Apparent Equilibrium Binding Constant Research Articles

Thermodynamics of cytochrome c adsorption on negatively charged gold nanoparticles

In this letter, we have probed the interaction of the heme protein cytochrome c from equine heart (ECc) with negatively charged gold nanoparticles (GNPs) by second harmonic light scattering (SHLS) in solution at pH 10.75 where the protein is also negatively charged. The giant SH response generated from the GNP surface gets quenched due to adsorption of ECc and the isotherm when modelled by a modified Langmuir adsorption model produces the apparent equilibrium binding constant (K), Gibbs free energy change (ΔG) and the maximum number of adsorbed ECc (nsat) on the GNP surface. The thickness of the protein layer on the surface obtained from dynamic light scattering (DLS) experiments show that only monolayer of ECc is formed on the surface and a simple calculation put the fraction of the surface coverage to 0.5 and 0.87 for 41 and 69 nm GNPs, respectively. The enthalpic (ΔH) and entropic (ΔS) contributions to the overall free energy change in adsorption obtained from a temperature dependent SHLS measurement show that the adsorption is primarily driven by enthalpy change indicating the insignificant role played by the electrostatic repulsion between the protein and the GNP surface in the process. Noncovalent interactions seem to dictate the adsorption process while displacement of the water molecules from the surface by the protein molecules do not affect the adsorption process in any major way as evidenced from the measured entropy change.

Sulfobutylether-β-cyclodextrin/5,10,15,20-tetrakis(1-methylpyridinium-4-yl)porphine nanoassemblies with sustained antimicrobial phototherapeutic action.

Nowadays, novel less-expensive nanoformulations for in situ-controlled and safe delivery of photosensitisers (PSs) against opportunistic pathogens in body-infections areas need to be developed. Antimicrobial photodynamic therapy (aPDT) is a promising approach to treat bacterial infections that are recalcitrant to antibiotics. In this paper, we propose the design and characterization of a novel nanophototherapeutic based on the trade cyclodextrin CAPTISOL® (sulfobutylether-beta-cyclodextrin, SBE-βCD) and 5,10,15,20-tetrakis(1-methylpyridinium-4-yl)porphine tetrakis(p-toluenesulfonate) (TMPyP) to fabricate efficient biocompatible systems for aPDT. Spherical nanoassemblies of about 360nm based on CAPTISOL®/TMPyP supramolecular complexes with 1:1 stoichiometry and apparent equilibrium binding constant (Kb≅1.32×105M-1) were prepared with entrapment efficiency of≅100% by simple mixing in aqueous media and freeze-drying. These systems have been characterized by complementary spectroscopy and microscopy techniques. Time resolved fluorescence pointed out the strong interaction of porphyrin monomer within nanoassemblies (τ2≅11ns with an amount of ca 90%) and scarce self-aggregation of porphyrins have been observed. Singlet oxygen comparative determination (ϕΔ CAPTISOL®/TMPyP=0.58) assessed their photodynamic potential. Release and photostability studies have been carried out under physiological conditions pointing out the role of CAPTISOL® to sustain porphyrin release for more than 2weeks and to protect PS from photodegradation. Finally, photoantimicrobial activity of nanoassemblies vs free porphyrin have been investigated against Gram-negative P. aeruginosa, E. coli and Gram-positive S. aureus. The proposed nanosystems were able to photokill both Gram-positive and -negative bacterial cells similarly to TMPyP at MBC90=6µM of TMPyP and at 42J/cm2 light dose. However, with respect to the less selective free TMPyP in biological sites, nanoassemblies exhibit sustained release properties and a higher photostability thus optimizing the PDT effect at the site of action. These results can open routes for in vivo translational studies on nano(photo)drugs and nanotheranostics based on less expensive formulations of CD and PS.

Bacterial Model Membranes Deform ( resp. Persist) upon Ni2+ Binding to Inner Core ( resp. O-Antigen) of Lipopolysaccharides.

The surface charge densities, apparent equilibrium binding constants, and free energies of binding of nickel ions to supported and suspended lipid membranes prepared from POPC and two types of lipopolysaccharide (LPS) are reported. Second- and third-order nonlinear optical mixing shows that rough LPS (rLPS)-incorporated bilayers carry the highest charge density and provide the most binding sites for nickel ions while LPS-free bilayers exhibit the lowest charge density and fewest binding sites. Ni2+ binding is almost fully reversible at low concentrations but less so at higher Ni2+ concentrations. Ni2+ adsorption isotherms exhibit hysteresis loops. The role of interfacial depth on the observed second harmonic generation (SHG) responses is discussed in the context of complementary dynamic light scattering, X-ray spectroscopy, and cryogenic transmission electron microscopy experiments. The latter reveal considerable Ni2+-induced structural deformations to the bacterial membrane models containing the short, O-antigen-free rLPS, consistent with complex formation on the vesicle surfaces that involve Ni2+ ions and carboxylate groups in the inner core of rLPS. In contrast, Ni2+ ion complexation to the charged groups (phosphates and carboxylate) of the considerably longer O-antigen units in sLPS appears to protect the phospholipid backbone against metal binding and thus preserve the vesicle structure.

Binding Kinetics of Methylene Blue on Monolayer Graphene Investigated by Multiparameter Surface Plasmon Resonance.

In this paper, we study the interaction of a small dye molecule, namely, methylene blue (MB) with graphene surfaces using surface plasmon resonance (SPR). We show that by utilizing all of the parameters of the SPR angular dip and exploiting the fact that MB absorbs light at the operating wavelength, it is possible to detect the binding of small molecules that would otherwise not give a significant signal. The binding of MB to unmodified graphene is found to be stronger than that for gold. By studying the interaction at modified surfaces, we demonstrate that electrostatic effects play a dominant role in the binding of MB on to graphene. Furthermore, following the binding kinetics at various concentrations allows us to estimate apparent equilibrium binding and rate constants for the interaction of MB with graphene.

Importance of hydrophobic interactions in the single-chained cationic surfactant-DNA complexation

The goal of this work was to understand the key factors determining the DNA compacting capacity of single-chained cationic surfactants. Fluorescence, zeta potential, circular dichroism, gel electrophoresis and AFM measurements were carried out in order to study the condensation of the nucleic acid resulting from the formation of the surfactant-DNA complexes. The apparent equilibrium binding constant of the surfactants to the nucleic acid, Kapp, estimated from the experimental results obtained in the ethidium bromide competitive binding experiments, can be considered directly related to the ability of a given surfactant as a DNA compacting agent. The plot of ln(Kapp) vs. ln(cmc), cmc being the critical micelle concentration, for all the bromide and chloride surfactants studied, was found to be a reasonably good linear correlation. This result shows that hydrophobic interactions mainly control the surfactant DNA compaction efficiency.

Simultaneous high-throughput determination of interaction kinetics for drugs and cyclodextrins by high performance affinity chromatography with mass spectrometry detection

The individual determination of the apparent dissociation rate constant (kd,app) using high performance affinity chromatography (HPAC) is a tedious process requiring numerous separate tests and massive data fitting, unable to provide the apparent association rate constant (ka) and equilibrium binding constant (Ka). In this study, a HPAC with mass spectrometry detection (HPAC-MS/MS) was employed to determine the drug-cyclodextrin (CD) interaction kinetics with low sample loading quantity (<10 ng per injection for single compound) and high-throughput yield as twenty drugs determined in one injection. The kd,app measured by HPAC-MS/MS approach were 0.89 ± 0.07, 4.34 ± 0.01, 1.48 ± 0.01 and 7.77 ± 0.04 s−1 for ketoprofen, trimethoprim, indapamide and acetaminophen, with kd,app for acetaminophen consistent with that from the HPAC method with UV detector in our previous studies. For twenty drugs with diverse structures and chemical properties, good correlationship was found between kd,app measured by single compound analysis method and high-throughput HPAC-MS/MS approach, with the correlation coefficient of 0.987 and the significance F less than 0.001. Comprehensive quantification of ka,app, kd,app and Ka values was further performed based on the measurement of kd,app by peak profiling method and Ka by the peak fitting method. And the investigation of the drug-CD interaction kinetics under different conditions indicated that the column temperature and mobile phase composition significantly affected the determination of ka,app, kd,app and Ka while also dependent on the acidity and basicity of drugs. In summary, the high-throughput HPAC-MS/MS approach has been demonstrated high efficiency in determination of the drug-CD primary interaction kinetic parameter, especially, kd,app, being proven as a novel tool in screening the right CD for the solubilization of the right drug.

Towards the development of artificial antigen-presenting cell (APP3P.116)

Abstract T cell programming into distinct T cell subsets depends in a large extent from the quality and quantity of the signal delivered to T cells by professional antigen presenting cells (APC). However, it is difficult to vary parameters of T cell stimulation using live APC. To calibrate the strength of naïve T cell stimulation, we are developing artificial antigen-presenting cell (APC) that display nanolipoprotein particles (NLPs) presenting pMHC and other ligands incorporated into lipid bilayers built on a glass beads. Soluble analogs of the pMHC assembled on NLPs permits to control the extent of the homotypic ligand clustering and to influence the strength of T cell stimulation. We have found that the apparent equilibrium binding constants of an agonist pMHC/NiNLPs were strongly dependent on pMHC density. A higher density of pMHC on NiNLPs resulted in a much stronger response even though the concentration of the pMHC/NiNLPs at the lower pMHC density was 10-fold higher. We have also demonstrated that bilayers on glass beads were fluid allowing the mobility of pMHC/NLPs on the surface of engineered APC. In a model experiment, T cells concentrated the ICAM-1/pMHC/NLPs conjugates at the contact surface indicating that these conjugates were able to move on the bilayers mimicking direction stimulation of the T cells with live APC. These and other data will be discussed to demonstrate the utility of engineered APC for stimulation of naïve T cells ex vivo.

Modeling immune receptor clustering with nano-scale discoidal membrane bilayers (P3350)

Abstract MHC proteins on antigen-presenting cells form clusters with each other and other cell surface ligands. The changes in the clustering serve as a sensitive mechanism to modulate T cell responses. We propose to mimic MHC membrane patches using nanolipoprotein particles (NLPs). NLPs are nano-scale discoidal lipid bilayers stabilized by apolipoproteins. Nickel-chelating lipids incorporated into the NLP bilayer capture soluble polyhistidine-tagged pMHC. We have studied the interactions of cognate (non-self) and non-cognate (self) pMHC/NLPs with CD8+ CTL. Both self and non-self pMHC/NLPs were bound to the T-cell surface but to different extents. The apparent equilibrium binding constants of both non-self and self pMHC/NLPs were strongly dependent on pMHC density. A 3-fold difference in pMHC density (from 10:1 to 30:1 per NLP) caused 10-fold and 30-fold increases in the apparent affinity, respectively. Surprisingly, the avidity of self and non-self multivalent interactions appears to be similar when the pMHC density on the NLP surface is high. The similar avidity may be explained by a very rapid on-rate of CD8 binding to clustered MHC and marginal difference in the dissociation rates at the high epitope density resulting from very efficient CD8-MHC rebinding. NLP-based model MHC clusters with pre-determined properties could be incorporated onto glass beads coated with a lipid bilayer to engineer artificial APC with the goal of inducing ex vivo long-lived T-cell effectors for adoptive T-cell therapy.

Mechanism of Binding of the Inhibitor (E)-3-(Furan-2-yl)-N-hydroxyacrylamide to a Histone Deacetylase-like Amidohydrolase

Histone deacetylases have proven to be attractive novel targets for the treatment of cancer. The first inhibitor of histone deacetylases was approved for the treatment of cutaneous T-cell lymphoma in 2006. The identification of new lead structures with improved effectiveness and fewer side effects is necessary. This report investigates the mechanism of inhibition of a histone deacetylase-like amidohydrolase by stopped-flow and equilibrium titration techniques. The interaction between the inhibitor (E)-3-(furan-2-yl)-N-hydroxyacrylamide and the enzyme generates a fluorescence resonance energy transfer from the intrinsic tryptophan residues of the enzyme to the chromophore of the inhibitor. The apparent equilibrium binding constant was determined to be 1.9 muM. Several independent experimental results provide evidence of the existence of solely one HDAH conformer. The association kinetics showed two phases representing two unimolecular processes. Kinetic arguments and accurate investigation of the very fast time range suggest a fast pre-equilibrium, in which the inhibitor binds to the surface of the enzyme. In the next step, the first complex undergoes a conformational change that allows the inhibitor to translocate into the active site. Finally, the intermediate complex is stabilized by another conformational rearrangement. All kinetic data are in agreement with a reversible three-step mechanism and analyzed using a global fit, yielding the association constant of the pre-equilibrium (K(1) = 0.28 x 10(6) M(-1)) and the forward and reverse rate constants of the consecutive conformational changes (k(2) = 6.6 s(-1), k(-2) = 1.5 s(-1), k(3) = 0.8 s(-1), and k(-3) = 0.3 s(-1)).

Competitive Binding of Off‐Flavor Compounds with Soy Protein and β‐Cyclodextrin in a Ternary System: A Model Study

AbstractRemoval of soy protein (SP)‐bound 2‐nonanone by β‐cyclodextrin (βCD) was studied using an equilibrium dialysis technique. It was observed that in the presence of βCD, a significant (p &lt; 0.05) amount of SP‐bound 2‐nonanone could be removed in a concentration‐dependent manner. Up to 94% of SP‐bound 2‐nonanone was stripped from SP when 6 mM βCD was present in the system. However, in thermodynamic terms, the net standard free energy change for transfer of 2‐nonanone from SP to βCD, i.e., , was essentially zero, implying that the apparent equilibrium binding constant for the formation of βCD–2‐nonanone complex was essentially same as that for the SP–2‐nonanone complex formation in the ternary system. This indicated that stripping‐off of 2‐nonanone from SP by βCD was driven by the mass action ratio. Based on these results, it is shown that βCD can be used effectively for removing SP‐bound off‐flavor carbonyl compounds.

The DNA-recognition mode shared by archaeal feast/famine-regulatory proteins revealed by the DNA-binding specificities of TvFL3, FL10, FL11 and Ss-LrpB

The DNA-binding mode of archaeal feast/famine-regulatory proteins (FFRPs), i.e. paralogs of the Esherichia coli leucine-responsive regulatory protein (Lrp), was studied. Using the method of systematic evolution of ligands by exponential enrichment (SELEX), optimal DNA duplexes for interacting with TvFL3, FL10, FL11 and Ss-LrpB were identified as TACGA[AAT/ATT]TCGTA, GTTCGA[AAT/ATT]TCGAAC, CCGAAA[AAT/ATT]TTTCGG and TTGCAA[AAT/ATT]TTGCAA, respectively, all fitting into the form abcdeWWWedcba. Here W is A or T, and e.g. a and a are bases complementary to each other. Apparent equilibrium binding constants of the FFRPs and various DNA duplexes were determined, thereby confirming the DNA-binding specificities of the FFRPs. It is likely that these FFRPs recognize DNA in essentially the same way, since their DNA-binding specificities were all explained by the same pattern of relationship between amino-acid positions and base positions to form chemical interactions. As predicted from this relationship, when Gly36 of TvFL3 was replaced by Thr, the b base in the optimal DNA duplex changed from A to T, and, when Thr36 of FL10 was replaced by Ser, the b base changed from T to G/A. DNA-binding characteristics of other archaeal FFRPs, Ptr1, Ptr2, Ss-Lrp and LysM, are also consistent with the relationship.

Positive and negative ion mode ESI-MS and MS/MS for studying drug–DNA complexes

We report systematic investigation of duplex DNA complexes with minor groove binders (Hoechsts 33258 and 33342, netropsin and DAPI) and intercalators (daunomycin, doxorubicin, actinomycin D, ethidium, cryptolepine, neocryptolepine, m-Amsacrine, proflavine, ellipticine and mitoxantrone) by ESI-MS and ESI-MS/MS in the negative ion mode and in the positive ion mode. The apparent solution phase equilibrium binding constants can be determined by measuring relative intensities in the ESI-MS spectrum. While negative ion mode gives reliable results, positive ion mode gives a systematic underestimation of the binding constants and even a complete suppression of the complexes for intercalators lacking functional groups capable of interacting in the grooves. In the second part of the paper we systematically compare MS/MS fragmentation channels and breakdown curves in the positive and the negative modes, and discuss the possible uses and caveats of MS/MS in drug–DNA complexes. In the negative mode, the drugs can be separated in three groups: (1) those that leave the complex with no net charge; (2) those that leave the complex with a negative charge; and (3) those that remain attached on the strands upon dissociation of the duplex due to their positive charge. In the positive ion mode, all complexes fragment via the loss of protonated drug. Information on the stabilization of the complex by drug–DNA noncovalent interactions can be obtained straightforwardly only in the case of neutral drug loss. In all other cases, proton affinity (in the positive ion mode), gas-phase basicity (in the negative ion mode) and coulombic repulsion are the major factors influencing the fragmentation channel and the dissociation kinetics.

Modulation, via Protein-Protein Interactions, of Glyceraldehyde-3-phosphate Dehydrogenase Activity through Redox Phosphoribulokinase Regulation

The activity of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) embedded in the phosphoribulokinase (PRK).GAPDH.CP12 complex was increased 2-3-fold by reducing agents. This occurred by interaction with PRK as the cysteinyl sulfhydryls (4 SH/subunit) of GAPDH within the complex were unchanged whatever the redox state of the complex. But isolated GAPDH was not activated. Alkylation plus mass spectrometry also showed that PRK had one disulfide bridge and three SH groups per monomer in the active oxidized complex. Reduction disrupted this disulfide bridge to give 2 more SH groups and a much more active enzyme. We assessed the kinetics and dynamics of the interactions between PRK and GAPDH/CP12 using biosensors to measure complex formation in real time. The apparent equilibrium binding constant for GAPDH/CP12 and PRK was 14 +/- 1.6 nm for oxidized PRK and 62 +/- 10 nm for reduced PRK. These interactions were neither pH- nor temperature-dependent. Thus, the dynamics of PRK.GAPDH.CP12 complex formation and GAPDH activity are modulated by the redox state of PRK.

Recoverin is a zinc-binding protein.

Recoverin is an N-myristoylated 23 kDa calcium-binding protein from retina, which modulates the Ca2+-sensitive deactivation of rhodopsin via Ca2+-dependent inhibition of rhodopsin kinase. It was shown by intrinsic and bis-ANS probe fluorescence, circular dichroism, and differential scanning calorimetry that myristoylated recombinant recoverin interacts specifically with zinc ions. Similar to the calcium binding, the binding of zinc to Ca2+-loaded recoverin additionally increases its alpha-helical content, hydrophobic surface area, and environmental mobility/polarity of its tryptophan residues. In contrast to the calcium binding, the binding of zinc decreases thermal stability of the Ca2+-loaded protein. Zn2+-titration of recoverin, traced by bis-ANS fluorescence, reveals binding of a single Zn2+ ion per protein molecule. It was shown that the double-mutant E85Q/E121Q with inactivated Ca2+-binding EF-hands 2 and 3 (Alekseev, A. M.; Shulga-Morskoy, S. V.; Zinchenko, D. V.; Shulga-Morskaya, S. A.; Suchkov, D. V.; Vaganova, S. A.; Senin, I. I.; Zargarov, A. A.; Lipkin, V. M.; Akhtar, M.; Philippov, P. P. FEBS Lett. 1998, 440, 116-118), which can be considered as an analogue of the apo-protein, binds Zn2+ ion as well. Apparent zinc equilibrium binding constants evaluated from spectrofluorimetric Zn2+-titrations of the protein are 1.4 x 10(5) M(-1) (dissociation constant 7.1 microM) for Ca2+-loaded wild-type recoverin and 3.3 x 10(4) M(-1) (dissociation constant 30 microM) for the E85Q/E121Q mutant (analogue of apo-recoverin). Study of the binding of wild-type recoverin to ROS membranes showed a zinc-dependent increase of its affinity for the membranes, without regard to calcium content, suggesting further solvation of a protein myristoyl group upon Zn2+ binding. Possible implications of these findings to the functioning of recoverin are discussed.

Cytotoxicity, DNA binding and localisation of novel bis-naphthalimidopropyl polyamine derivatives

Bis-naphthalimidopropyl spermidine (BNIPSpd), spermine (BNIPSpm) and oxa-spermine (BNIPOSpm) showed high in vitro cytotoxicity against human breast cancer MCF-7 cells with IC 50 values of 1.38, 2.91 and 8.45 μM, respectively. These compounds were found to effectively displace the intercalating agent ethidium bromide bound to the calf thymus DNA using fluorimetric methods ( C 50 0.08–0.12 μM) and their apparent equilibrium binding constants ( K app) were calculated to be in the range of 10.5–18×10 7 M −1. Furthermore, strong stabilisation of calf thymus DNA duplex in the presence of bis-naphthalimidopropyl polyamine derivatives (BNIPSpd, BNIPSpm and BNIPOSpm) was observed by UV spectrophotometric analysis ( T m=93.3–97°C compared with 75°C for calf thymus DNA without drug). Because of their inherent fluorescence, these compounds were localised preferentially inside the nucleus as evidenced by their direct observation under the fluorescence microscope. The results obtained suggest that the cytotoxic activity of the bis-naphthalimidopropyl polyamines may be in part, caused by their effects on DNA.

Purification of a Soluble UmuD′C Complex from Escherichia coli: COOPERATIVE BINDING OF UmuD′C TO SINGLE-STRANDED DNA

The Escherichia coli UmuD' and UmuC proteins play essential roles in SOS-induced mutagenesis. Previous studies investigating the molecular mechanisms of mutagenesis have been hindered by the lack of availability of a soluble UmuC protein. We report the extensive purification of a soluble UmuD'C complex and its interactions with DNA. The molecular mass of the complex is estimated to be 70 kDa, suggesting that the complex consists of one UmuC (46 kDa) and two UmuD' (12 kDa) molecules. In contrast to its inability to bind to double-stranded DNA, UmuD'C binds cooperatively to single-stranded DNA as measured by agarose gel electrophoresis and confirmed by steady-state fluorescence depolarization. A Hill coefficient, n = 3, characterizes the binding of UmuD'C to M13 DNA and to a 600 nucleotide DNA oligomer, suggesting that at least three protein complexes may interact cooperatively when binding to DNA. The apparent equilibrium binding constant of UmuD'C to single-stranded DNA is approximately 300 nM. Binding of the complex to a short, 80 nucleotide, DNA oligonucleotide was detectable by fluorescence depolarization, but it did not appear to be cooperative. Binding of UmuD'C to single-stranded M13 DNA causes an acceleration of the protein-DNA complex, suggesting that the longer DNA may undergo compaction. The UmuD'C complex associates with RecA-coated DNA, and the UmuD'C complex remains bound to DNA in the presence of RecA.

Estimation of intracellular calcium activity in confluent monolayers of primary cultures of quail medullary bone osteoclasts.

The present study was concerned with an investigation of the relative utilities of the fluorescent indicators fura-2 and fluo-3 for spectrofluorimetric estimation of the intracellular calcium concentration ([Ca2+]i) in confluent osteoclast monolayers that had been isolated from medullary bone of quail hens and maintained in tissue culture for 6-8 days. Additionally, we have determined the effects of raised extracellular calcium ([Ca2+]o) and chicken calcitonin (CT) on [Ca2+]i in this preparation. Relative to fura-2, fluo-3 was poorly incorporated into the osteoclasts and had a high apparent equilibrium binding constant (Kd) for Ca2+ binding (809 nM). The osteoclasts were only weakly sensitive to the calcium ionophore, ionomycin. It is concluded that fura-2 is of greater utility than furo-3 in this preparation. In contrast to its lack of effect in freshly isolated cells, elevated [Ca2+]o up to 20 mM stimulated a concentration-dependent increase in [Ca2+]i in cultured osteoclasts, but CT was without effect. These findings further support the idea that quail osteoclasts are able to acquire a Ca2+ sensor or 'receptor' and thus to respond to [Ca2+]o in a similar manner to mammalian osteoclasts when they are removed from the bone microenvironment, but retain a refractoriness to CT under these conditions.

Systemic absorption and anticholinergic activity of topically applied tropicamide.

We studied the plasma levels and systemic anticholinergic activity of tropicamide after ocular administration in eight women. Two 40 microliters drops of 0.5% tropicamide were instilled into the lower cul-de-sac of one eye of the subjects and concentrations and respective muscarinic receptor occupancy of tropicamide in plasma were monitored using radioligand binding techniques. Tropicamide was rapidly absorbed systemically with the mean peak concentration in plasma being 2.8 +/- 1.7 ng/ml (mean +/- SD) at five minutes after instillation. Tropicamide disappeared rapidly from the systemic circulation: drug concentration in plasma was 0.46 +/- 0.51 ng/ml (mean +/- SD) at 60 minutes and below 240 pg/ml at 120 minutes after instillation. Tropicamide bound to muscarinic receptors of rat brain with an apparent equilibrium binding constant (Ki-value in plasma) 220 +/- 25 nM (mean +/- SD, n = 3). Tropicamide occupied maximally 8% of muscarinic receptors in plasma after ocular application. The low affinity of tropicamide for muscarinic receptors and its negligible receptor occupancy in plasma can explain the low incidence of systemic side-effects of tropicamide eyedrops.

Comparative study of adsorption behavior of copper, lead, and zinc onto goethite in aqueous systems

The adsorption of lead(II) and zinc(II) onto goethite was studied as a function of pH, total dissolved metal concentration, surface area of goethite, and ionic strength. The results for zinc and lead were compared with those for copper reported earlier. The adsorption edge of lead ranges from pH 4 to 7, similar to that of copper, but the adsorption edge of zinc is displaced by 1.5 pH units toward higher pH. A fourfold increase in goethite surface area had a significant effect on the adsorption edge of lead, but a tenfold increase in the ionic strength of the medium did not affect the adsorption edge of lead and zinc. At neutral pH, 50 percent of the zinc was still available for transport and reactions in aqueous solution, whereas almost 100 percent of the lead and copper were bound to the goethite surface. The distribution coefficients increase sharply with the increase in pH and ranged from 60 to 30,000 ml/g in 2.5 pH units for lead and from 60 to 3000 ml/g in 1.5 pH units for zinc, depending on the goethite surface area and metal concentration. Distribution coefficients were used to calculate the number of protons released per mole of metal adsorbed during the adsorption process, with the average number of protons released per mole of lead and zinc adsorbed estimated to be 0.97±0.07 and 1.32±0.06, respectively. Proton coefficients of copper, lead, and zinc were correlated to their ionic radii and apparent equilibrium binding constant. Although the adsorption behavior of copper and lead were similar and both have the same charge, the drop in pH per mole of metal adsorbed is more in copper than in lead.

Determination of Rate and Equilibrium Binding Constants for Macromolecular Interactions Using Surface Plasmon Resonance: Use of Nonlinear Least Squares Analysis Methods

Surface plasmon resonance (SPR) is a label-free, real time, optical detection method which has recently been commercialized as the BIAcore (Pharmacia). The technique relies on the immobilization of one of the interactants, the ligand, onto a dextran-coated gold surface. The second interactant, the ligate, is then injected across the surface and the interaction of the soluble ligate with the immobilized ligand is observed continuously and directly. The process of dissociation of bound ligate may also be observed directly after the sample plug has traversed the layer. Thus, the data generated contain information on the kinetic rate and equilibrium binding constants for the interaction under investigation. Historically, data from this instrument have been analyzed in terms of linear transformations of the primary data and requires that data from several ligate concentrations be analyzed to determine a single value for the association and dissociation rate constants. Here we discuss the analysis of untransformed BIAcore data by nonlinear least squares methods. The primary data are analyzed according to the integrated rate equations which describe the kinetics of the interaction of soluble ligate with immobilized ligand and the dissociation of the formed complex from the surface, respectively. Such analyses allow the direct determination of the association and dissociation rate constants for each binding experiment and, further, allow the analysis of data over a wider concentration range with lower associated errors compared to previously described methods. Through the use of modeling these interactions, we also demonstrate the limitations in determining the dissociation rate constant from the association phase of the interaction, thereby requiring that the dissociation process be analyzed. Indeed, the dissociation phase should be analyzed first to yield a relatively precise and unambiguous value of the dissociation rate constant, k d , which can then be used to constrain the analysis of the association phase to yield a better estimate of the association rate constant, k a . We further demonstrate that, at least for the interaction investigated, the apparent rate and equilibrium binding constants determined using SPR are concentration independent and can be determined with good reproducibility.