QTAIM and ETS-NOCV analyses of intramolecular CH···HC interactions in metal complexes

The topological analysis, based on the quantum theory of atoms in molecules (QTAIM) of Bader and the ETS-NOCV charge and energy decomposition method have been used to characterize coordination bonds, chelating rings, and additional intramolecular interactions in the ZnNTA and ZnNTPA complexes in solvent. The QTAIM and ETS-NOCV studies have conclusively demonstrated that the H-clashes (they are observed only in the ZnNTPA complex and classically are interpreted as steric hindrance destabilizing a complex) are characterized by (i) the electron flow channel between the H-atoms involved, as discovered by the ETS-NOCV analysis (on average, ΔE(orb) = -1.35 kcal mol(-1)) and (ii) QTAIM-defined a bond path that indicates the presence of a preferred quantum-mechanical exchange channel, hence, they should be seen as H-H intramolecular bonding interactions. The main reason for the formation of a weaker ZnNTPA complex was attributed to the strain energy (from both QTAIM and ETS-NOCV techniques) and the larger Pauli repulsion contribution found from the ETS-NOCV analysis. An excellent agreement between physical properties controlling the stability of the two complexes was found from the two techniques, QTAIM and ETS-NOCV.     

On characterization of probability distributions by means of independent statistics

Summary The probability density functions f_k(x_k)=A_k|x_k|^p _k−1 e^−aφ _k(x_k) of independent random variables x₀, x₁, ..., x_n, are characterized by independence of two functions of them. Entrata in Redazione il 12 aprile 1969.     

LINEAR DICHROISM STUDY OF METALLOPORPHYRIN TRANSITION MOMENTS IN VIEW OF RADIATIONLESS INTERACTIONS WITH TRYPTOPHAN IN HEMOPROTEINS

We measured the linear dichroism of several metalloporphyrins embedded in stretched polyvinyl alcohol (PVA) films to estimate the orientation of the absorption transition moments, which in hemoproteins are relevant to the radiationless energy transfer between tryptophan and heme. The metalloporphyrins were derivatives of protoporphyrin IX (PPIX), namely Fe³⁺-PPIX (ferric-heme) and Fe²⁺CO-PPIX (CO-heme), Mg-PPIX (Mg-heme) and Zn-PPIX (Zn-heme). Measurements were conducted between 300 and 700 nm. In all cases the linear dichroism was wavelength dependent, indicating the presence of several transition moments with different orientations. We focused our attention on the near-UV (300–380 nm) and Soret (380450 nm) absorption bands. Deconvolution in terms of Gaussian components gave three components between 380 and 450 nm and only one in the 300–380 nm region. Deconvolution of the near-UV and Soret spectra of oxy-, deoxy- and carbonmonoxyhemoglobin gave very similar results, suggesting a very similar orientation of the various transition moments in the free and protein-embedded hemes. It should be stressed that the single 300–380 nm band is the only one responsible for the overlap integral that regulates the energy transfer from tryptophan to heme in hemoproteins (Gryczynski et al., Biophys. J . 63, 648–653, 1992). The dichroism of this single band indicated that its transition moment is oriented at about 60 from the α-γ meso-axis of the heme moiety. We conclude that the heme should be considered a linear oscillator when it acts as acceptor of energy transfer from tryptophans.     

Elimination of autofluorescence in fluorescence correlation spectroscopy using the AzaDiOxaTriAngulenium (ADOTA) fluorophore in combination with time-correlated single-photon counting (TCSPC)

Fluorescence correlation spectroscopy (FCS) is a frequently applied technique that allows for the precise and sensitive analysis of molecular diffusion and interactions. However, the potential of FCS for in vitro or ex vivo studies has not been fully realized due in part to artifacts originating from autofluorescence (fluorescence of inherent components and fixative-induced fluorescence). Here, we propose the azadioxatriangulenium (ADOTA) dye as a solution to this problem. The lifetime of the ADOTA probe, about 19.4 ns, is much longer than most components of autofluorescence. Thus, it can be easily separated by time-correlated single-photon counting methods. Here, we demonstrate the suppression of autofluorescence in FCS using ADOTA-labeled hyaluronan macromolecules (HAs) with Rhodamine 123 added to simulate diffusing fluorescent background components. The emission spectrum and decay rate of Rhodamine 123 overlap with the usual sources of autofluorescence, and its diffusion behavior is well known. We show that the contributions from Rhodamine 123 can be eliminated by time gating or by fluorescence lifetime correlation spectroscopy (FLCS). While the pairing of ADOTA and time gating is an effective strategy for the removal of autofluorescence from fluorescence imaging, the loss of photons leads to erroneous concentration values with FCS. On the other hand, FLCS eliminates autofluorescence without such errors. We then show that both time gating and FLCS may be used successfully with ADOTA-labeled HA to detect the presence of hyaluronidase, the overexpression of which has been observed in many types of cancer.     

Voltammetry as Virtual Potentiometric Sensor in Modelling of a Metal/Ligand System and Refinement of Stability Constants. Part 5

The concept of virtual potential (employed here in modelling operations), a unique experimental setup designed and built in our laboratories, and new regression equations derived for nonlinear fitting of quasi‐reversible direct‐current polarograms were combined with the existing rigorous treatment and refinement of polarographic data to establish reliable metal/ligand models and accurate stability constants for the lead(II)/glycine/OH^? and lead(II)/sarcosine/OH^? systems (sarcosine = N‐methylglycine). In the case of glycine, the complexes [M(HL)], [ML], [ML₂], and [ML₃] were identified, and their stability constants (as log β) were established to be 10.51 ± 0.06, 4.58 ± 0.02, 7.19 ± 0.10, and 9.27 ± 0.02, respectively, the complex [ML₃] being reported here for the first time (Table 2). The system with sarcosine involving [M(HL)], [ML], [ML₂], [ML₃], and [ML₂(OH)₂], with the stability constants (as log β) 11.01 ± 0.04, 4.18 ± 0.03, 7.23 ± 0.03, 9.1 ± 0.3, and 15.97 ± 0.07, respectively, is reported for the first time (Table 3). The log K₁ value for Pb^II with sarcosine is a fraction of a log unit smaller when compared with the Pb^II complex with glycine, in agreement with the literature data for Cu^II, Ni^II, and Zn^II showing the same trend for these two ligands. The proposed nonlinear curve‐fitting operations expand the applicability of polarography to study reliably and conveniently quasi‐reversible, on the polarographic time scale, metal/ligand systems (systems with involved heterogeneous kinetics).     

Silver nanoparticle-enhanced fluorescence in microtransponder-based immuno- and DNAhybridization assays

The aim of this study is to improve assay sensitivity in common solid-phase bioassay configurations as the result of using silver nanoparticles. The solid phase was provided by numerically indexed, silicon-based electronic chips, microtransponders (p-Chips) that have previously been used in multiplexed assays. Assay configurations investigated included an ELISA-type immunoassay and a DNA hybridization assay. The surface of p-Chips was derivatized with the silver island film (SIF) and a polymer, and then characterized with AFM and SEM. Silver nanoparticle sizes were in the range of 100 to 200 nm. Four fluorophores were tested for fluorescence enhancement; namely, green fluorescent protein, phycoerythrin, Cy3 and Alexa Fluor 555. We consistently observed significant fluorescence enhancement and sensitivity improvement in the p-Chip-based assays: the sensitivity in the cytokine IL-6 immunoassay was 4.3 pg/ml, which represented a 25-fold increase over the method not involving a SIF; and 50 pM in the hybridization assay, a 38-fold increase. The greatest enhancement was obtained for p-Chip surfaces derivatized first with the polymer and then coated with SIF. In conclusion, we show that the SIF-p-Chip-based platform is a highly sensitive method to quantify low-abundance biomolecules in nucleic acid-based assays and immunoassays.     

Binding of 8-anilino-1-naphthalenesulfonate to lecithin:cholesterol acyltransferase studied by fluorescence techniques

The solvatochromic fluorescent probe 8-anilino-1-naphthalenesulfonate (ANS) has been used to study the hydrophobicity and conformational dynamics of lecithin:cholesterol acyltransferase (LCAT). The ANS to LCAT binding constant was estimated from titrations with ANS, keeping a constant concentration of LCAT (2 microM). Apparent binding constant was found to be dependent on the excitation. For the direct excitation of ANS at 375 nm the binding constant was 4.7 microM(-1) and for UV excitation at 295 nm was 3.2 microM(-1). In the later case, not only ANS but also tryptophan (Trp) residues of LCAT is being excited. Fluorescence spectra and intensity decays show an efficient energy transfer from tryptophan residues to ANS. The apparent distance from Trp donor to ANS acceptor, estimated from the changes in donor lifetime was about 3 nm and depends on the ANS concentration. Steady-state and time-resolved fluorescence emission and anisotropies have been characterized. The lifetime of ANS bound to LCAT was above 16 ns which is characteristic for it being in a hydrophobic environment. The ANS labeled LCAT fluorescence anisotropy decay revealed the correlation time of 42 ns with a weak residual motion of 2.8 ns. These characteristics of ANS labeled LCAT fluorescence show that ANS is an excellent probe to study conformational changes of LCAT protein and its interactions with other macromolecules.     

Surface plasmon-coupled polarized emission of N-acetyl-l-tryptophanamide

We report an observation of ultraviolet (UV) surface plasmon-coupled emission (SPCE) of N-acetyl-l-tryptophanamide (NATA). The sample was spin coated from poly(vinyl alcohol) (PVA) solution on 20 nm aluminum film deposited on a quartz substrate. The directional UV SPCE occurs within a well-defined narrow angle at 52 degrees from the normal to the coupling hemicylinder quartz prism. The NATA directional emission is highly p polarized as expected for surface plasmon-coupled radiation. The 10 nm protective SiO2 layer deposited on top of the aluminum film significantly neutralized the fluorophore quenching by the metal surface. SPCE of NATA demonstrates a remarkable intrinsic dispersive property-the maximum of the emission spectrum depends on the observation angle. The efficient spectral resolution of SPCE can be used in the construction of miniaturized spectrofluorometers. The observation of SPCE of tryptophan opens a new possibility for the study of many unlabeled proteins with the technique complementary to surface plasmon resonance analysis.     

A Novel Approach to Monitoring of the Diffusion Junction Potential in Speciation Studies by Polarography under very Acidic Conditions. Part II: The Quasi‐Reversible Cu(II)‐Picolinic Acid System

The use of polarography to accurately determine stability constants of complexes formed under very acidic conditions (below pH 2) is demonstrated. The diffusion junction potentials, which must be accounted for below pH 2, were evaluated by applying protocols developed where Tl(I) is used as an internal reference. The Cu(II)‐picolinic acid (2‐pyridinecarboxylic acid) system studied was chosen since the CuL⁺ species only exists in solution below pH 2 under the conditions used and literature data exists to confirm the accuracy of procedure. Additionally, the reduction of Cu(II) was quasi‐reversible and procedures to determine the reversible half‐wave potentials were investigated. Average log β values of 7.75±0.09 for CuL⁺ and 14.8±0.1 for CuL₂ were obtained, which compared well to literature data.     

Challenges in Modelling and Optimization of Stability Constants in the Study of Metal Complexes with Monoprotonated Ligands. Part II

The influence of 2‐hydroxy‐3‐[(2‐hydroxy‐1,1‐dimethylethyl)amino]propane‐1‐sulfonic acid (AMPSO=HL) on systems containing copper(II) was studied by glass‐electrode potentiometry (GEP) and direct‐current polarography (DCP), at fixed total‐ligand‐to‐total‐metal‐concentration ratios and various pH values (25°, 0.1M KNO₃ medium). The predicted model ([CuL]⁺, [CuL(OH)], [CuL₂], [CuL₂(OH)]^?, [CuL₂(OH)₂]^2?, and [CuL₃]^?) and the overall stability constants for species found were obtained by combining results from both electrochemical techniques. The last five complexes are reported for the first time. For the species [CuL]⁺, [CuL₂], [CuL₃]^?, and [CuL₂(OH)₂]^2?, it was possible to determine stability constants with reasonable certainty and their values, as log β, were found to be 4.62±0.04, 9.5±0.1, 13.4±0.1, and 21.2±0.1, respectively. For the species [CuL(OH)] and [CuL₂(OH)]^?, stability constants 11.7±0.2 and 15.6±0.2, respectively, are presented as indicative values. It was demonstrated that AMPSO buffer may decrease the Cu²⁺ concentration by ten orders of magnitude by forming complexes with Cu²⁺. For the first time, the correction in DCP waves for the adsorption of the ligand and quasi‐reversibility of the metal allowed to determine stability‐constant values that are in good agreement with the values obtained by GEP. The importance of graphic analysis of data and significance of employing two analytical techniques was demonstrated; neither GEP nor DCP would be able to provide the correct M/L/OH^? model and reliable stability constants when used independently.