Job's Titration Research Articles

Ratiometic fluorescent detection of Au(III) and fluorescent “turn-off” identification of Cu(II)/Hg(II) with a coumarin-based probe

A coumarin-based fluorescent probe CHA functionalized with a 2-hydrazone pyridine unit displayed different photophysical responses to Au3+, Cu2+ and Hg2+. It exhibited ratiometric fluorescent response to Au3+, whereas remarkable fluorescence “turn-off” response to Cu2+/Hg2+ in aqueous solution. Au3+ and Cu2+ respectively induced clear blue-shift and red-shift in the absorption maximum, while Hg2+ had little impact on the absorption spectrum. The results of NMR, Job's titration and competition assay reveal that CHA was hydrolyzed to aldehyde under catalysis of Au3+, while it formed complex with Cu2+ and Hg2+. The ratio of fluorescence intensities I490/I540 of CHA exhibited linear relationship to Au3+ concentration in the range of 0.5–5.0 μM with a detection limit of 0.02 μM. The fluorescence intensity at 540 nm was in linearly correlative with Cu2+/Hg2+ concentration. Therefore, CHA could be used for detection of Au3+, Cu2+/Hg2+ in different channels as well as visual differentiation of exogenous Au3+ and Cu2+/Hg2+ in living HeLa cells.

The combination of rare earth upconversion nanocrystals with rhodamine dyes for the selective detection of amino acids

The following paper designed two rhodamine-based chemosensors for cysteine optical recognition. In order to decrease their photobleaching, up-conversion NaYF4 nanocrystals were used as excitation host. There was an efficient energy transfer from these NaYF4 nanocrystals to these rhodamine chemosensors, which was confirmed by spectral analysis, energy transfer radius calculation and emission decay lifetime comparison. Job's titration experiment suggested that these chemosensors followed a simple sensing mechanism towards Cys with binding stoichiometry of 1:1. Chemosensor emission was increased by Cys, showing emission turn on effect. Good selectivity and linear response were observed. It was found that sulfur modification on chemosensor improved sensitivity and selectivity.

Biomimetic Taste Receptors with Chiral Recognition by Photoluminescent Metal-Organic Frameworks Chelated with Polyaniline Helices.

The adsorption of phenylaniline (Phe) enantiomers on (+)-polyaniline (PAN)-chelated [In(OH)(bdc)]n microcrystals was carefully designed and studied by using the Job titration, circular dichroism, X-ray photoelectron spectroscopy, and photoluminescence to mimic heterotrimeric guanine nucleotide-binding protein (G protein)-coupled receptors in selective, but not specific, ligand binding with chiral recognition and signal transduction. Six essential working principles across different length scales are unraveled: 1) a chiral (+)-PAN (host), 2) specific sites for Phe-(+)/PAN (guest-host) binding, 3) a conformational change of (+)-PAN after binding with Phe enantiomers, 4) different degrees of packing for (+)-PAN, 5) interactions between (+)-PAN and the underlying signal-generating framework (i.e., [In(OH)(bdc)]n microcrystals), and 6) a systematic photoluminescent signal combination by using principal-component analysis from the other three polymer-chelated metal-organic frameworkds (MOFs), such as poly(acrylic acid) (PAA), sodium alginate (SA), and polyvinylpyrrolidone (PVP) to enhance the selectivity and discrimination capabilities.

Resolution of Multiple Substrate Binding Sites in Cytochrome P450 3A4: The Stoichiometry of the Enzyme−Substrate Complexes Probed by FRET and Job's Titration

To explore the mechanism of homotropic cooperativity in human cytochrome P450 3A4 (CYP3A4) we studied the interactions of the enzyme with 1-pyrenebutanol (1-PB), 1-pyrenemethylamine (PMA), and bromocriptine by FRET from the substrate fluorophore to the heme, and by absorbance spectroscopy. These approaches combined with an innovative setup of titration-by-dilution and continuous variation (Job's titration) experiments allowed us to probe the relationship between substrate binding and the subsequent spin transition caused by 1-PB or bromocriptine or the type-II spectral changes caused by PMA. The 1-PB-induced spin shift in CYP3A4 reveals prominent homotropic cooperativity, which is characterized by a Hill coefficient of 1.8 +/- 0.3 (S50 = 8.0 +/- 1.1 microM). In contrast, the interactions of CYP3A4 with bromocriptine or PMA reveal no cooperativity, exhibiting KD values of 0.31 +/- 0.08 microM and 7.1 +/- 2.3 microM, respectively. The binding of all three substrates monitored by FRET in titration-by-dilution experiments at an enzyme:substrate ratio of 1 reveals a simple bimolecular interaction with KD values of 0.16 +/- 0.09, 4.8 +/- 1.4, and 0.18 +/- 0.09 microM for 1-PB, PMA, and bromocriptine, respectively. Correspondingly, Job's titration experiments showed that the 1-PB-induced spin shift reflects the formation of a complex of the enzyme with two substrate molecules, while bromocriptine and PMA exhibit 1:1 binding stoichiometry. Combining the results of Job's titrations with the value of KD obtained in our FRET experiments, we demonstrate that the interactions of CYP3A4 with 1-PB obey a sequential binding mechanism, where the spin transition is triggered by the binding of 1-PB to the low-affinity site, which becomes possible only upon saturation of the high-affinity site.

Terpyridine‐substituted, fluorescent polymers and their chelation with zinc ion: The ligand‐to‐metal ratio and optical properties

AbstractSoluble, fluorescent, terpyridine‐substituted, conjugated polymers were prepared and characterized. The polymer chains included a defined oligo(phenylenevinylene) fragment, on which the terpyridine‐functional group was attached. The polymers were blue‐fluorescent with emission peaks at 400–427 nm in tetrahydrofuran solutions. Upon chelation with the Zn(II) cation, the emission maxima were shifted to a longer wavelength by as much as 113 to 506–526 nm. A model compound was also prepared to aid the structural characterization. The ratio of terpyridine to Zn2+ in the polymer complex was found to be 1:1 on the basis of spectroscopic evidence, which included mass spectrometry, 1H NMR, and Job titration. © 2006 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 44: 2338–2345, 2006

Electrochemistry of Co–Ru hetero-dinuclear porphyrin complex in a Nafion matrix

The metallo porphyrins possessing four ionic substituents of (meso-tetrakis(N-methyl-4-pyridiniumyl)porphyrinato)ruthenium(II) (RuTMPyP) and (meso-tetrakis(4-sulfonatophenyl)porphyrinato)cobalt(II) (CoTPPS) spontaneously associate. The equilibrium constant of dissociation (Kd) of the Co–Ru porphyrin was estimated to be 5 × 10−7 mol dm−3 by Job's titration. A single redox wave based on a 2-electron transfer was observed at 0.19 V s. SCE using cyclic voltammetry in a Nafion matrix. Potential-step chronocoulometry (PSCC) and potential-step chronoamperometry (PSCA) determined the kinetics of the electron transfer (k0 = 1.47 cm s−1) from the electrode to the complex in the Nafion film, and the apparent diffusion coefficient of electrons (Dapp = 8 × 10−8 cm2 s−1) in the Nafion film. Dimerization of the Co and Ru porphyrins results in an accelerated 2-electron transfer because the electrons are transferred ia overlapping π orbitals on the porphyrin ring. The film containing the dinuclear porphyrins shows sufficient catalytic activity toward dioxygen with a 4-electron reduction, based on successive electron transfer from the electrode to dioxygen.

Synthesis of phthalocyaninatocobalt(II) and complex formation with imidazole

Two kinds of phthalocyaninatocobalt(II) with different substituents have been synthesized. Complexation of imidazole was observed to proceed in two steps to form a mono- and, subsequently, a di-imidazole complex in solution. It is thought that the steric effect of the substituents on the ring, control the number of coordinated imidazole molecules. The stoichiometry of the complexes was determined by Job's titration method and the formation constants were then evaluated. The absorption spectrum of phthalocyaninatocobalt(II) exhibits a very broad band in thin films due to molecular aggregation while its di-imidazole adduct showed a sharp and red-shifted absorption band which is critical for optical recording materials.