive probe.

Transient NOE-exchange-relay experiment: Application to ligand–protein binding under slow exchange conditions

A new version of one-dimensional 1H experiment has been developed to probe ligand binding to macromolecular targets. The experiment, called transient NOE-exchange relay, is similar to the ‘reverse NOE pumping’ technique [A. Chen, M.J. Shapiro, J. Am. Chem. Soc. 122 (2000) 414–415]. The T 2 filter is used to erase protein magnetization, and the saturation then spreads from protein to bound ligand (via NOE) and further to a free ligand (via on–off exchange). The ligand signals, monitored as a function of mixing time, present a familiar ‘dip’ pattern characteristic of transient NOE or transient exchange experiments. In addition to the T 2 filter, we have also implemented a T 1 filter which makes use of the fact that the selective T 1 - 1 rates in macromolecules are much higher than those in small ligands. To model the experiment, complete relaxation and exchange matrix analysis has been invoked. This formalism was further used as a starting point to develop a simplified treatment where the relaxation and exchange components are represented by 2 × 2 matrix and, in addition, there is a special term responsible for coupling of ligand magnetization to the protein spin bath. The proposed experimental scheme has been tested on a system of peanut agglutinin complexed with Me-β- d-galactopyranoside, which is known to be in a slow exchange regime. The results suggest that the NOE-exchange-relay experiment can be used at the advanced stages of the drug development process to confirm high-affinity ligand binding.

Bridging Chemical and Biological Space: “Target Fishing” Using 2D and 3D Molecular Descriptors

Bridging chemical and biological space is the key to drug discovery and development. Typically, cheminformatics methods operate under the assumption that similar chemicals have similar biological activity. Ideally then, one could predict a drug's biological function(s) given only its chemical structure by similarity searching in libraries of compounds with known activities. In practice, effectively choosing a similarity metric is case dependent. This work compares both 2D and 3D chemical descriptors as tools for predicting the biological targets of ligand probes, on the basis of their similarity to reference molecules in a 46,000 compound, biologically annotated chemical database. Overall, we found that the 2D methods employed here outperform the 3D (88% vs 67% success) in correct target prediction. However, the 3D descriptors proved superior in cases of probes with low structural similarity to other compounds in the database (singletons). Additionally, the 3D method (FEPOPS) shows promise for providing pharmacophoric alignment of the small molecules' chemical features consistent with those seen in experimental ligand/ receptor complexes. These results suggest that querying annotated chemical databases with a systematic combination of both 2D and 3D descriptors will prove more effective than employing single methods.

Fluorescent Indicators Distributed throughout the Pharmacophore of Cholecystokinin Provide Insights into Distinct Modes of Binding and Activation of Type A and B Cholecystokinin Receptors

Ligand probes with fluorescent indicators positioned throughout the pharmacophoric domain can provide important insights into the molecular basis of receptor binding and activation as reflected in the microenvironment of each indicator while docked at a receptor. We developed three cholecystokinin-like probes with Aladan situated at the N terminus, in the mid-region, and at the C terminus (positions 24, 29, and 33, respectively). These were studied in solution and docked at type A and B cholecystokinin receptors. This study demonstrated clear differences in mechanisms of cholecystokinin binding and activation of these structurally related receptors with distinct agonist structure-activity relationships. The fluorescence characteristics of Aladan are highly sensitive to the polarity of its microenvironment. The mid-region probe was least accessible to the aqueous milieu as determined by fluorescence emission spectra and iodide quenching, which was not altered by changes in conformation from active to inactive. Accessibility of the N- and C-terminal probes was affected by receptor conformation. The position 24 probe was more easily quenched in the active than in the G protein-uncoupled conformation for both receptors. However, the position 33 probe docked at the type A cholecystokinin receptor was more easily quenched in the active conformation, whereas the same probe docked at the type B cholecystokinin receptor was more easily quenched in the inactive conformation. Fluorescence anisotropy and red edge excitation shift determinations confirmed these observations and supported the proposed movements. Although both type A and B cholecystokinin receptors bind cholecystokinin with high affinity, resulting in fully efficacious biological responses, these receptors utilize distinct molecular modes of binding.

High-Affinity Ligand Probes of CD22 Overcome the Threshold Set by cis Ligands to Allow for Binding, Endocytosis, and Killing of B Cells

CD22 (Siglec-2) is a key regulator of B cell signaling whose function is modulated by interaction with extracellular glycan ligands mediated through its N-terminal Ig domain. Its preferred ligand is the sequence Sia alpha2-6Gal that is abundantly expressed on N-linked glycans of B cell glycoproteins, and by binding to CD22 in cis causes CD22 to appear "masked" from binding to synthetic sialoside probes. Yet, despite the presence of cis ligands, CD22 redistributes to sites of cell contact by binding to trans ligands on neighboring cells. In this study, we demonstrate the dynamic equilibrium that exists between CD22 and its cis and trans ligands, using a high-affinity multivalent sialoside probe that competes with cis ligands and binds to CD22 on native human and murine B cells. Consistent with the constitutive endocytosis reported for CD22, the probes are internalized once bound, demonstrating that CD22 is an endocytic receptor that can carry ligand-decorated "cargo" to intracellular compartments. Conjugation of the sialoside probes to the toxin saporin resulted in toxin uptake and toxin-mediated killing of B lymphoma cell lines, suggesting an alternative approach for targeting CD22 for treatment of B cell lymphomas.

Determination of the Two-Dimensional Interaction Rate Constants of a Cytokine Receptor Complex

Ligand-receptor interactions within the plane of the plasma membrane play a pivotal role for transmembrane signaling. The biophysical principles of protein-protein interactions on lipid bilayers, though, have hardly been experimentally addressed. We have dissected the interactions involved in ternary complex formation by ligand-induced cross-linking of the subunits of the type I interferon (IFN) receptors ifnar1 and ifnar2 in vitro. The extracellular domains ifnar1-ectodomain (EC) and ifnar2-EC were tethered in an oriented manner on solid-supported lipid bilayers. The interactions of IFN α2 and several mutants, which exhibit different association and dissociation rate constants toward ifnar1-EC and ifnar2-EC, were monitored by simultaneous label-free detection and surface-sensitive fluorescence spectroscopy. Surface dissociation rate constants were determined by measuring ligand exchange kinetics, and by measuring receptor exchange on the surface by fluorescence resonance energy transfer. Strikingly, approximately three-times lower dissociation rate constants were observed for both receptor subunits compared to the dissociation in solution. Based on these directly determined surface-dissociation rate constants, the surface-association rate constants were assessed by probing ligand dissociation at different relative surface concentrations of the receptor subunits. In contrast to the interaction in solution, the association rate constants depended on the orientation of the receptor components. Furthermore, the large differences in association kinetics observed in solution were not detectable on the surface. Based on these results, the key roles of orientation and lateral diffusion on the kinetics of protein interactions in plane of the membrane are discussed.

X-ray absorption methods for the determination of Ru–Cl bond covalency in olefin metathesis catalysts: On the normalization of chlorine K-edges in ruthenium complexes

A systematic approach to quantitative analysis of Cl K-edge XAS spectra in ruthenium complexes is outlined and applied to ruthenium carbene complexes involved in olefin metathesis catalysis. This method is generally well-suited for probing ligand donor contributions into empty transition metal d orbitals, but overlap of the Ru L 2, 3 edge makes this very difficult in these complexes. A method for removing the Ru L 2, 3 edges from the Cl K-edge spectrum is developed and tested. The normalization procedure makes use of the well-separated Ru L 2 edge and an appropriate L 3/L 2 branching ratio to unambiguously remove contributions from these edges and allow for accurate normalization of the Cl K-edge spectrum. A generalized background subtraction that accounts for variability over the large energy range involved is also described. The methodology should be general for any similarly overlapping edges, as long as one component of the spin–orbit split metal (L- or M-) edge is unperturbed. The results of this study indicate a substantial difference in Ru–Cl bonding depending on the nature of the catalyst.

Ultrafast photoinduced deligation and ligation dynamics: DCM in micelle and micelle-enzyme complex

We report studies on diffusion controlled deligation and ligation dynamics of a probe ligand 4-(dicyanomethylene)-2-methyl-6-( p-dimethylamino-styryl) 4H-pyran (DCM) with cationic cetyltrimethylammonium bromide (CTAB) micelles. In order to investigate the effect of spatial heterogeneity on the dynamics we study the DCM labeled micelle upon complexation with an enzyme α-chymotrypsin (CHT). The variation of fluorescence line-width ( Γ( t)) of DCM in the complex and also in the micelle indicates the diffusion dynamics of DCM through various environments of different polarities. The temporal behavior of Γ( t) reveals that at 50 mM CTAB concentration the excited DCM traverses 6.5 Å distance from the surface of a host micelle (deligation) before entering to a stern layer of another adjacent micelle (ligation). From neutron scattering experiment the distance 6.5 Å is found to be the thickness of a stern layer of CTAB micelle. No indication of ligation has been found at 2 mM CTAB concentration as the intermicellar distance is estimated to be very large (416 Å) compared to the previous case. The dynamical behavior of Γ( t) is also indicative of significantly slower diffusion of the ligand molecules (DCM) at the surface of the micelle in presence and absence of the enzyme compared to that in the bulk buffer. We have also studied the dynamics of solvation and local geometrical restriction on the probe DCM at the micellar surface with and without CHT. With picosecond time resolution, we found time constants of the solvation relaxation processes of the DCM labeled enzyme-micelle complex to be 230 ps (45%) and 870 ps (55%), which were comparable to those of the micelle without the enzyme. The time dependent anisotropy revealing local orientational motions of the probe in the complex was also found to be similar to that of DCM at the micellar surface in absence of CHT. These studies attempt to link the dynamical features for insight into the ligand mediated intercellular communication and the biological function of the enzyme α-chymotrypsin upon complexation with the CTAB micelle.

New fluorometric enzyme immunoassay for 17β-estradiol by homogeneous reaction using biotinylated estradiol

A new fluorometric enzyme immunoassay for 17β-estradiol (E2) using biotinylated estradiol (BE) as a probe ligand, is described. In this method, E2 is detected indirectly by a solid-phase avidin–biotin binding assay, in which the biotin is immobilized on a microtiter plate (biotin-plate). After the competitive reaction between E2 and BE for the anti-E2 antibody in solution, the free E2 and BE are separated from the bound forms by means of ultrafiltration. The concentration of BE in the solution is determined from the reaction between the biotin immobilized on the plate and the free BE for the limited biotin binding sites of avidin conjugated with horseradish peroxidase (avidin–HRP), which is added to the solution. The enzymatic reaction of HRP was measured by a fluorometric analysis with the QuantaBlu™ Fluorogenic Peroxidase Substrate (QFPS) in order to detect of the avidin–biotin binding with a high degree of sensitivity. The detection limit and linear range for the determination of E2 were 0.12 nM and from 0.12 to 25 nM, respectively. The relative standard deviations (R.S.D.) for the E2 assay were between 2.2 and 9.1% ( n = 3). The cross-reactivity for several other estrogens was also evaluated.

Observation of Ligand Binding to Cytochrome P450 BM-3 by Means of Solid-State NMR Spectroscopy

A broad understanding of the binding modes of ligands and inhibitors to cytochrome P450 is vital for the development of new drugs. We investigated ligand binding in a site-specific fashion on cytochrome P450 BM-3 from Bacillus megaterium, a 119 kDa paramagnetic enzyme, using solid-state magic angle spinning nuclear magnetic resonance methods. Selective labeling and longitudinal relaxation effects were utilized to identify the peaks in a site-specific fashion and to provide evidence for binding. Well-resolved one-dimensional and two-dimensional NMR spectra of cytochrome P450 BM-3 reveal shifts upon binding of its substrate, N-palmitoylglycine. These data are consistent with the crystallographic result that a biochemically important amino acid residue, Phe87, moves upon ligation. This experimental scheme provides a tool for probing ligand binding for complex systems.

Use of Phosphorus Ligand NMR Probes To Investigate Electronic and Second-Sphere Solvent Effects in Ligand Substitution Reactions at Manganese(II) and Manganese(III)

Manganese/ligand association dynamics were studied using a series of structurally related anionic phosphorus ester ligand probes [CH(3)OP(O)(X)(Y)(-), where X = CH(3)O, CH(3)CH(2), or H and Y = O, S, or BH(3)]. Reactions of the probe ions with Mn(H(2)O)(6)(2+) and a manganese(III) porphyrin (Mn(III)TMPyP(5+)) were studied in aqueous solution by paramagnetic (31)P NMR line-broadening techniques. A satisfactory linear free energy relationship for reactions of the probe ions with Mn(H(2)O)(6)(2+) and Mn(III)TMPyP(5+) required consideration of both the basicity and solvent affinity of the probe ligands: log(k(app)) = log(k(0)) + alpha pK(a) + beta log(K(ext)), where k(0), alpha, and beta are metal complex dependent parameters and pK(a) and K(ext) represent the measured Bronsted acidity and water/n-butanol extraction constant for the probe anions, respectively. Reactions of Mn(H(2)O)(6)(2+) were relatively insensitive to changes in ligand basicity (alpha = -0.04) and favored the more hydrophilic anions (beta = -0.54). These observations are consistent with a dissociative ligand exchange mechanism wherein the outer-sphere complex is stabilized by hydrogen bonding between Mn(H(2)O)(6)(2+) and the incoming ligand. In contrast, reactions with Mn(III)TMPyP(5+) are accelerated by decreases in both the basicity (alpha = -0.43) and the hydrophilicity (beta = +0.97) of the probe. We conclude that reactions of Mn(III)TMPyP(5+) are also dissociative but that the aromatic groups of the porphyrin provide a hydrophobic environment surrounding the ligand binding site in Mn(III)TMPyP(5+). Thus, the probe/water solvent interactions must be significantly weakened in order to form the outer-sphere complex that leads to ligand substitution. This work demonstrates the utility of phosphorus relaxation enhancement (PhoRE) techniques for characterizing the second coordination sphere environment of metal complexes leading to ligation and will allow comparison of the second coordination spheres of Mn(H(2)O)(6)(2+) and Mn(III)TMPyP(5+) to those of other metal complexes.

Probing the nature of the Co(iii) ion in cobalamins: a comparison of the reaction of aquacobalamin (vitamin B12a) and aqua-10-chlorocobalamin with some anionic and N-donor ligands

To probe the cis effect of the corrin macrocycle in vitamin B12 derivatives, equilibrium constants for the substitution of coordinated H2O in aquacobalamin (vitamin B12a, H2OCbl+) and in aqua-10-chlorocobalamin, H2O-10-ClCbl+, (in which Cl has replaced the C10 H) by an exogenous ligand, L (L = an anion, NO2-, SCN-, N3-, OCN-, S2O3(2-), NCSe- or a neutral N-donor, CH3NH2, pyridine, imidazole) have been determined. The cis influence reported in the electronic spectra of the cobalamins is observed in the spectra of L-10-ClCbls as well. Anionic ligands bind more strongly to H2O-10-ClCbl+ than to H2OCbl+ with log K values between 0.10 and 0.63 (average 0.26) larger; the converse is true for the neutral N-donor ligands, where log K is between 0.17 and 0.3 (average 0.25) smaller. Semi-empirical molecular orbital (SEMO) calculations using the ZINDO/1 model on the hydroxo complexes show that charge density is delocalised from the axial donor atom to the metal and Cl. This explains why coordinated OH- is a poorer base in HO-10-ClCbl than in HOCbl and the pK(a) of H2O-10-ClCbl+ is lower than that of H2OCbl+. It further explains why, because of the ability of the metal in concert with the C10 Cl to accept charge density from the ligand, an anionic ligand will bind more strongly to Co(III) in H2O-10-ClCbl+ than in H2OCbl+. The kinetics of the replacement of coordinated H2O by two probe ligand, pyridine and azide, were determined. The rate constants for substitution of H2O in H2O-10-ClCbl+ by pyridine show saturation, whilst those for substitution by N3- do not; this is inconsistent with a purely dissociative mechanism and the reactions proceed through an interchange mechanism. The values of the activation parameters are more positive for the reaction between these ligands and H2OCbl+, than for their reaction with H2O-10-ClCbl+. This is interpreted to mean that the transition state in the reaction of H2O-10-ClCbl+ occurs earlier along the reaction coordinate. In the temperature range studied, H2O-10-ClCbl+ reacts more slowly with py and N3- than does H2OCbl+. SEMO calculations indicate that as the Co-O bond to the departing H(2)O molecule is stretched, the charge density on Co in H2OCbl+ is always lower than on Co in H2O-10-ClCbl+. This suggests that the former is a better electrophile towards the incoming ligand, and offers an explanation for the kinetics observations.