Determination Of Association Constants Research Articles

Organic-soluble cytosine derivatives for studying base-pair interactions in nonaqueous solution

The presence of higher order G-quadruplex-DNA (G4) structures in human cells in guanine-rich DNA sequences has been well established. In cases of G4 helicase impairment or G4 stabilization via association with G4 ligands, it has been determined that arrest and/or termination of DNA transcription/replication occurs because these structures act as physical barriers to both helicase and polymerase mobility along the DNA. Truncation of transcription or replication may result in the loss of genetic material expression leading to disorders. To ameliorate G4-induced barriers, agents that destabilize or “unwind” the quadraplex structures are sought. Earlier work has shown that the cytosine analogue phenylpyrrolocytosine has potential G4-destabilizing ability. It was hypothesized that the unwinding activity was related to its ability to base pair with guanosine. To facilitate the design and evaluation of the hydrogen bonding ability of cytosine analogues, solubility in aprotic, and low-polarity solvents, e.g., deutero-chloroform (CDCl3), is needed; then, determination of association constants ( Ka) with guanine via 1H nuclear magnetic resonance (NMR) spectroscopy or isothermal titration calorimetry (ITC) will be possible. Herein, we report on the synthesis of three new chloroform-soluble, cytosine analogues: N1-cyclohexylmethyl-7-phenylimidazolocytosine, N1-cyclohexylmethyl-7-(6-(methoxycarbonyl)pyridin-2-yl)imidazolocytosine, and N1-cyclohexylmethyl-7-(6-carbamoylpyridin-2-yl)imidazolocytosine. We further report the binding interactions of each of these analogues with the guanosine derivative 2′,3′,5′- O-tris( tert-butyldimethylsilyl)guanosine through the use of both ITC and 1H NMR titration experiments in chloroform and compare the binding to persilyated ribo-cytidine.

Modulating the ion-transfer electrochemistry of perfluorooctanoate with serum albumin and β-cyclodextrin.

Per- and polyfluoroalkyl substances (PFAS) are durable synthetic pollutants that persist in the environment and resist biodegradation. Ion-transfer electrochemistry at aqueous-organic interfaces is a simple strategy for the detection of ionised PFAS. Herein, we investigate the modulation of the ion transfer voltammetry of perfluorooctanoate (PFOA) at liquid-liquid micro-interface arrays by aqueous phase bovine serum albumin (BSA) or β-cyclodextrin (β-CD) and examine the determination of association constants for these binding interactions. By tracking the ion transfer current due to ionised, uncomplexed PFOA as a function of BSA or β-CD concentration, titration curves are produced. Fitting of a binding isotherm to these data provides the association constants. The association constant of PFOA with the BSA determined in this way was ca. 105 M-1 assuming a 1 : 1 binding. Likewise, the association constant for PFOA with β-CD was ca. 104 M-1 for a 1 : 1 β-CD-PFOA complex. Finally, the simultaneous effect of both BSA and β-CD on the ion transfer voltammetry of PFOA was studied, showing clearly that PFOA bound to BSA is released (de-complexed) upon addition of β-CD. The results presented here show ion transfer voltammetry as a simple strategy for the study of molecular and biomolecular binding of ionised PFAS and is potentially useful in understanding the affinity of different PFAS with aqueous phase binding agents such as proteins and carbohydrates.

P-259 - Determination of Association Constants of Macropa-based Chelators for the Stable Complexation of Barium-131 and Lanthanum-133

P-259 - Determination of Association Constants of Macropa-based Chelators for the Stable Complexation of Barium-131 and Lanthanum-133

Tracking metal enhanced fluorescence from Eschericcia coli nano-bio-assemblies within colloidal dispersions by static and 3D spectra emissions

It was studied the interactions of single Ultraluminescent 40 nm gold Core shell Nanoparticles (d40 Au@SiO2-Fl) with Eschericcia Coli bacteria (Esc. Col). By this manner, it was tracked from initial Nano-Bio-assembly formation (Au@SiO2-Fl-Esc. Col) towards inter-Nano-Biostructure interactions. In order to do that it was determined association constants between d40 Au@SiO2-Fl and Biostructures by 3D Fluorescence, fluorescence spectroscopy measurements. The association constant was related with non-covalent Nano-Biostructure interactions of 45 ± 2 biostructure-1; accompanied with a Quantum Yield ratio between the Bio-assembly and Nano-labeller of 3.6 ± 0.1 times, with the maximal concentration of bacteria addition. This phenomenon of interaction was tracked from the bulk of the colloidal dispersion. And, from these individual interactions, it was possible to generate Bioimaging by Laser Fluorescence Microscopy in real time of the dynamic of Bio-assembly formation as well as the by inter-Nano-Biostructures. These data leaded to the correlation of 3D Fluorescence spectroscopy within different bacteria concentrations. The recorded spectra showed increased values of intensities that fitted to a linear equation as model of the experimental data records. About analytical performances, it could be noted that optimized sensitivity by 3D Fluorescence was unless 65% higher than by static Fluorescence. Moreover, it should be mentioned that two distributions of emissions were recorded that correlated with Plasmonics coupling with natural emitters within the UV and MEF Nanolabeller emissions. Thus, optimizing conditions, LODs of 0.3x10 3 bacterium*mL−1 were determined. Moreover, by In Flow Cytometry was recorded different sensitivity values depending of the Laser excitation applied. These phenomena were explained by the development of different Nano-Bio-emitters species. In addition, in order to validate the methodology, it was quantified Biostructures from fortified aqueous samples. By 3D Fluorescence it was determined recoveries of (97 ± 8)x103, and (93 ± 5)x103 from upper and lower distributions of emissions recorded in 3D Fluorescence topographies. The reproducibility in all cases was by %CV of 98–99 %; therefore the proposed Bioassays showed interesting insights in Nano-Biotechnology with impact in varied Research fields.In this way, it was showed and discussed about Bio-analytical applications for Eschericcia Coli detection and quantification within colloidal dispersion with relative simple, accessible and portable fluorescence spectroscopy set ups. Therefore, it was given insights for accessible Nano-Biodetection based on Ultra-sensitive MEF Nanoplatforms for Bio-labeling using standard spectroscopic and compared by In Flow Citometry analysis as well.

Synergistic use of siderophores and weak organic ligands during zinc transport in the rhizosphere controlled by pH and ion strength gradients

Citrate (Cit) and Deferoxamine B (DFOB) are two important organic ligands coexisting in soils with distinct different affinities for metal ions. It has been theorized that siderophores and weak organic ligands play a synergistic role during the transport of micronutrients in the rhizosphere, but the geochemical controls of this process remain unknown. Here we test the hypothesis that gradients in pH and ion strength regulate and enable the cooperation. To this end, first we use potentiometric titrations to identify the dominant Zn(II)–Cit and Zn(II)–DFOB complexes and to determine their ionic strength dependent stability constants between 0 and 1 mol dm−3. We parametrise the Extended Debye-Hückel (EDH) equation and determine accurate intrinsic association constants (logβ0) for the formation of the complexes present. The speciation model developed confirms the presence of [Zn(Cit)]−, [Zn(HCit)], [Zn2(Cit)2(OH)2]4−, and [Zn(Cit)2]4−, with [Zn(Cit)]− and [Zn2(Cit)2(OH)2]4− the dominant species in the pH range relevant to rhizosphere. We propose the existence of a new [Zn(Cit)(OH)3]4− complex above pH 10. We also verify the existence of two hexadentate Zn(II)–DFOB species, i.e., [Zn(DFOB)]− and [Zn(HDFOB)], and of one tetradentate species [Zn(H2DFOB)]+. Second, we identify the pH and ionic strength dependent ligand exchange points (LEP) of Zn with citrate and DFOB and the stability windows for Zn(II)–Cit and Zn(II)–DFOB complexes in NaCl and rice soil solutions. We find that the LEPs fall within the pH and ionic strength gradients expected in rhizospheres and that the stability windows for Zn(II)–citrate and Zn(II)–DFOB, i.e., low and high affinity ligands, can be distinctly set off. This suggests that pH and ion strength gradients allow for Zn(II) complexes with citrate and DFOB to dominate in different parts of the rhizosphere and this explains why mixtures of low and high affinity ligands increase leaching of micronutrients in soils. Speciation models of soil solutions using newly determined association constants demonstrate that the presence of dissolved organic matter and inorganic ligands (i.e., bicarbonate, phosphate, sulphate, or chlorides) do neither affect the position of the LEP nor the width of the stability windows significantly. In conclusion, we demonstrate that cooperative and synergistic ligand interaction between low and high affinity ligands is a valid mechanism for controlling zinc transport in the rhizosphere and possibly in other environmental reservoirs such as in the phycosphere. Multiple production of weak and strong ligands is therefore a valid strategy of plants and other soil organisms to improve access to micronutrients.

Thermodynamic and Kinetic Studies on the Conversion of Solvent-Shared to Contact Ion Pairs in Sparingly Soluble MF2 (M = Mg2+ and Ca2+) Aqueous Solutions: Implications for Understanding Supersaturated Behavior and Association Constant Determination.

The thermodynamic and kinetic behaviors of Mg2+-F- ion pairing in aqueous solution are investigated theoretically and experimentally and are contrasted to those of Ca2+-F-. Thermodynamically, similar to CaFx(H2O)142-x (x = 1 and 2), MgF(H2O)y+ (y = 14-20) contact ion pairs (CIPs) are more stable than their solvent-shared ion pairs (SSIPs), whereas the CIPs and SSIPs of MF2(H2O)y are almost isoenergetic. However, in kinetics, the conversion of SSIPs to CIPs for M2+-F- (M = Mg2+ and Ca2+) ion pairing must overcome a high energy barrier due to the strong hydration of Mg2+ and F-. The kinetics dominate after the thermodynamics and kinetics are balanced, which hinders the formation of M2+-F- CIPs in practical MF2 aqueous solutions (less than or equal to saturated concentrations). This result is also supported by the 19F nuclear magnetic resonance spectra of saturated MF2 solutions. Although the interaction between Mg2+ and F- is slightly stronger than that between Ca2+ and F- due to the smaller radius of Mg2+, the formation of Mg2+-F- CIPs needs to go through two rate-limiting steps, the dehydration and entrance of F- (i.e., via exchange mode) with a higher energy barrier, due to the ability of strongly bound water molecules and rigorous octahedral coordinated configuration of Mg2+, while the formation of Ca2+-F- CIPs only goes through a single rate-limiting step, the entrance of F- (i.e., via swinging mode) with a lower energy barrier, due to the flexible coordination configuration of Ca2+. This is responsible for precipitation in MgF2 aqueous solution requiring a larger supersaturation degree and a lower precipitation rate than in CaF2. These kinetic factors lead to the association constants previously reported for MF+ determined by a fluoride ion-selective electrode (ISE) combined with the titration method, where the MF2 solutions were always unsaturated at the titration end point, which actually corresponds to those of the ligand process going from completely free M2+ and F- to their SSIPs. A possible strategy to accurately determine the association constants of MF+ and MF2(aq) CIPs by fluoride ISEs is proposed. The present results suggest that judging the formation of M2+-F- CIPs in practical solutions from a theoretical calculation perspective requires significant consideration of the kinetic factors, except for the thermodynamic factors.

Evaluating Halogen-Bond Strength as a Function of Molecular Structure Using Nuclear Magnetic Resonance Spectroscopy and Computational Analysis.

Halogen bonding (XB) is a highly directional, non-covalent intermolecular interaction between a molecule (XB donor) presenting a halogen with an electron-deficient region or sigma hole (σ-hole) and an electron-rich or Lewis-base molecule (XB acceptor). A systematic, experimental, and theoretical study of solution-phase XB strength as a function of the molecular structure for both XB donor and acceptor molecules is presented. The impact of specific structural features is assessed using 19F and 1H nuclear magnetic resonance (NMR) titrations to determine association constants, density functional theory calculations for interaction energies and bond lengths, as well as 19F-1H HOESY NMR measurements of intermolecular cross-relaxation between the interacting XB donor-acceptor adducts. For XB donor molecules (perfluoro-halogenated benzenes), results indicate the critical importance of iodine coupled with electron-withdrawing entities. Prominent structural components of XB acceptor molecules include a central atom working in conjunction with a Lewis-base atom to present high electron density directed at the σ-hole (e.g., tributylphosphine oxide). Additionally, larger surrounding aliphatic R groups (e.g., butyl and octyl) were found to significantly stabilize strong XB, particularly in solvents that promote the interaction. With a more thorough understanding of structure-optimized XB, one can envision harnessing XB interactions more strategically for specific design of optimal materials and chemical applications.

Dynamic enzymatic synthesis of γ-cyclodextrin using a photoremovable hydrazone template

Dynamic enzymatic synthesis of γ-cyclodextrin using a photoremovable hydrazone template

Supramolecular properties of amphiphilic adamantylated azo dyes

Despite environmental and health risks, azo dyes are still very popular colourising agents; therefore, methods for removing the dyes and/or related pollutants from wastewater have been developed. We have incorporated a lipophilic adamantane cage into two polar disulphonatonaphthalene-1-azobenzene dyes to prove the concept that modified dyes can be treated via host–guest supramolecular interactions using suitable cyclodextrin (CD) and cucurbit[n]uril (CBn) hosts. We conducted 1H NMR experiments to demonstrate that both dyes form specific inclusion complexes with an adamantane cage buried inside the CB7 or β-CD cavity. Using isothermal titration calorimetry, we determined association constants with β-CD and CB7 in the range of (0.7–1.3) × 106 and (1.4–3.4) × 108, respectively. As the dye@CB7 complexes were sparingly soluble in water, the dye can be efficiently removed from water by precipitation, even in the presence of β-CD.

Investigation of labile protein-ligand interactions by native electrospray ionization mass spectrometry

The investigations of noncovalent interaction between proteins (P) and small ligands (L) have played an important role for drug discovery and therapy. Electrospray ionization mass spectrometry (ESI-MS) has been typically applied to analysis of P-L interactions. However, some labile complexes are problematic for determination of association constants (Ka) by ESI-MS due to their in-source dissociation. Here, a strategy based on native MS for analysis of the labile complexes was developed. The reference ligand (Lref) with specific binding to P and a nonspecific binding ligand were employed. The equations for Ka calculation of labile complexes were developed. According to mathematical fitting curves, the Ka values of partially dissociated lysozyme-icariin complex and completely dissociated lysozyme-berberine complex were obtained. The obtained Ka values were consistent with those by fluorescence method. This strategy can be applied to the labile complexes of other small ligands to protein.

Binding energy and photostability of the β-cyclodextrin encapsulates of lornoxicam and tenoxicam drugs: A combined experimental and theoretical study

The lornoxicam (LRX) and tenoxicam (TNX) drugs form a stable 1:1 inclusion complex with b-cyclodextrin (β-CD) in aqueous solution. The experimentally determined association constants (K) of LRX- β-CD and TNX- β-CD are 13.4 and 10.3 M-1, respectively. Quantum chemical computations simulated the preferred orientation of guest molecules in the host. Geometry optimized results using the ONIOM technique provided more in-depth insights and identified the structure and showed that both drugs were partially encapsulated within the β-CD cavity. The calculated inclusion binding energy (BE, kcal mol-1) reveals the noticeable thermal stability of LRX-β-CD (-24.19 kcal/mol) over the TNX-β-CD (-13.45 kcal/mol) capsulate. Furthermore, the photostabilities of the encapsulated drugs were tested. Drug encapsulation did not result in any additional photostability. Moreover, encapsulation of the drugs in the β-CD resulted in noticeable changes in the electronic characteristics of the drugs, as reflected in their reactivity indices. The fact that the water-soluble β-CD formed inclusion complexes with water-insoluble LRX and TNX enables the drug delivery vehicle for oral administration.

Molecular Imprinted Polymer for Ethylmorphine with Methacrylic Acid and Acrylamide as Functional Monomer in Butanol Using Two Polymerization Method

Ethylmorphine is an opioid that has therapeutic effects as narcotic analgesic and antitussive, which has low levels and can be misused. Hence, it is crucial to monitor by analyze the levels of ethylmorphine in blood selectively. The preparation method that can be used to extract ethylmorphine from the sample is using molecular imprinting solid-phase extraction (MI-SPE) due to its sensitivity and selectivity. This study aims to compare the result of synthesis using two different polymerization methods, and also to examine the analytical performance and characteristics of imprinted polymers from two distinct functional monomers: methacrylic acid (MAA) and acrylamide (AM). The stages of this study include the determination of association constants, synthesis of polymer MI-SPE ethylmorphine using bulk and precipitation polymerization method, extracted template from the polymer, and determined the adsorption ability, capacity, and selectivity of the polymer. MI-SPE that has been made then characterized by using Fourier-Transform Infrared (FTIR) and Scanning Electron Microscope (SEM). The results showed that MIP with acrylamide (MIP-AM) as functional monomer and made by precipitation polymerization had better analytic performances than MIP that made by bulk polymerization, with affinity value 0.072 mg/g and homogeneity value -0.77. It is also selective toward ethylmorphine with imprinting factor value 27.43. In addition, the result of characterization using FTIR and SEM showed that MIP-AM 2, MIP-MAA 1, and MIP-MAA 2 might have a low degree of polymerization due to the presence of vinyl peaks, besides MIP-AM 2 and MIP-MAA 2 had smaller particle size than the NIP with an average value of 0,31 ± 0,21 mm and 0.28 ± 0.05 mm. Based on the result of this study, MIP-AM made by precipitation polymerization could be used to extract ethylmorphine on solid-phase extraction.

Determination of association constants and FRET in hydrazide-based molecular duplex strands

The association constants for the hydrazide-based molecular duplex strands can be determined via monitoring the pyrene excimer emission. By mixing pyrene and perylene labelled oligomers, supramolecular substitution reactions induced efficient FRET.

Quaternary protoberberine alkaloids and their interactions with DNA

The G-quadruplex DNA is commonly present in several protooncogenic-DNA promoters and participates in many important biological processes such as replication, transcription, and translation. Because of their supposed role in cancer, G-quadruplex DNA is often studied as a target for anti-cancer drugs. Quaternary protoberberine and tetrahydroprotoberberine alkaloids (corysamine, coptisine, stylopine), which are supposed to selectively bind these structures, have been compared in terms of stability with selected types of DNA. Influence of selected alkaloids on the stability of double-stranded DNA and non-canonical forms of DNA was observed by determining association constants of alkaloid–DNA complexes using spectroscopic methods—molecular absorption spectrometry, fluorescence spectrometry, and mass spectrometry. Furthermore, the effect of given alkaloids on the melting temperature of these DNA structures was determined using CD spectrometry. Competitive dialysis and electrospray mass spectrometry were performed for affinity comparison of certain alkaloids to different DNA structures including G-quadruplexes. These experiments have proven that corysamine and coptisine prefer interaction with G-quadruplexes in comparison to dsDNA and ssDNA, whereas tetrahydroprotoberberine alkaloid stylopine does not interact with any DNA whatsoever.

Synthesis of Atenolol-Imprinted Polymers with Methyl Methacrylate as Functional Monomer in Propanol Using Bulk and Precipitation Polymerization Method.

Atenolol is one of the beta-1 blocker drugs that is misused by athletes to increase their performance during competition. Therefore, it is important to analyze atenolol levels in blood selectively. The preparation method that can be used in separating atenolol in sample is molecular imprinting solid-phase extraction (MI-SPE) because it has good selectivity and sensitivity. This study aims to examine the characteristics and analytical performance of imprinted polymers synthesized from functional monomer methyl methacrylate. The stages of this study include the determination of association constants, synthesis of sorbent MI-SPE atenolol using the bulk polymerization method, and precipitation with atenolol as the template, methyl methacrylate as the functional monomer, and propanol as the porogen. The template was extracted from a polymer, and then, the adsorption ability, capacity, and selectivity of MI-SPE and finally the application of the best MI-SPE to spiked serum samples were determined. MI-SPE was also characterized by using Fourier-transform instrument infrared (FTIR) and scanning electron microscope (SEM). The result of characterization with FTIR and SEM showed that MIP made by the precipitation polymerization method was completely polymerized, more porous, and produced smaller particle size with an average value of 0.274 μm. It had better analytic performances than MIP made by bulk polymerization, with affinity value 0.3607 mg/g and homogeneity value 1.3246, and good selectivity toward atenolol with imprinting factor value 22.519. Application of MI-SPE to spiked serum samples has an excellent recovery percentage of 95.46% over 0% for the nonimprinting one. Based on the result of study, MIP made by precipitation polymerization could be used to extract atenolol on serum samples toward drug analysis.

SELECTIVE ATENOLOL DETERMINATION IN BLOOD USING MOLECULAR IMPRINTED POLYMER WITH ITACONIC ACID AS FUNCTIONAL MONOMER

Objective: This study was aimed to determine analytical performance and physical character of MI-SPE (Molecular Imprinted Solid Phase Extraction) atenolol using itaconic acid as the functional monomer and to implement the material for the extraction of atenolol in blood serum.Methods: This experiment was performed by determining association constants between monomer-template with UV-Vis spectrophotometer, the synthesis of MI-SPE atenolol using bulk polymerization method, template extraction, evaluation of the adsorption ability and capacity of sorbent, evaluation of sorbent selectivity, and determining their physical character using Fourier Transform Infrared (FTIR) and Scanning Electron Microscope (SEM). In the end, the sorbent then was implemented to extract atenolol in blood serum.Results: Analytical performance showed that MI-SPE sorbent has Imprinting Factor (IF) 10.632 which is the largest number compared to IF when using another beta blocker compound. Physical characterization obtained by MI-SPE using Scanning Electron Microscope (SEM) method showed that MI-SPE morphology has homogeneous pore and number of cavities than its blank. MI-SPE has recovery percentage 92.22 % atenolol when it applied to blood serum spiked with atenolol standard.Conclusion: MI-SPE sorbent made from the itaconic acid monomer in methanol porogen potential to be used for the extraction of atenolol from the blood sample by selectively bind to atenolol.

Computational Modeling of the Interactions of Drugs with Human Serum Albumin (HSA)

Human serum albumin (HSA) is the most abundant protein in the circulatory system that shows a remarkable capacity to bind a wide range of drugs impacting their therapeutic effect. Therefore, the binding to HSA represents a fundamental factor to consider when designing and developing new drugs. Although biophysical techniques (e.g. spectroscopy) are commonly employed to measure the extent to which drugs bind to HSA, these methods are time consuming and usually extremely expensive. Hence, there is an urgent need to incorporate more efficient methods in an attempt to streamline the development of new drugs. Here we present the implementation of a robust and cost-effective computational method to the prediction of the binding affinity of drugs towards HSA. Our method incorporates the program AutoDock Vina to perform in silico molecular docking of a highly diverse set of drugs against the 3D crystal structure of HSA. The 3D structure of HSA was retrieved from the Protein Data Bank and prepared to be used as receptor in our docking simulations. 3D structures of drugs were generated and optimized using Open Babel. Our protocol using AutoDock Vina as the docking engine was capable of reproducing the binding mode of indoxyl sulfate within the X-ray crystal structure of HSA (RMSD < 2.0 A). In addition, our protocol correlated accurately predicted affinity values with experimentally determined association constants (r2=0.61). Our computational-based molecular docking approach incorporating AutoDock Vina may prove useful to the prediction of the binding affinities of drugs towards human serum albumin, and thus, could help alleviate a major bottleneck of the drug discovery process.

Fluorinated Bambusurils as Highly Effective and Selective Transmembrane Cl−/HCO3− Antiporters

Summary The exchange of chloride and bicarbonate across lipid bilayers is an important biological process. Synthetic molecules can act as mobile carriers for these anions, although most show little selectivity. Here we report on three bambus[6]uril macrocycles functionalized with fluorinated benzyl groups, which are able to exchange Cl− and HCO3− efficiently. Remarkably, rates for Cl−/NO3− exchange are two orders of magnitude lower. The higher rates of Cl−/HCO3− transport can be explained by the ability of the bambusurils to complex Cl− and HCO3− simultaneously, facilitating their exchange at the bilayer interface. Furthermore, the exceptionally high affinity and selectivity of these systems for NO3− appear to contribute to the poor Cl−/NO3− exchange. This work not only demonstrates the importance of anion binding characteristics on anion transport but also the potential relevance of bambusurils for anion transport applications considering the high rate observed for Cl−/HCO3− exchange.

Determination of association constants between water-soluble phospholipid polymer bearing phenylboronic acid group and polyol compounds for reversible formation of three-dimensional networks

To develop a reversible forming polymer network system, a polymer complexation was conducted between water-soluble and cytocompatible polymers, namely poly(2-methacryloyloxyethyl phosphorylcholine-co-n-butyl methacrylate-co-p-vinylphenylboronic acid) (PMBV) and poly(vinyl alcohol) (PVA). The two polymers spontaneously form hydrogel upon mixing. The association constant depended on the chemical structure of the polyol compounds and the aggregation of the PMBV. Compared to the association constants of PVA to PMBV, d-glucose and D-sorbitol have smaller and larger association constants, respectively, which is in good accord with the dissociation phenomenon of PMBV/PVA hydrogels. In conclusion, consideration of the association constants between the PMBV and polyol compounds is important for obtaining good reversibility of a polymer hydrogel system.

Fluorescence anisotropy of fluorescein derivative varies according to pH: Lessons for binding studies

Fluorescence spectroscopy is a powerful tool to comprehend macromolecules interaction. The characteristics of the fluorophore as well its behavior under the experimental conditions are central to ascertain the results. The present work characterizes the fluorescence emission and anisotropy of a fluorescein derivative linked to a double strand DNA containing 67 base pairs (named as 6-FAM-67-mer) under different pH conditions. The results show that fluorescence emission and anisotropy of this fluorescein derivative varies according to pH (as the pH becomes more acidic, the total fluorescence emission decreases). The decrease in fluorescence emission intensity is accompanied by an increase in anisotropy. The monoanionic form of fluorescein derivative is the predominant species from pH 7.5 to 9.2. Our main conclusion is that a constant fluorescein derivative emission is a prerequisite to determine association constants between macromolecules.