Thermodynamics of porphyrin binding to human serum albumin using affinity capillary electrophoresis

Summary The interaction thermodynamics of heptacarboxylporphyrin (HCP) and protoporhyrin (PP) with human serum albumin (HSA) was studied by affinity capillary electrophoresis (ACE) over the temperature range of 25–50°C, where HCP and PP bound to HSAvia 1:1 molecular association. The binding equilibrium constants (pH 7.4, phosphate buffer) for the binding of HCP with HSA were found to decrease with an increase in temperature, whereas the binding constants of the PP/HSA system appeared to be independent of temperature changes over the range studied. The van’t Hoff relationship (25–50°C) was found to be linear for the interaction of either HCP or PP with HSA. However, the interaction thermodynamics for both of these porphyrins with HSA were found to be quite different. In particular, the interaction of HCP (a hydrophilic porphyrin) with HSA appeared to be based on an enthalpy-driven process, whereas the binding between PP (a hydrophobic porphyrin) and HSA driven by a favorable change in entropy. The ability of using ACE to evaluate the interaction thermodynamics of serum proteins (e.g., HSA) with ligands (e.g., porphyrins and related compounds) should aid in the development of new and more effective photosensitizers in the photodynamic therapy of cancer.     

Applications of affinity interactions in capillary electrophoresis

Capillary electrophoresis (CE) has proven useful for the study of reversible molecular interactions. This is because highly efficient and reproducible separations take place in an environment where molecular interactions may contribute to selectivity without being inhibited by adverse buffer conditions. Affinity CE may be used to estimate quantitative binding data (binding constants and in some cases binding stoichiometries and rate constants) for various molecular interactions. Specific binding interactions (e.g., based on antibodies or aptamers) may also be utilized to quantitatively measure specific analytes using CE. Applications within these areas are here reviewed with focus on the last three years and with emphasis on novel concepts as well as innovative methodology and technology. It is concluded that the affinity CE approach is of growing versatility and will continue to play an integral role in discovering, characterizing, and exploiting biomolecular interactions.     

Molecular recognition: comparative study of a tunable host-guest system by using a fluorescent model system and collision-induced dissociation mass spectrometry on dendrimers

Host-guest interactions between the periphery of adamantylurea-functionalized dendrimers (host) and ureido acetic acid derivatives (guest) were shown to be specific, strong and spatially well-defined. The binding becomes stronger when using phosphonic or sulfonic acid derivatives. In the present work we have quantified the binding constants for the host-guest interactions between two different host motifs and six different guest molecules. The host molecules, which resemble the periphery of a poly(propylene imine) dendrimer, have been fitted with an anthracene-based fluorescent probe. The two host motifs differ in terms of the length of the spacer between a tertiary amine and two ureido functionalities. The guest molecules all contain an acidic moiety (either a carboxylic acid, a phosphonic acid, or a sulfonic acid) and three of them also contain an ureido moiety capable of forming multiple hydrogen bonds to the hosts. The binding constants for all 12 host-guest complexes have been determined by using fluorescence titrations by monitoring the increase in fluorescence of the host upon protonation by the addition of the guest. The binding constants could be tuned by changing the design of the acidic part of the guest. The formation of hydrogen bonds gives, in all cases, higher association constants, demonstrating that the host is more than a proton sensor. The host with the longer spacer (propyl) shows higher association constants than the host with the shorter spacer (ethyl). The gain in association constants are higher when the urea function is added to the guests for the host with the longer spacer, indicating a better fit. Collision-induced dissociation mass spectrometry (CID-MS) is used to study the stability of the six motifs using the corresponding third generation dendrimer. A similar trend is found when the six different guests are compared.     

Toward bioinspired galectin mimetics: identification of ligand-contacting peptides by proteolytic-excision mass spectrometry

Clinically relevant bioactivities of human galectins (adhesion/growth-regulatory galactoside-specific lectins) inspired the design of peptides as new tools to elicit favorable effects (e.g., in growth control) or block harmful binding (e.g., in tissue invasion). To obtain the bioinspired lead compounds, we combined a proteolytic fragmentation approach without/with ligand contact (excision) with mass spectrometric identification of affinity-bound protein fragments, using galectin-1 and -3 as models. Two peptides from the carbohydrate recognition domains were obtained in each case in experimental series rigorously controlled for specificity, and the [157-162] peptide of galectin-3 proved to be active in blocking lectin binding to a neoglycoprotein and to tumor cell surfaces. This approach affords peptide sequences for structural optimization and intrafamily/phylogenetic galectin comparison at the binding-site level with a minimal requirement of protein quantity, and it is even amenable to mixtures.     

Applications of on-line weak affinity interactions in free solution capillary electrophoresis

The impressive selectivity offered by capillary electrophoresis can in some cases be further increased when ligands or additives that engage in weak affinity interactions with one or more of the separated analytes are added to the electrophoresis buffer. This on-line affinity capillary electrophoresis approach is feasible when the migration of complexed molecules is different from the migration of free molecules and when separation conditions are nondenaturing. In this review, we focus on applying weak interactions as tools to enhance the separation of closely related molecules, e.g., drug enantiomers and on using capillary electrophoresis to characterize such interactions quantitatively. We describe the equations for binding isotherms, illustrate how selectivity can be manipulated by varying the additive concentrations, and show how the methods may be used to estimate binding constants. On-line affinity capillary electrophoresis methods are especially valuable for enantiomeric separations and for functional characterization of the contents of biological samples that are only available in minute quantities.     

Protein-ligand NOE matching: a high-throughput method for binding pose evaluation that does not require protein NMR resonance assignments

Given the three-dimensional (3D) structure of a protein, the binding pose of a ligand can be determined using distance restraints derived from assigned intra-ligand and protein-ligand nuclear Overhauser effects (NOEs). A primary limitation of this approach is the need for resonance assignments of the ligand-bound protein. We have developed an approach that utilizes data from 3D 13C-edited, 13C/15N-filtered HSQC-NOESY spectra for evaluating ligand binding poses without requiring protein NMR resonance assignments. Only the 1H NMR assignments of the bound ligand are essential. Trial ligand binding poses are generated by any suitable method (e.g., computational docking). For each trial binding pose, the 3D 13C-edited, 13C/15N-filtered HSQC-NOESY spectrum is predicted, and the predicted and observed patterns of protein-ligand NOEs are matched and scored using a fast, deterministic bipartite graph matching algorithm. The best scoring (lowest "cost") poses are identified. Our method can incorporate any explicit restraints or protein assignment data that are available, and many extensions of the basic procedure are feasible. Only a single sample is required, and the method can be applied to both slowly and rapidly exchanging ligands. The method was applied to three test cases: one complex involving muscle fatty acid-binding protein (mFABP) and two complexes involving the leukocyte function-associated antigen 1 (LFA-1) I-domain. Without using experimental protein NMR assignments, the method identified the known binding poses with good accuracy. The addition of experimental protein NMR assignments improves the results. Our "NOE matching" approach is expected to be widely applicable; i.e., it does not appear to depend on a fortuitous distribution of binding pocket residues.     

Bayesian analysis of heterogeneity in the distribution of binding properties of immobilized surface sites

Once a homogeneous ensemble of a protein ligand is taken from solution and immobilized to a surface, for many reasons the resulting ensemble of surface binding sites to soluble analytes may be heterogeneous. For example, this can be due to the intrinsic surface roughness causing variations in the local microenvironment, nonuniform density distribution of polymeric linkers, or nonuniform chemical attachment producing different protein orientations and conformations. We previously described a computational method for determining the distribution of affinity and rate constants of surface sites from analysis of experimental surface binding data. It fully exploits the high signal/noise ratio and reproducibility provided by optical biosensor technology, such as surface plasmon resonance. Since the computational analysis is ill conditioned, the previous approach used a regularization strategy assuming a priori all binding parameters to be equally likely, resulting in the broadest possible parameter distribution consistent with the experimental data. We now extended this method in a Bayesian approach to incorporate the opposite assumption, i.e., that the surface sites a priori are expected to be uniform (as one would expect in free solution). This results in a distribution of binding parameters as close to monodispersity as possible given the experimental data. Using several model protein systems immobilized on a carboxymethyl dextran surface and probed with surface plasmon resonance, we show microheterogeneity of the surface sites in addition to broad populations of significantly altered affinity. The distributions obtained are highly reproducible. Immobilization conditions and the total surface density of immobilized sites can have a substantial impact on the functional distribution of the binding sites.     

Surprises in the design of anion receptors: calorimetry prevents false reasoning

Supplementing bicyclic guanidinium anion receptors with four sec-carboxamido groups leads to enhanced affinity for oxoanions, however, for a different reason than originally planned. Calorimetric analysis reveals that better binding is due to higher association entropies rather than more negative enthalpies. Thus, molecular design following geometric and functional complementarity principles may misguide supramolecular constructions aimed at a unique host-guest binding mode, as required, e.g., by self-assembly or catalysis.     

The Hammett relationship and reactions in the excited electronic state: hemithioindigo Z/E-photoisomerization

The photochemical reaction dynamics of a set of photochromic compounds based on thioindigo and stilbene molecular parts (hemithioindigos, HTI) are presented. Photochemical Z/E isomerization around the central double bond occurs with time constants of 216 ps (Z --> E) and 10 ps (E --> Z) for a 5-methyl-hemithioindigo. Chemical substitution on the stilbene moiety causes unusually strong changes in the reaction rate. Electron-donating substituents in the position para to the central double bond (e.g., para-methoxy) strongly accelerate the reaction, while the reaction is drastically slowed by electron-withdrawing groups in this position (e.g., para-nitrile). We correlate the experimental data of seven HTI-compounds in a quantitative manner using the Hammett equation and present a qualitative explanation for the application of ground-state Hammett constants to describe the photoisomerization reaction.     

Sequential isomerism of activated complexes: Interfering versus noninterfering intermediate

A unimolecular process is considered that was treated experimentally as a concerted one; however, subsequent quantum-chemical analysis has proved that the process is realized via an intermediate (double sequential isomerism of activated complexes). The intermediate is supposed to contribute directly to values of the primary observed data (e.g., spectral absorbances). Within this approach of interfering intermediate any degree of this interference is allowed including the limit case of noninterfering intermediate serving as a reference standard. A technique is suggested enabling correct comparison of the quantum-chemical outputs with the experimental overall values for rate constants and activation enthalpy and entropy. The technique employs mere amount of information currently obtainable from quantum-chemical study of a rate process (i.e., representation of potential energy hypersurface by its stationary points and activated-complex theory). Properties of the derived formulae are illustrated with model examples. The results are important for meaningful comparison of experimental and theoretical data in the case of processes with sequential isomerism of activated complexes disclosed only theoretically.     

Voltammetric measurements of the interaction of metal complexes with nucleic acids

Cyclic voltammetry and differential-pulse voltammetry at mm-sized electrodes were used to measure the decrease in the rate of diffusion of metal complexes upon binding to DNA and to extract the binding constants and effective binding site sizes. A linear correlation was observed between the site size determined electrochemically and the diameter of the complexes [site size: Cu(phen)2(2+) > Fe(phen)3(2+) > Co(bipy)3(3+) approximately Fe(bipy)3(2+) > Ru(NH3)6(3+)]. The binding constants were found to be influenced by the charge of the metal complex, the nature of ligand and the geometry about the metal centre. Competition experiments, in which differential pulse voltammetry was used to observe the release of bound metal complex on addition of a second DNA-binding molecule to the solution, were sensitive to the nature and location of the binding sites for the two species. Steady-state voltammetric experiments at microelectrodes are shown to have a number of advantages over cyclic voltammetry and differential pulse voltammetry at mm-sized electrodes for determination of binding constants. In particular, the steady-state diffusion limited current is directly proportional to the diffusion coefficient, rather than its square root, which improves the discrimination between DNA-bound and freely diffusing metal complex. Further, the kinetics of the binding process do not affect the steady state measurement, whereas for transient techniques, e.g., cyclic voltammetry, only a range of values can be extracted corresponding to the limits of fast and slow binding kinetics compared to the experimental timescale.     

Development of a fitting model suitable for the isothermal titration calorimetric curve of DNA with cationic ligands

A novel curve fitting model was developed for the isothermal titration calorimetry (ITC) of a cationic ligand binding to DNA. The ligand binding often generates a DNA conformational change from an elongated random coil into a compact collapsed form that is referred to as "DNA condensation". The ligand binding can be classified into two regimes having different binding constants Ki, i.e., the binding to an elongated DNA chain with a binding constant K1 and with K2 that occurred during the conformational transition. The two-variable curve fitting models are usually bound by a strict regulation on the difference in the values of the binding constants K1 > K2. For the DNA condensation, however, the relationships for K1 and K2 are still unclear. The novel curve fitting model developed in this study takes into account this uncertainty on the relationship of the binding constants and is highly flexible for the two-variable binding constant system.     

Ultraviolet difference spectroscopic study on the interactions of cellulase fromTrichoderma reesei with cellodextrins

The formation of cellodextrin-cellobiohydrolase complex was studied by ultraviolet difference spectroscopy. Upon the binding of cellodextrins (G₇‐G₃), cellobiohydrolase (EC 3.2.1.91) purified fromTrichoderma reesei produced difference spectra having maxima at 289‐293 nm and 283‐286 nm. These spectra are consistent with prior observations reported for lysozyme and amylase. In this case, water soluble cellulose oligomers (i.e., cellodextrins) are shown to interact with tryptophan residue(s) on cellobiohydrolase. The difference spectral maxima observed at acidic or alkaline pH were shifted. This was accompanied by a marked decrease of binding ability of cellobiohydrolase for cellodextrins. The standard free energy change for the association of cellodextrins to the cellobiohydrolase was an order of 4 kcal/gmol. The association constant of enzyme for substrate decreases by 15‐20% as temperature increases from 20 to 48°C. At 25°C, the dissociation constants for the enzyme with respect to cellohexose and cellotriose were estimated to be 1.19 and 1.37 mM, respectively. A decrease in dissociation constants was observed with an increase in the number of glucosyl units from 3 to 6. This suggests that there may be six or more subsites in the active center of cellobiohydrolase.     

The binding behavior of cyclodextrins toward a nitroxide spin probe in the presence of different alcohols as studied by EPR

Stability constants, rates of association and dissociation, and thermodynamic and activation parameters for the formation of inclusion complexes between the radical guest, N-benzyl- tert-butyl- d 9-nitroxide and beta- or 2,6- O-dimethyl-beta-cyclodextrin (CDs), have been determined by EPR spectroscopy in water in the presence of 14 different alcohols, differing in size and lipophilicity. In all cases, it was found that addition of alcohol, depending on its structure and concentration, causes a reduction of the stability of the paramagnetic complex. Global analysis of EPR data allowed us to explain the CDs binding behavior: we discarded the formation of a ternary complex, where alcohol and radical guest are coincluded into CD cavity, while data were found more consistent with the formation of a binary complex alcohol:CD competing with the monitored complex nitroxide:CD. Both kinetic and thermodynamic analysis of the experimental results have revealed that the presence of alcohols affects to a larger extent the dissociation rather then the association of radical probe and CD and that the former process is of greater importance in determining the stability of the complex, this confirming the reliability of the competition model proposed. This competition has been used for the indirect determination of the stability constants of complexes between CD and examined alcohols. By using a similar approach, we showed EPR spectroscopy can be considered a rapid and accurate technique to investigate the CDs binding behavior toward different nonradical guest.     

Application of automated docking to the binding of naphthalenes to βCD in water: correlation with spectrofluorimetric data

Automated semi-rigid docking has been explored as an alternative approach for the theoretical study of the inclusion complexes with cyclodextrins. To this purpose we have chosen as a model for the binding to βCD some naphthalene derivatives (naphthalene, 2-ethylnaphthalene, 2-acetylnaphthalene, 1-naphthyl acetate, 2-naphthyl acetate and 1-naphthol). For comparison purposes, the binding constants in water and the associated thermodynamic parameters have been obtained under the same experimental conditions by steady-state fluorescence spectroscopy. The calculations of the automated docking regarding the topology of the guest inside the cavity produce a cluster of structures that qualitatively agrees with fluorescence results and literature data. However, the predicted values of the free energy of binding are lower than the experimental ones by ca. −10 kJ mol⁻¹, and very close to the experimental enthalpy of binding deduced from the temperature dependence of the association constants. The differences are ascribed mainly to the assumption of rigidity of the CD into the auto-docking scheme.     

Antihydrophobic solvent effects: an experimental probe for the hydrophobic contribution to enzyme-inhibitor binding

The hydrophobic component to the binding affinities of one acyclic phosphinate (4) and three macrocyclic phosphonamidate inhibitors (1-3) to the zinc peptidase thermolysin was probed by varying the solvent composition. Increasing the percentage of ethanol in the buffer solution over the range 0-9% increases the inhibition constants, K(i), by up to an order of magnitude. This approach represents an experimental method for distinguishing solvation from conformational or other effects on protein-ligand binding. The size of the "antihydrophobic effect" is correlated with the amount of hydrophobic surface area sequestered from solvent on association of the inhibitor and enzyme, although it is attenuated from that calculated from the surface tension of ethanol-water mixtures. The results are consistent with the Lum-Chandler-Weeks explanation for the size dependence of the hydrophobic effect.     

GVVPT2 multireference perturbation theory description of diatomic scandium, chromium, and manganese

With relatively simple model spaces derived from valence bond models, a straightforward zero-order Hamiltonian, and the use of moderate-sized Dunning-type correlation consistent basis sets (cc-pVTZ, aug-cc-pVTZ, and cc-pVQZ), the second order generalized Van Vleck perturbation theory (GVVPT2) method is shown to produce potential energy curves (PECs) and spectroscopic constants close to experimental results for both ground and low-lying excited electronic states of Sc(2), Cr(2) and Mn(2). In spite of multiple quasidegeneracies (particularly for the cases of Sc(2) and Mn(2)), the GVVPT2 PECs are smooth with no discontinuities. Since these molecules have been identified as ones that widely used perturbative methods are inadequate for describing well, due to intruder state problems, unless shift parameters are introduced that can obfuscate the physics, this study suggests that the conclusion about the inadequacy of multireference perturbation theory be re-evaluated. The ground state of Sc(2) is predicted to be X(5)∑(u)(-), and its spectroscopic constants are close to the ones at the MRCISD level. Near equilibrium geometries, the 1(3)∑(u)(-) electronic state of Sc(2) is found to be less stable than the quintet ground state by 0.23 eV. The Cr(2) PEC has several features of the Rydberg-Klein-Rees (RKR) experimental curve (e.g., the pronounced shelf at elongated bond lengths), although the predicted bond length is slightly long (R(e) = 1.80 ? with cc-pVQZ compared to the experimental value of 1.68 ?). The X(1)∑(g)(+) ground state of Mn(2) is predicted to be a van der Waals molecule with a long bond length, R(e), of 3.83 ? using a cc-pVQZ basis set (experimental value = 3.40 ?) and a binding energy, D(e), of only 0.05 eV (experimental value = 0.1 eV). We obtained R(e) = 3.40 ? and D(e) = 0.09 eV at the complete basis set (CBS) limit for ground state Mn(2). Low lying excited state curves have also been characterized for all three cases (Cr(2), Mn(2), and Sc(2)) and show similar mathematical robustness as the ground states. These results suggest that the GVVPT2 multireference perturbation theory method is more broadly applicable than previously documented.     

Determination of association constants for alcohols based on ethers as homomorphs

Hofman, T. and Nagata, I., 1986. Determination of association constants for alcohols based on ethers as homomorphs. Fluid Phase Equilibria, 25: 113–128.A new, improved version of the method for determining the association constants of alcohols from the properties of pure substances is presented. The proposed procedure assumes both the isomeric ether as the alcohol's homomorph and ceases ignoring the volume change of mixing. Obtained values of enthalpies of association with are closer to the experimental ones than those calculated by means of standard methods, confirm both the credibility of accepted assumptions and the usefulness of the present approach.