Spectral Titration Data Research Articles

Filling two needs with one deed: Differential sensing of anthrax biomarker dipicolinic acid and bovine serum albumin using binary ensemble based on 1,8-naphthalimide Schiff base and cationic surfactant

Keeping in mind the severities of anthrax disease caused by bacteria Bacillus anthracis and side effects of abnormal levels of a nonmetalloprotein bovine serum albumin (BSA), their early diagnosis was highly required. The existing lanthanide based sensors for their detection are not up to the mark due to their potential disadvantages of genetic toxicity, bio-migration, supply shortages, low atom utilization and poor stability. To address these issues, a binary ensemble OHNP@CTAB was fabricated in aqueous medium by encapsulation of 1,8-naphthalimide derived Schiff base (OHNP) in the micelles of a cationic surfactant CTAB. Significant changes were observed in the absorption and emission spectrum of OHNP@CTAB in the presence of DPA and BSA mainly due to electrostatic interaction among positively charged receptor and negatively charged guest species. Limit of detection (LOD) values of OHNP@CTAB towards DPA and BSA were calculated to be 1.19 and 0.11 µM, respectively from fluorescence spectral titration data. Moreover, the binary ensemble OHNP@CTAB efficiently detected BSA and DPA in real samples viz. packaged milk, gulkand and candy.

A Monochromophoric Approach to Succinct Ratiometric Fluorescent Probes without Probe-Product Crosstalk

Ratiometric probes facilitate quantitative studies via self-calibration and are cherished for bioimaging. Often, a small probe-product spectral separation leads to crosstalk, but the rational devel...

Enhancing the student experience: the role of the Midwifery Practice Coordinator (MPC)

In this paper, a new dual optical behavior Cu2+ selective Schiff base (L) was developed by reacting thiophene carbaldehyde with diamino malononitrile. The sensing ability of L was studied with different metal ions by visual test, UV–Vis and spectrofluorometric methods in MeOH:H2O (8:2, v/v) media. Accordingly, the visual test was confirmed by changing initial yellow to colorless upon interaction with Cu2+ while for other it did not respond in natural light while the same solution when it observed under UV light, it exhibits emission color (cyan) only with Cu2+. Spectrophotometrically, L alone exhibits a strong absorption band at 420 nm, upon interaction with Cu2+, it exhibits an additional new peak at 290 nm while spectrofluorimetrically, a new emission peak was observed at 360 nm while other metal ions it does not exhibits this optical behavior. The spectral titration data reveals that the LOD limit down to of 6.21 × 10−6 and 1.45 × 10−8 M by UV–vis and fluorescence respectively which is lower than the WHO guidelines (0.02 μM) in drinking water. The formation 1:1 binding stoichiometry of host-guest complexation between L and Cu2+ is studied by using Jobs plot and the binding capacity is estimated by Benesi-Hilderbrand method (Ka = 7.31 × 104 M-1). The coordination behavior of L with Cu2+ is well supported by FTIR, 1H NMR, 13C NMR, ESI mass spectral data and theoretical calculations using Gaussian 09 W program.

Dual optical properties of new schiff base based on bisthiophene for sensing of Cu2+ in protic media

In this paper, a new dual optical behavior Cu2+ selective Schiff base (L) was developed by reacting thiophene carbaldehyde with diamino malononitrile. The sensing ability of L was studied with different metal ions by visual test, UV–Vis and spectrofluorometric methods in MeOH:H2O (8:2, v/v) media. Accordingly, the visual test was confirmed by changing initial yellow to colorless upon interaction with Cu2+ while for other it did not respond in natural light while the same solution when it observed under UV light, it exhibits emission color (cyan) only with Cu2+. Spectrophotometrically, L alone exhibits a strong absorption band at 420 nm, upon interaction with Cu2+, it exhibits an additional new peak at 290 nm while spectrofluorimetrically, a new emission peak was observed at 360 nm while other metal ions it does not exhibits this optical behavior. The spectral titration data reveals that the LOD limit down to of 6.21 × 10−6 and 1.45 × 10−8 M by UV–vis and fluorescence respectively which is lower than the WHO guidelines (0.02 μM) in drinking water. The formation 1:1 binding stoichiometry of host-guest complexation between L and Cu2+ is studied by using Jobs plot and the binding capacity is estimated by Benesi-Hilderbrand method (Ka = 7.31 × 104 M-1). The coordination behavior of L with Cu2+ is well supported by FTIR, 1H NMR, 13C NMR, ESI mass spectral data and theoretical calculations using Gaussian 09 W program.

A new colorimetric and fluorescent chemosensor based on Schiff base-phenyl-crown ether for selective detection of Al3+ and Fe3+

A novel chemosensor (L) based on Schiff base-phenyl-crown ether was synthesized and evaluated as a chemoselective Al3+ and Fe3+ sensor in ethanol through “turn-on” fluorescence enhancement and sharp colorimetric responses. Adding 10 equiv. Al3+ or Fe3+ could change the color of the sensor L from colorless to orange yellow, and increase the fluorescence intensity of L at 596 nm by 160-fold or 130-fold, respectively. The selective response of L to Al3+ and Fe3+ was not interfered by other ions, and their bonding was reversible. The results of fluorescence spectral titration data showed that the association constants (lgKs) of L for Al3+ and Fe3+ were 8.95 and 8.67 in ethanol, and the corresponding detection limits were 0.33 and 0.36 μM, respectively. The Job’s plot and mass spectrometry revealed that the sensor L formed a 2:1 complex with Al3+ or Fe3+. The proposed binding modes of L with Al3+ and Fe3+ were demonstrated by means of the infrared spectroscopy and 1H NMR titration. The results of DFT/B3LYP calculations showed that the binding with Al3+ and Fe3+ makes the sensor L accompanied a mechanism with the inhibition of CN isomerization (cis-trans) and the activation of chelation enhanced fluorescence (CHEF).

Synthesis and biological evaluation of a new dysprosium(III) complex containing 2,9-dimethyl 1,10-phenanthroline

The binding of [Dy(dmp)2Cl3(OH2)], where dmp is 2,9-dimethyl 1,10-phenanthroline, with Fish salmon DNA (FS-DNA) is investigated by absorption and emission spectroscopy, quenching studies, salt dependent, and gel electrophoresis. The binding constant (Kb) of the interaction is calculated as (1.27 ± .05) × 105 M−1 from absorption spectral titration data. The Stern–Volmer constant (KSV), thermodynamic parameters involves ΔG°, ∆H°, and ∆S° are calculated by fluorescent data and Van’t Hoff equation. The thermodynamic studies show that the reaction for the binding of the complex with FS-DNA is endothermic and entropically driven (ΔS° > 0, ΔH° > 0). The effect of the complex concentration on FS-DNA cleavage reactions is also investigated by gel electrophoresis. Furthermore, the Dy(III) complex has been screened for its antibacterial activity. The experimental results suggest that the Dy(III) complex binds significantly to FS-DNA by hydrophobic groove binding mode and the complex has more efficient antibacterial activity compared to its metal salt.

Synthesis, Chirality and Complexation Phenomena of Two Diastereoisomeric Dinuclear Lanthanide(III) Complexes

AbstractTwo diastereoisomeric ditopic ligands have been synthesised by linking (SSS)‐heptadentate triamide derivative of cyclen with a central (SS)‐ or (SR)‐1,2‐diphenyl‐1,2‐diaminoethane derivative. Complexes of selected LnIII ions (Ln = Eu, Tb, Yb) have been characterized and circularly polarised luminescence spectra of the Eu systems revealed significant differences, consistent with a change in overall complex chirality associated with the local helicity at the metal centre. Stepwise binding constants for the formation of 1:1 and 2:1 ternary adducts with (S)‐lactate and (S)‐O‐phospho‐tyrosine have been determined by least‐squares analysis of spectral titration data, and emission and 1H NMR spectroscopic data compared with the mononuclear analogue.

Association Equilibrium of Methylene Blue by Spectral Titration and Chemometrics Analysis: A Thermodynamic Study

AbstractThe monomer‐dimer equilibrium of methylene blue (MB, Scheme I) has been investigated by means of UV‐Visible spectroscopy in aqueous solutions. The self aggregation of MB in water has been investigated by recording absorption spectra in the wavelength range of 450–750 nm, and in different ionic strengths using concentrated KCl solutions in the temperature range of 20–90°C. Chemometrics analysis of the spectral data gave a dimerization constant, individual spectra of the monomer and dimer forms of the dye molecule. The quantitative analysis of the data of the undefined mixture was carried out by simultaneous resolution of the overlapping spectral bands in the whole set of absorption spectra. The dimerization constants of MB determined by mathematical deconvolution of the thermometric spectral titration data show dependency on temperature variations. The concentration range of MB was 6.00 × 10−5‐3.00 × 10−4 M. Utilizing the van't Hoff relation, which describes the dependence of the equilibrium constant on temperature, the thermodynamics parameters ΔH° and ΔS° of the aggregation process were determined. The compensation effect was verified by the thermodynamics results of the dimerization process of the dye.

DNA intercalation of methylene blue and quinacrine: new insights into base and sequence specificity from structural and thermodynamic studies with polynucleotides

The binding of the known DNA intercalators methylene blue and quinacrine with four sequence specific polynucleotides, viz. poly(dG-dC).poly(dG-dC), poly(dG).poly(dC), poly(dA-dT).poly(dA-dT) and poly(dA).poly(dT), have been compared using absorbance, fluorescence, competition dialysis and thermal melting and the thermodynamic aspects of the interaction studied. In all the cases, non-cooperative binding phenomena obeying neighbor exclusion principle was observed though the affinity was remarkably higher for quinacrine and the nature of the binding was characterized to be true intercalation. The data on the salt dependence of binding derived from the plot of log Kvs. log[Na(+)] revealed a slope of around 1.0, consistent with the values predicted by the theories for the binding of monovalent cations, and contained contributions from polyelectrolytic and non-polyelectrolytic forces. The bindings were characterized by strong stabilization of the polynucleotides against thermal strand separation in both optical melting as well as differential scanning calorimetry studies. The data analyzed from the thermal melting and isothermal titration calorimetry studies were in close proximity to those obtained from absorption spectral titration data. Isothermal titration calorimetry results revealed the bindings to poly(dG-dC).poly(dG-dC), poly(dG).poly(dC) and poly(dA-dT).poly(dA-dT) to be exothermic and favoured by both negative enthalpy and large favourable positive entropy changes, while that to poly(dA).poly(dT) was endothermic and entropy driven. The heat capacity changes obtained from temperature dependence of enthalpy gave negative values to all polynucleotides. New insights on the molecular aspects of interaction of these molecules to DNA have emerged from these studies.

DNA Intercalation by Quinacrine and Methylene Blue: A Comparative Binding and Thermodynamic Characterization Study

There is compelling evidence that cellular DNA is the target of many anticancer agents. Consequently, elucidation of the molecular nature governing the interaction of small molecules to DNA is paramount to the progression of rational drug design strategies. In this study, we have compared the binding and thermodynamic aspects of two known DNA-binding agents, quinacrine (QNA) and methylene blue (MB), with calf thymus (CT) DNA. The study revealed noncooperative binding phenomena for both the drugs to DNA with an affinity one order higher for QNA compared to MB as observed from diverse techniques, but both bindings obeyed neighbor exclusion principle. The data of the salt dependence of QNA and MB from the plot of log K versus log [Na+] revealed a slope of 1.06 and 0.93 consistent with the values predicted by theories for the binding of monovalent cations, and have been analyzed for contributions from polyelectrolytic and nonpolyelectrolytic forces. The binding of both drugs was further characterized by strong stabilization of DNA against thermal strand separation in both optical melting and differential scanning calorimetry studies. The binding data analyzed from the thermal denaturation and from isothermal titration calorimetry (ITC) were in close proximity to those obtained from spectral titration data. ITC results revealed the binding to be exothermic and favored by both negative enthalpy and positive entropy changes. The heat capacity changes obtained from temperature dependence of enthalpy indicated -146 and -78 cal/(mol.K), respectively, for the binding of QNA and MB to CT DNA. Circular dichroism study further characterized the structural changes on DNA upon intercalation of these molecules. Molecular aspects of interaction of these molecules to DNA are discussed.

Spectrophotometric Determination of Acidity Constants of Group B Vitamins in Different Ionic Strengths at 25±0.1°C

The apparent acid dissociation constants of four water-soluble vitamins, folic acid (vitamin B 9 or B 0 ), thiamine (vitamin B 1 ), riboflavin (vitamin B 2 ) and pyridoxal (vitamin B 6 ) were determined spectrophotometrically in different ionic strengths at 25 °C±0.1. An accurate and sophisticated method based on chemometrical concepts was applied in order to determine acidity constants at different ionic strengths. For this purpose, spectral titration data were used. The spectra were recorded in the range 225-500 nm at different ionic strengths. The acidity constants of all vitamins at various ionic strengths were calculated by means of computer fitting of the pH-absorbance data with appropriate mass balance equations according to mono-, dior triprotic acids. The computer program DATAN was used to extract the desired information from the spectral data. The outputs of the fitting processes were acidity constants, spectral profiles of pure forms, distribution diagrams, and other factor analysis data. The effect of ionic strength on the acidity constants is discussed.

Principal component analysis applied to the study of successive complex formation data in Cu(II)-ethanolamine systems

Application of principal component analysis to Cu(II)–ethanolamine complex formation data is shown. Determination of the number of complex species is obtained from the rank of the matrix of spectral data using either Gauss elimination or factorial analysis. Relevant information concerning species distribution versus pH is obtained from the plot of the signficant factors upsurging from the evolution of spectral titration data.

Interactions of Substrate and Product with Cytochrome P450: P4502B4versus P450cam

In the present study, two P450s (P4502B4and P450cam) have been examined with regard to their interactions with their substrates and products utilizing the characteristic spectral perturbations as criteria for their binding. The results indicate that although there are differences between the two P450s (E) in regard to their precise interactions with their substrates (S) and products (P), the spectral titration data were consistent with the two-site model—E + S ↔ ES (K1), E + P ↔ EP (K2); EP + S ↔ ESP (K3); ES + P ↔ ESP(K4) in which S and P bind to E forming ESP. The data were inconsistent with the two-site model in which S and P compete for the same site. As required by the two-site model, the relationshipK2K3=K1K4was maintained with both P450s at all product concentrations tested, althoughK3andK4decreased considerably when product concentration was increased. The relationshipK3⪢K4was also maintained, indicating that with both enzymes' ESP is formed predominantly by binding of S to EP rather than binding of P to ES, and that ESP dissociates predominantly to ES and P rather than EP and S. In other words, binding of S to EP facilitates the dissociation of P. This indicates that the relative parameter values are compatible for ESP to have functional significance. The possible role of ESP in controlling catalytic rate and catalytic efficiency is discussed.

Interactions of substrate and product with cytochrome P450 2B4.

Interactions of the substrate(s) benzphetamine and the product (P) desmethylbenzphetamine with cytochrome P450 2B4 were studied by difference spectrophotometry. A two-sites model in which site 1 binding, causing Type I transition (low- to high-spin) must precede site 2 binding, causing Type II transition (high- to low-spin), gave an acceptable fit to the spectral titration data. The equilibrium association constant of substrate for site 1 (K1) was greater than that for site 2 (K2), and the K2 for the product was greater than K1, indicating that the substrate binds preferentially to site 1 and the product prefers site 2. In addition, competition between P and a strong Type II ligand (1-benzylimidazole) and a noncompetitive type of interaction between S and the same Type II ligand was observed. This indicates that P binds to the same site as the Type II ligand and S binds to a different site. The observed high-spin maxima for both P (EP1HSmax) and S (ES1HSmax) were similar to those calculated using the K1 and K2 values obtained from the curve-fitting procedure, indicating that the equilibrium concentration of the high-spin species is controlled entirely by K1 and K2. Simultaneous presence of the substrate and product decreased K1 of the substrate and K2 of the product, indicating that there is interaction between the substrate-preferred and the product-preferred sites. A possible functional significance of the differences in the site preferences of the substrate and product is discussed.

Fluorescence techniques for metal-humic interactions

Two fluorescence techniques to study metal-humic interactions are presented. In the first technique, Lanthanide Ion Probe Spectroscopy (LIPS), the humic samples are titrated by Eu3+ ions. The ratio of the intensities of two emission lines of Eu3+, R=I592/I616, is used to estimate the amount of bound and free species of the probe ions. The titration plot is presented as R versus the logarithm of total added Eu3+. In the second technique, fluorescence quenching of the humic material by Cu2+ is used to produce titration curves of intensity versus the logarithm of total added Cu2+. The two techniques are used in conjunction with a model that treats the various ligands in humic substances as continuous distributions of binding sites in which individual ligand concentrations are normally distributed with respect to the individual stability constants for metal binding. The model includes the effects of pH, ionic strength, and competing metal ions. The parameters of the model are estimated by fitting the spectral titration data to the calculated titration plot. Some simulation and experimental data are presented and discussed.

6-Nitro- l-tryptophan: A novel spectroscopic probe of trp aporepressor and human serum albumin

The binding of 6-nitro- l-tryptophan to trp aporepressor and human serum albumin has been examined by visible difference spectroscopy and circular dichroism. 6-Nitro- l-tryptophan, prepared by nitration of l-tryptophan with nitric acid in glacial acetic acid, exhibits a visible and near-uv absorption spectrum with λ max at about 330 nm ( ϵ = 7 × 10 3M −1 cm −1) and a shoulder near 380 nm in H 2O. In the presence of frp aporepressor, the visible absorption intensity is sharply diminished. Visible difference spectral titration data give K D = 1.27 × 10 −4M and n = 0.95 per subunit at 25 °C. While 6-nitro- l-tryptophan exhibits no significant circular dichroism between 300 and 500 nm, the complex with frp aporepressor exhibits strong circular dichroism signals, with a negative maximum at 386 nm (Δ ϵ = −7.5 M −1 cm −1) and a positive maximum at 310 nm (Δ ϵ = +6 M −1 cm −1). Circular dichroism titration data give K D = 1.69 × 10 −4M and n = 0.90 per subunit at 25 °C. The K D values determined spectroscopically are in excellent agreement with that determined by equilibrium dialysis, K D = 1.5 × 10 −4M at 25 °C. In the presence of human serum albumin, the spectrum of 6-nitro- l-tryptophan exhibits a blue shift and an increase in absorption intensity; similar changes are observed in solvents of low dielectric contrast such as 80% aqueous dioxane. Visible difference spectral titration data give K D = 8.0 × 10 −5M and n = 0.95 for human serum albumin. The complex of 6-nitro- l-tryptophan with human serum albumin exhibits a strong positive circular dichroism maximum at 380 nm (Δ ϵ = +9.8 M −1 cm −1) with a shoulder at 310–320 nm. Circular dichroism titration data give K D = 6.4 × 10 −5M and n = 0.83, in good agreement with the visible difference spectral results. Taken together, our results demonstrate the utility of 6-nitro- l-tryptophan as a spectroscopic probe for tryptophan-binding proteins.

Interaction des ATP:Guanidine phosphotransférases avec leurs substrats, étudiée par spectrophotometrie différentielle

Differential spectrophotometry of ATP:guanidine phosphotransferase-substrate complexes 1. 1. The difference spectra which result from the binding of nucleotide and guanidine substrates to creatine kinase and arginine kinase has been studied. p] ADP produces specific shift in the absorption spectrum towards higher wave lengths when interacting with both enzymes. The change observed with ATP is quite different and results from a smaller absorption of the adenine ring. When present, the metal Mg 2+ does not change the spectra. Spectrophotometric titrations with nucleotides indicate a stoichiometry of one mole of nucleotide per mole of arginine kinase and two moles of nucleotide per mole of creatine kinase. The dissociation constants for the nucleotide-phosphagene kinase complexes derived from spectral titration data are similar to those obtained from kinetic studies. 2. 2. The binding of creatine to creatine kinase provides no difference spectrum. But a specific one accompanies the binding of l-arginine and l-arginine phosphate to argnine kinase with tyrosyl peaks at 280 and 287 mμ and a hypochromic phase at 239 mμ. 3. 3. Identical difference spectra are also obtained with various hydrophobic l-amino acids such as isoleucine, valine, etc., suggesting interaction between the enzyme active site and the structure ▪ of the studied derivatives. This fact is supported by the protecting effect afforded by the above amino acids against sulfhydryl reagents inhibition. On the other hand, alkylation of arginine kinase with iodoacetamide provides a difference spectrum showing the tyrosyl peaks produced with l-arginine and l-amino acids. The S-carboxylmethyl enzyme fails to bind arginine or amino acids while it still reacts with nucleotides. These experiments suggest that the process of binding l-arginine and some l-amino acids to the arginine kinase active site involves the interaction of the essential sulfhydryl group with the ▪ function of these compounds.

The Interaction of Nucleotides with the Active Site of Staphylococcal Nuclease: SPECTROPHOTOMETRIC STUDIES

Abstract A number of nucleotides that inhibit enzymatic activity of staphylococcal nuclease and bind in a 1:1 molar ratio to its active site produce specific shifts in the absorption spectrum toward higher wave lengths when interacting with the enzyme. The difference spectrum, which results from the binding of deoxythymidine 3',5'-diphosphate to nuclease at neutral pH, has tyrosyl peaks at 280 and 287 mµ, and a broad hypochromic phase at 240 to 270 mµ. The latter change results from perturbations of the nucleotide chromophore. Binding of deoxythymidine 3',5'-diphosphate to nuclease facilitates ionization of the nucleotide, which probably accounts for the change in nucleotide absorption. No changes of tryptophyl absorption accompany binding of nucleotides to nuclease. The magnitudes of the spectral differences, measured at 287 or 247 mµ, vary with the concentration of Ca++ and the nature of the nucleotide in parallel with changes in catalytic activity. The dependence of nucleotide binding on pH suggests that a residue with a pK of 6 to 7 is involved in the binding process. Binding is almost maximal at pH 7, where catalytic activity is virtually absent. Spectrophotometric titrations with nucleotide indicate a stoichiometry of 1 mole of nucleotide per mole of enzyme. The dissociation constants for the nucleotide-nuclease complexes derived from spectral titration data are similar to those obtained from studies of inhibition of enzyme activity, gel filtration, and fluorescence properties of the protein. The spectral changes are dependent on an intact tertiary structure, as suggested by studies on the effects of urea, or of chemical modifications of the nuclease. The spectrophotometric titration curve, in the region of phenolic hydroxyl ionization, is shifted to considerably higher pH values when the titration is carried out on the nuclease nucleotide complex. Solvent perturbation studies reveal that four to five of the seven tyrosyl groups of native nuclease are exposed to the aqueous solvent. In the presence of deoxythymidine 3',5'-diphosphate, however, only one to two groups are similarly exposed. These experiments suggest that the process of binding nucleotides to the active site of nuclease involves burying of 3 to 4 tyrosyl residues. It cannot be determined from these studies whether this occurs by direct interaction of the nucleotide with tyrosyl groups or whether a conformational change is induced which, secondarily, affects these residues.