Determination Of Binding Constants Research Articles

Single-Molecule Fluorescence Spectroscopy of the Structure and Dynamics of the Spliceosomal Complex

Binding reactions can be ideally investigated by Dual-Color Fluorescence Cross-Correlation Spectroscopy (FCCS). Here, this method is used to study the recruitment and dissociation of proteins to and from the spliceosome. The spliceosome is the cellular machinery responsible for removing non-coding introns from precursor mRNA (pre-mRNA). The spliceosome assembles on a pre-mRNA, which consists of two exons and one intron, stepwise by the binding of five snRNPs and several other proteins. This leads to the formation of the B complex which has no active catalytic center yet. Structural rearrangements lead to the activated Bact complex which is formed by further rearrangements in the catalytically active B* complex. The B* complex catalyzes the first splicing step in which the branch site (BS) attacks the 5' splice site (5'SS). The 5' exon is cleaved and the intron-3'-exon lariat is formed. The resulting C complex then catalyzes the second splicing step. The intron is cleaved and both exons are joined together forming mature mRNA. During both catalytic steps, the spliceosome undergoes many changes in its protein composition, and several structural rearrangements occur. These changes and rearrangements can be investigated by standard biochemical methods. However, these methods often do not provide data about dynamics, which are necessary for understanding the recruitment and release of particular molecules. One method which provides these information is FCCS. In contrast to methods like mass spectrometry, in FCCS measurements it is possible to observe reactions in real-time and at equilibrium without further biochemical perturbation of the sample. FCCS works at low nanomolar concentrations and only small sample volumes are necessary. Using FCCS, it could be determined how changes in the spliceosome composition and conformation occur (simultaneously or consecutively). The roles of certain spliceosomal RNA helicases in the restructuring of the complex could be investigated. FCCS enables the observation of protein-protein interactions and the determination of binding constants for proteins to the spliceosome. In order to better understand the dynamic nature of the spliceosome during its catalytic activation, the step 1 factor Cwc25 and the step 2 factors Slu7, Prp18, and Prp16 were investigated. Their role in the maturation process, the fundamental question of the time point and manner of their recruitment has not been answered yet. With FCCS, the binding of the step 1 factor Cwc25 was observed. It was shown that Cwc25 has a high-binding affinity to the spliceosome after the Prp2-mediated rearrangements into the catalytically activated B* complex. The high-binding affinity was reflected in a strong binding constant of 30 pM which was measured with FCCS. By using several mutant pre-mRNAs it could be demonstrated that Cwc25's release does not depend on the second catalytic step per se. Its dissociation depends on the docking of the 3'SS to the active site and the action of Prp16 and Slu7/Prp18. It was also observed that the distance between the branch site and 3'SS influences the release of Cwc25 and the second catalytic step. If the distance between BS and 3'SS is short (e.g. 7 nucleotides long), the distance between the 3'SS and the active site is also small. The 3'SS can dock into the active site without the further stabilization by Slu7/Prp18. The activity of Prp16 and the docking event lead to the release of Cwc25. Slu7/Prp18 can stabilize the system and thereby induce further release of Cwc25. If the distance between BS and 3'SS is longer (e.g. 38 nucleotides), Slu7/Prp18 has to stabilize the interaction of the 3'SS and the active site and the step 2 conformation. In this case, Prp16 alone cannot induce the release of Cwc25. Cwc25 does dissociate from the spliceosome after the action of Prp16 and in the presence of Slu7/Prp18. It was shown further that Prp16 and its ATPase activity are necessary for the formation of a functional step 2 active site. Slu7/Prp18 are required for the efficient docking of the 3' splice site (3'SS) to the active site. The FCCS experiments showed that Prp16 and Slu7/Prp18 have distinct binding sites in the spliceosome which are formed during the catalytic activation. During activation their first low-affinity binding sites are transformed into high-affinity binding sites. Both proteins are bound to the spliceosome at an early stage so that they are present before their catalytic function is actually required in the process. Here, it was shown that FCCS is ideally suited to investigate macromolecular protein complexes like the spliceosome and is a powerful tool for studying quantitatively spliceosomal protein dynamics at equilibrium.

Evidence of Direct Binding of G-Actin and Calmodulin to PMCA by Surface Plasmon Resonance

Previous studies of our laboratory suggest that the main isoform of the plasma membrane calcium ATPase (PMCA) from erythrocytes, PMCAh4b, associates with the actin cytoskeleton and its catalytic activity can be regulated through this interaction. Apparently, G-actin and/or short oligomers enhance the catalytic activity of the pump, while F-actin seems to inhibit it. To further explore the interaction between PMCA and G-actin, we performed binding experiments based on Surface Plasmon Resonance technology (SPR) using purified PMCAh4b from erythrocytes and purified rabbit muscle G-actin on a BIAcore T100 system. Our strategy consisted in immobilizing G-actin to the sensor chip surface while PMCA constituted the analyte (125 nM to 2 μM). The sensorgrams obtained showed an increase in the response as a function of PMCA concentration with a saturable binding. The unspecific binding values were subtracted from the response obtained in the control flow cell (without immobilized G-actin). The apparent KD (1.25 μM) was determined by kinetic analysis with parameters kon (1.33±0.07) 104 M-1s-1 and koff (1.66±0.03) 10-2 s-1.Conversely, we immobilized PMCAh4b to the sensor chip surface. The analytes were (i) G-actin (0.6 to 5 μM) or (ii) Calmodulin, a known activator of PMCAh4b (1.8 to 15 nM). Results show that G-actin binds to the activated sensor with KD=3μM, kon (2.98±0.45) 104 M-1s-1 and koff (8.98±0.67) 10-2 s-1 and calmodulin binds with an apparent KD of (1.14±0.6) 10-8 M. This last value agrees with the known value of calmodulin binding affinity determined by other methods.These results show a specific and high apparent affinity binding between PMCA and G-actin and reveal the usefulness of SPR technology to determine binding constants between small molecules and a large membrane protein like the plasmatic calcium pump. With grants of ANPCYT/CONICET/UBACYT.

Spectroscopic Exploration of Mode of Binding of ctDNA with 3-Hydroxyflavone: A Contrast to the Mode of Binding with Flavonoids Having Additional Hydroxyl Groups

Binding interaction of 3-hydroxyflavone (3HF), a bioactive flavonoid, with calf-thymus DNA (ctDNA) has been explored exploiting various experimental techniques. The dual fluorescence of 3HF resulting from the excited state intramolecular proton transfer (ESIPT) is modified remarkably upon binding with the biomacromolecule. The determined binding constant, fluorescence quenching experiment, circular dichroism (CD) study, comparative binding study with the known intercalative binder ethidium bromide and thermometric experiment relating to the helix melting of ctDNA confirm the groove binding of 3HF with the DNA. This is in contrast to two other members of the flavonoid group, namely, fisetin and quercetin, where the bindings are established to be intercalative. The structural difference of 3HF from the other two probes with respect to the absence/presence of the additional hydroxyl groups is ascribed to be responsible for the difference in the mode of binding.

Cover Picture: Electrophoresis 24'2011

AbstractIssue no. 24 is in one major part a Focus Issue on Glycomics and Glycoproteomics that includes 12 contributions “describing the development of new analytical methods or improvement of existing methods, permitting enhanced and sensitive analysis of glycans and glycoproteins.” The first part of the Focus Issue “includes two review articles critically evaluating the current status of glycan analysis by both hydrophilic interaction liquid chromatography and capillary electrophoresis‐mass spectrometry.” The second part of this Focus Issue “includes 4 research articles describing improved methods for the analysis of glycans by capillary electrophoresis with laser‐induced fluorescence detection.” The third part “includes 4 research articles describing new mass spectrometry and tandem mass spectrometry methods for the sensitive characterization of glycans and glycopeptides. Last but not least, 2 research articles are included in the fourth part, describing the analysis of glycoproteins by SDS‐urea agarose polyacrylamide composite gel electrophoresis and magnetic bead array‐coupled to tandem mass spectrometry.” Issue 24 has also an additional eight regular contributions emphasizing interesting studies on proteins and proteomics.Featured articles include:Site‐specific characterisation of densely O‐glycosylated mucin‐type peptides using electron transfer dissociation ESI‐MS/MS (10.1002/elps.201100294))Evaluation of non‐reductive ?‐elimination/Michael‐addition for glycosylation site determination in mucin‐like O‐glycopeptides (10.1002/elps.201100393))High‐throughput lectin magnetic bead array‐coupled tandem mass spectrometry (LeMBA‐MS) for glycoprotein biomarker discovery (10.1002/elps.201100341))Rapid determination of protein binding constant by a pressure‐mediated affinity capillary electrophoresis method (10.1002/elps.201100211))Plasma proteomics for biomarker discovery: A study in blue (10.1002/elps.201100307))

Rapid determination of protein binding constant by a pressure‐mediated affinity capillary electrophoresis method

A new pressure-mediated affinity capillary electrophoresis method for the rapid and accurate determination of drug-protein binding constants is described. A special combination of pressure and electrophoresis is used to shorten the electrophoresis and the overall analysis time to only a few minutes. At the same time, the suitability of this method is checked against a traditional fluorescence spectroscopy method. The binding constants of bovine serum albumin and a total of eight drugs with different pK(a) have been evaluated and compared with those determined by the fluorescence spectroscopy method and other methods in literature. The results indicate that the P-ACE method is well suited for the determination of binding constants with weak interaction (K(b) <10(5) M(-1)).

Generally Applicable NMR Titration Methods for the Determination of Equilibrium Constants for Coordination Complexes: Syntheses and Characterizations of Metallacrown Ethers with α,ω-Bis(phosphite)-polyether Ligands and Determination of Equilibrium Binding Constants to Li+

A new metallacrown ether, cis-PdCl2{(2,2′-C12H8O2)P(OCH2CH2)4OP(2,2′-O2H8C12)}, has been synthesized and fully characterized by NMR spectroscopy and X-ray crystallography. A new synthetic method and complete NMR characterization for the previously reported and closely related cis-Mo(CO)4(2,2′-C12H8O2)P(OCH2CH2)4OP(2,2′-O2H8C12) metallacrown ether are also reported. Both complexes are monomeric, exhibiting cis coordination geometries both in solution and in the solid state, and the cis-Mo(CO)4(2,2′-C12H8O2)P(OCH2CH2)4OP(2,2′-O2H8C12) metallacrown ether does not appear to undergo cis–trans isomerization in the presence of HgCl2. These results are surprising because the closely related PdCl2(Ph2P(CH2CH2O)nCH2CH2PPh2) (n = 3–5) metallacrown ethers exhibit both cis–trans and monomer–cyclic oligomer equilibria in solution, and cis-Mo(CO)4 (Ph2P(CH2CH2O)nCH2CH2PPh2) (n = 3–5) metallacrown ethers rapidly undergo cis–trans isomerization in the presence of HgCl2. The coordination of Li+ cations to the cis-Mo(CO)4(2...

Binding of benzodiazepine drugs to bovine serum albumin: A second derivative spectrophotometric study

The binding constants ( K values) of three benzodiazepine drugs to bovine serum albumin were determined by a second derivative spectrophotometric method. Despite the sample and reference samples were prepared in the same way to maintain the same albumin content in each sample and reference pair, the absorption spectra show that the baseline compensation was incomplete because of the strong background signals caused by bovine serum albumin. Accordingly, further quantitative spectral information could not be obtained from these absorption spectra. On the other hand, the calculated second derivative spectra clearly show isosbestic points indicating the complete removal of the residual background signal effects. Using the derivative intensity differences (Δ D values) of the studied benzodiazepine drugs before and after the addition of albumin, the binding constants were calculated and obtained with R.S.D. of less than 8%. The interactions of drugs with bovine serum albumin were investigated using Scatchard's plot. In addition, the consistency between the fractions of bound benzodiazepine calculated from the obtained K values and the experimental values were established. The results indicate that the second derivative method can be advantageously applicable to the determination of binding constants of drugs to serum albumin without prior separation. Moreover, the validity of the proposed method was confirmed.

Luminescent Lanthanide-Functionalized Gold Nanoparticles: Exploiting the Interaction with Bovine Serum Albumin for Potential Sensing Applications

As luminescent surface-functionalized gold nanoparticles emerged as potential powerful analytical tools in the biomedical fields, understanding the interaction of such systems with proteins has become crucial. In the present study, the interaction of luminescent water-soluble gold nanoparticles (AuNP-1·Eu-nta), obtained through the self-assembly of a naphthalene β-diketone antenna with a Eu(III) cyclen complex tethered to the gold surface via a C(12) alkyl thiol spacer, with bovine serum albumin (BSA) was investigated. The changes in the UV-visible absorption and fluorescence spectra of both the antenna and protein, as well as in the time-resolved Eu(III)-centered emission, of the resulting self-assembly were monitored, at physiological pH, as a function of the BSA concentration. We demonstrate that the Eu(III) emission arising from the self-assembly on the AuNP surface is almost completely quenched upon addition of BSA. Binding constant determination clearly showed that the sensitizing antenna was not displaced and that the quenching was the result of the interaction between the antenna and BSA. Detailed spectroscopic studies performed on the nta-BSA system brought a better insight in the strength of such interaction as well as its effect on the protein secondary structure. Finally, the information gathered on each system resulted in applying AuNP-1·Eu-nta-BSA for the luminescent detection of drugs via the perturbation of the nta-BSA interaction. Competitive titrations using ibuprofen and warfarin showed that nta was located in the binding site II of BSA and that the presence of warfarin, a site I drug, did not interfere with the detection of site II ibuprofen.

Energetics of Halogen Bonding of Group 10 Metal Fluoride Complexes

A study is presented of the thermodynamics of the halogen-bonding interaction of C(6)F(5)I with a series of structurally similar group 10 metal fluoride complexes trans-[Ni(F)(2-C(5)NF(4))(PCy(3))(2)] (2), trans-[Pd(F)(4-C(5)NF(4))(PCy(3))(2)] (3), trans-[Pt(F){2-C(5)NF(2)H(CF(3))}(PR(3))(2)] (4a, R = Cy; 4bR = iPr) and trans-[Ni(F){2-C(5)NF(2)H(CF(3))}(PCy(3))(2)] (5a) in toluene solution. (19)F NMR titration experiments are used to determine binding constants, enthalpies and entropies of these interactions (2.4 ≤ K(300) ≤ 5.2; -25 ≤ ΔH(o) ≤ -16 kJ mol(-1); -73 ≤ ΔS(o) ≤ -49 J K(-1) mol(-1)). The data for -ΔH(o) for the halogen bonding follow a trend Ni < Pd < Pt. The fluoropyridyl ligand is shown to have a negligible influence on the thermodynamic data, but the influence of the phosphine ligand is significant. We also show that the value of the spin-spin coupling constant J(PtF) increases substantially with adduct formation. X-ray crystallographic data for Ni complexes 5a and 5c are compared to previously published data for a platinum analogue. We show by experiment and computation that the difference between Pt-X and Ni-X (X = F, C, P) bond lengths is greatest for X = F, consistent with F(2pπ)-Pt(5dπ) repulsive interactions. DFT calculations on the metal fluoride complexes show the very negative electrostatic potential around the fluoride. Calculations of the enthalpy of adduct formation show energies of -18.8 and -22.8 kJ mol(-1) for Ni and Pt complexes of types 5 and 4, respectively, in excellent agreement with experiment.

Methods for determination of all binding parameters in systems with simultaneous borate and cyclodextrin complexation

Two novel methods for determination of binding constants in the systems with borate and cyclodextrin complexation were developed. The methods enable to determine all binding parameters in these systems and even the binding constants of interaction of a neutral analyte with a neutral cyclodextrin. The first method is based on nonlinear fitting of experimental data and further evaluation of fitting parameters. The second method requires a multiple regression. The methods provide identical results with low experimental error. Only one set of measurements is required for both methods. Thus the binding parameters can be mutually compared. The binding parameters for neutral analytes ((R,R)-(+)-hydrobenzoin and (S,S)-(−)-hydrobenzoin) and neutral cyclodextrin (heptakis(2,6-di-O-methyl)-β-cyclodextrin) were evaluated and the effect of individual types of interaction was revealed. The interaction of the analytes with cyclodextrin governs the chiral recognition, while the complexation of analyte with borate is responsible for electromigration. Very low values of the binding constants of mixed analyte–cyclodextrin–borate complexes indicate that this type of complexation has negligible effect on enantioseparation.

Some theoretical and practical aspects in the separation of humic substances by combined liquid chromatography methods

Permanent need to understand nature, structure and properties of humic substances influences also separation methods that are in a wide scope used for fractionation, characterization and analysis of humic substances (HS). At the first glance techniques based on size-exclusion phenomena are the most useful and utilized for relating elution data to the molecular mass distribution of HS, however, with some limitations and exceptions, respectively, in the structural investigation of HS. The second most abundant separation mechanism is reversed-phase based on weak hydrophobic interactions beneficially combined with the step gradients inducing distinct features in rather featureless analytical signal of HS. Relatively great effort is invested to the developments of immobilized-metal affinity chromatography mimicking chelate-forming properties of HS as ligands in the environment. Surprisingly, relatively less attention is given to the ion-ion interactions based ion-exchange chromatography of HS. Chromatographic separation methods play also an important role in the examination of interactions of HS with pesticides. They allow us to determine binding constants and the other data necessary to predict the mobility of chemical pollutants in the environment. HS is frequently adversely acting in analytical procedures as interfering substance, so more detailed information is desired on manifestation of its numerous properties in analytical procedures. The article topic is covered by the review emphasizing advances in the field done in the period of last 10 years from 2000 till 2010.

Large-scale integrated super-computing platform for next generation virtual drug discovery

Traditional drug discovery starts by experimentally screening chemical libraries to find hit compounds that bind to protein targets, modulating their activity. Subsequent rounds of iterative chemical derivitization and rescreening are conducted to enhance the potency, selectivity, and pharmacological properties of hit compounds. Although computational docking of ligands to targets has been used to augment the empirical discovery process, its historical effectiveness has been limited because of the poor correlation of ligand dock scores and experimentally determined binding constants. Recent progress in super-computing, coupled to theoretical insights, allows the calculation of the Gibbs free energy, and therefore accurate binding constants, for usually large ligand–receptor systems. This advance extends the potential of virtual drug discovery. A specific embodiment of the technology, integrating de novo, abstract fragment based drug design, sophisticated molecular simulation, and the ability to calculate thermodynamic binding constants with unprecedented accuracy, are discussed.

New potential antitumoral fluorescent tetracyclic thieno[3,2-b]pyridine derivatives: interaction with DNA and nanosized liposomes

Fluorescence properties of two new potential antitumoral tetracyclic thieno[3,2-b]pyridine derivatives were studied in solution and in liposomes of DPPC (dipalmitoyl phosphatidylcholine), egg lecithin (phosphatidylcholine from egg yolk; Egg-PC) and DODAB (dioctadecyldimethylammonium bromide). Compound 1, pyrido[2',3':3,2]thieno[4,5-d]pyrido[1,2-a]pyrimidin-6-one, exhibits reasonably high fluorescence quantum yields in all solvents studied (0.20 ≤ ΦF ≤ 0.30), while for compound 2, 3-[(p-methoxyphenyl)ethynyl]pyrido[2',3':3,2]thieno[4,5-d]pyrido[1,2-a]pyrimidin-6-one, the values are much lower (0.01 ≤ ΦF ≤ 0.05). The interaction of these compounds with salmon sperm DNA was studied using spectroscopic methods, allowing the determination of intrinsic binding constants, Ki = (8.7 ± 0.9) × 103 M-1 for compound 1 and Ki = (5.9 ± 0.6) × 103 M-1 for 2, and binding site sizes of n = 11 ± 3 and n = 7 ± 2 base pairs, respectively. Compound 2 is the most intercalative compound in salmon sperm DNA (35%), while for compound 1 only 11% of the molecules are intercalated. Studies of incorporation of both compounds in liposomes of DPPC, Egg-PC and DODAB revealed that compound 2 is mainly located in the hydrophobic region of the lipid bilayer, while compound 1 prefers a hydrated and fluid environment.

Alizarin Red S as an electrochemical indicator for saccharide recognition

In addition to the well-established spectroscopic Alizarin Red S (ARS) assay for the determination of binding constants between arylboronic acids and different saccharides, we report the use of ARS as a reporter in an electrochemical set-up. The electrochemical properties of ARS, the binding to phenyl boronic acid (PBA) and the competition with fructose in phosphate buffer at pH 7.4 were investigated by cyclic voltammetry (CV). By choosing a negative scan direction (starting at +0.2 V), a quasi-reversible process was detected at E 0′ = −0.59V with Δ E p = 0.1V. An irreversible oxidation peak at +0.42 V could also be detected. These peaks are characterised both as a 2-proton-2-electron transfer and corresponds to the oxidation and reduction of the anthraquinone or the ortho-quinone moiety. After addition of phenylboronic acid a new oxidation peaks occurred at −0.42 V which correlates with the ARS–PBA interaction. The peak current increased with increasing phenylboronic acid concentration according to the release of BA and formation of the ARS–PBA ester. After addition of fructose the peak current decreases again, in proportion to the fructose concentration, enabling the use of ARS as an electrochemical reporter for fructose detection up to 50 mM. Also the interaction with other cis-diol containing compounds such as sorbitol, mannitol, glucose and mannose was investigated and a dependence based on already published binding constants to phenylboronic acid could be shown.

Soluble Synthetic Analogues of Malaria Pigment: Structure of Mesohematin Anhydride and its Interaction with Chloroquine in Solution

Changing the vinyl groups of hematin anhydride to either ethyl or hydrogen groups results in increased solubility (Por=porphyrin). Determination of the weak binding constants of the antimalarial drug chloroquine to dimers of these hematin anhydride analogues suggests that solution-phase heme/drug interactions alone are unlikely to be the origin of the action of the drug.

Characterization of drug interactions with serum proteins by using high-performance affinity chromatography.

The binding of drugs with serum proteins can affect the activity, distribution, rate of excretion, and toxicity of pharmaceutical agents in the body. One tool that can be used to quickly analyze and characterize these interactions is high-performance affinity chromatography (HPAC). This review shows how HPAC can be used to study drug-protein binding and describes the various applications of this approach when examining drug interactions with serum proteins. Methods for determining binding constants, characterizing binding sites, examining drug-drug interactions, and studying drug-protein dissociation rates will be discussed. Applications that illustrate the use of HPAC with serum binding agents such as human serum albumin, α(1)-acid glycoprotein, and lipoproteins will be presented. Recent developments will also be examined, such as new methods for immobilizing serum proteins in HPAC columns, the utilization of HPAC as a tool in personalized medicine, and HPAC methods for the high-throughput screening and characterization of drug-protein binding.

DNA-binding study of anthraquinone derivatives using Chemometrics methods

This work presents the interaction of two anthraquinones including quinizarin (1, 4-dihydroxyanthraquinone) and danthron (1, 8-dihydroxyanthraquinone) with CT-DNA in a mixture of Brittone–Robinson buffer (pH = 7) with 50% of ethanol by UV–vis absorption, circular dichroism spectroscopic methods and viscosity measurements. The PCA and INDICES methods were used for predicting the number of light-absorbing components. Partially intercalative and hydrogen binding were found to be the dominant binding modes between anthraquinones and CT-DNA. The effect of ionic strength and pH on the behavior of the above system and also the interaction of anthraquinones with ds and ss-DNA were used to confirm the mentioned binding modes. The EQUISPEC software and nonlinear least-squares analysis were applied for binding constant determination.

Importance of extracellular polysaccharides on proton and Cd binding to bacterial biomass: A comparative study

The importance of extracellular polysaccharide (EPS) on proton and Cd binding was examined by comparing the adsorption behaviors of 4 bacterial species ( Pseudomonas putida, Shewenella oneidensis, Rhizobium tropici, and Agrobacterium sp. [ATCC# 21680]) with intact capsular EPS to corresponding adsorption behaviors with the EPS enzymatically removed from the biomass. Potentiometric titrations were conducted to detect any differences in proton binding of the biomass with and without the presence of EPS. Enzymatic removal of the EPS from each of the bacterial species in this study resulted in no significant differences in biomass proton binding behavior. Batch Cd adsorption experiments also showed no significant differences in the adsorption capacities between the EPS and EPS-free systems for all 4 species of bacteria. Our results suggest that EPS contains proton-active functional groups that are similar to those on the cell wall, and that, on a mass-normalized basis, EPS and bacterial cell walls exhibit similar site concentrations and affinities for adsorbing protons and Cd from solution. Because EPS exhibits similar Cd and proton binding properties to bacterial cell walls, and because of the similarity in binding properties between species, it may be possible to model metal and proton binding to biofilms in general using a single set of stability constants. This general modeling approach would obviate the impossible task of determining binding constants for protons and each metal of interest with each EPS component and each bacterial cell wall of interest.

Binding Constants of Mercury and Dissolved Organic Matter Determined by a Modified Ion Exchange Technique

Ion-exchange techniques have been widely used for determining the conditional stability constants (logK) between dissolved organic matter (DOM) and various metal ions in aqueous solution. An exception is mercuric ion, Hg2+, whose exceedingly strong binding with reduced sulfur or thiol-like functional groups in DOM makes the ion exchange reactions difficult. Using a Hg-selective thiol resin, we have developed a modified ion-exchange technique which overcomes this limitation. This technique allows not only the determination of binding constants between Hg2+ and DOM of varying origins, but also the discrimination of complexes with varying coordination numbers [i.e., 1:1 and 1:2 Hg:thiol-ligand (HgL) complexes]. Measured logK values of four selected DOM isolates varied slightly from 21.9 to 23.6 for 1:1 HgL complexes, and from 30.1 to 31.6 for 1:2 HgL(2) complexes. These results suggest similar binding modes that are likely occurring between Hg2+ and key thiolate functional groups in DOM particularly at a relatively low Hg to DOM ratio. Future studies should further elucidate the nature and precise stoichiometries of binding between Hg2+ and DOM at environmentally relevant concentrations.

Interaction of Caffeine with Bovine Serum Albumin: Determination of Binding Constants and the Binding Site by Spectroscopic Methods

AbstractThe interaction of caffeine with bovine serum albumin (BSA) under physiological condition was investigated by fluorescence, UV‐vis absorption and circular dichroism (CD) spectroscopy. Fluorescence data revealed that the fluorescence quenching of BSA by caffeine was a result of the formation of BSA‐caffeine complex. The binding constants Ka at different temperatures and corresponding thermodynamic parameters ΔH, ΔG and ΔS were calculated. The spectroscopic measurements and the thermodynamic parameters suggested that van der Waals interaction and hydrogen bonds were the predominant intermolecular forces to stabilize the complex. The conformational change of BSA induced by caffeine has been analyzed by means of CD and synchronous fluorescence spectroscopy. Furthermore, it is observed from the probe of competitive experiments that the binding location of caffeine with BSA could be the same as warfarin binding site I of BSA, which was also revealed by fluorescence anisotropy.