Molecular spectroscopic studies on the interaction of glycosaminoglycans with brilliant cresol blue and its analytical application.

The interaction of brilliant cresol blue (BCB) with glycosaminoglycans (GAGs), such as heparin (Hep) and chondroitin 4-sulfate (CS), in aqueous solution has been studied by spectrophotometry and light scattering spectroscopy. Absorbance of BCB at 632 and 594 nm decreased on addition of Hep or CS with the appearance of a new blue-shifted absorption band at 550 nm, which indicated that new metachromatic complex formed. The linear decrease in absorbance of BCB at 632 nm was observed. In addition, Hep was more effective than CS (1.7 times) in decreasing absorbance of BCB. The stoichiometry of Hep or CS with BCB was determined by spectrophotometric titration and the MacIntosh extraction method. The result showed that the stoichiometry of BCB/Hep was 1.8 times that of BCB/CS. These results suggested that the interaction between GAGs and BCB was the result of electrostatic forces, and the differences between Hep and CS were attributed to the different negative charge numbers on repetitive disaccharides unit. Studies on the effects of alcohol and urea indicated that GAGs only interacted with the aggregates of BCB. Moreover, a strong light scattering signal was observed after mixing BCB with GAGs. Furthermore, the light scattering intensity at light scattering bands was proportional to the concentration of Hep or CS added when the concentration of BCB was constant.     

Spectrophotometric and Voltammetric Studies on the Interaction of Heparin with Phenosafranine

The interaction of phenosafranine （PSF） with a glycosaminoglycans of heparin （Hep） in aqueous solution has been characterized by UV-Vis absorption spectrophotometry and cyclic voltammetry in pH 1.5 Britton-Robinson （B-R） buffer solution. The addition of Hep caused decrease of the absorbance of PSF at 532 nm and the redox peak current of PSF. The study showed that an supramolecular complex of PSF-Hep was formed because of the electrostatic attraction of negatively charged Hep with the positively charged PSF, which resulted in the decrease of the equilibrium concentration of PSF in solutions, and the decrease of the absorbance or the peak current of PSF. The stoichiometry of the Hep/PSF complex was further calculated by voltammetric data with the result of 1：1 complex.     

The SARS-CoV-2 Entry Inhibition Mechanisms of Serine Protease Inhibitors,OM-85, Heparin and Soluble HS Might Be Linked to HS Attachment Sites

This article discusses the importance of D-xylose for fighting viruses (especially SARS-CoV-2) that use core proteins as receptors at the cell surface, by providing additional supporting facts that these viruses probably bind at HS/CS attachment sites (i.e., the hydroxyl groups of Ser/Thr residues of the core proteins intended to receive the D-xylose molecules to initiate the HS/CS chains). Essentially, the additional supporting facts, are: some anterior studies on the binding sites of exogenous heparin and soluble HS on the core proteins, the inhibition of the viral entry by pre-incubation of cells with heparin, and additionally, corroborating studies about the mechanism leading to type 2 diabetes during viral infection. We then discuss the mechanism by which serine protease inhibitors inhibit SARS-CoV-2 entry. The biosynthesis of heparan sulfate (HS), chondroitin sulfate (CS), dermatan sulfate (DS), and heparin (Hep) is initiated not only by D-xylose derived from uridine diphosphate (UDP)-xylose, but also bioactive D-xylose molecules, even in situations where cells were previously treated with GAG inhibitors. This property of D-xylose shown by previous anterior studies helped in the explanation of the mechanism leading to type 2 diabetes during SARS-CoV-2 infection. This explanation is completed here by a preliminary estimation of xyloside GAGs (HS/CS/DS/Hep) in the body, and with other previous studies helping to corroborate the mechanism by which the D-xylose exhibits its antiglycaemic properties and the mechanism leading to type 2 diabetes during SARS-CoV-2 infection. This paper also discusses the confirmatory studies of regarding the correlation between D-xylose and COVID-19 severity.     

布洛芬与人血白蛋白位点II动态结合过程的分子模拟:一种结合途径分析

人血白蛋白（HSA）主要有两个药物结合位点,位点I和位点Ⅱ,许多小分子优先结合在位点Ⅱ上,包括抗炎类药物布洛芬。本文采用分子模拟方法研究了布洛芬小分子与HSA位点Ⅱ结合的动态过程,探讨了二者的结合机制。首先构建了50个随机分布的布洛芬与HSA复合物体系,经50 ns分子动力学模拟,其中一个布洛芬分子稳定结合于位点Ⅱ。基于该分子的运动轨迹分析,发现布洛芬的结合可分为四个阶段,即远程吸引、表面结合调整、进入位点Ⅱ空腔和稳定结合。比较范德华和静电相互作用能,发现初期以静电吸引为主,中期在HSA表面的两个极性区域间调整,逐步转移至位点Ⅱ附近;然后在位点Ⅱ入口处的极性残基和附近疏水残基的共同作用下,布洛芬进入位点Ⅱ空腔;进入空腔后,静电和疏水共同作用形成稳定结合。在结合过程中,位点Ⅱ附近的蛋白表面发生明显改变,体现出一定的“诱导契合”作用,同时分子模拟得到的结合模式和布洛芬-HSA结合的晶体结构类似。结果表明,分子模拟可以辅助研究小分子和蛋白结合的动态过程,从分子水平阐述相关结合机制。     

Electrochemical Studies on the Interaction of Heparin with Phenosafranine and Its Analytical Application

The interaction of phenosafranine (PSF) with a glycosaminoglycan of heparin (Hep) in aqueous solution was characterized by cyclic voltammetry (CV) and linear sweep voltammetry (LSV) in this paper and further used for Hep detection. In pH 1.5 Britton‐Robinson (B‐R) buffer solution PSF had a well‐defined voltammetric reductive peak at ?0.256 V (vs SCE), and the electrochemical response was faded by the addition of Hep. Cyclic voltammetric experiments indicated that the electrochemical behaviors of free PSF didn't change no matter whether Hep was presented in PSF solution or not. Based on the decrease of the peak current, a second order derivative linear sweep voltammetry was used to establish a sensitive electroanalytical method for Hep. The peak current was proportional to the concentration of Hep in the range of 0.7～20.0 mg L^?1, demonstrating that this method was suitable for routine Hep detection. Under optimal conditions, the linear regression equation for the Hep determination was ΔIp”(nA) = 46.30 C (mg L^?1) + 343.31 (n = 11, γ = 0.991) with a detection limit of 0.08 mg L^?1 (3σ). The established method was further successfully applied to heparin sodium injection samples determination. The interaction mechanism was discussed based on the electrostatic attraction of the negatively charged Hep with the positively charged PSF, and the stoichiometry of Hep‐PSF was calculated by the voltammetric method.     

Chitosan and a modified chitosan as agents to improve performances of mucoadhesive vaginal gels

New mucoadhesive formulations were designed and studied in order to improve local vaginal therapy by increasing formulation retention prolonging thus drug-mucosa contact time. Some gels were prepared using hydroxyethylcellulose (HEC) alone or mixed with chitosan (CS) or its derivative 5-methyl-pyrrolidinone-chitosan (MPCS) and were loaded with the antibacterial metronidazole (MET) (0.75%). All formulations showed pseudoplastic flow and viscosity increase was observed proportionally to chitosan content (CS>MPCS). Prepared gels showed better extrusion properties (yield stress) than market formulation Zidoval. Mucoadhesion force studies permitted to point out that: (i) CS decreases mucoadhesion force; (ii) MPCS addition increases the mucoadhesion force at high percentage; (iii) all gels containing chitosan showed better mucoadhesive performances than Zidoval. Gels containing MPCS showed higher and faster drug release than those containing CS. All the preparations were able to release higher drug amounts if compared to market formulation. In conclusion MPCS improved gel characteristics in terms of mucoadhesion force, rheological behaviour and drug release pointing out that this modified chitosan is very suitable to obtain manageable and more acceptable vaginal formulation.     

Electrochemical methodology to study labile trace metal/natural organic matter complexation at low concentration levels in natural waters

A new electrochemical methodology to study labile trace metal/natural organic matter complexation at low concentration levels in natural waters is presented. This methodology consists of three steps: (i) an estimation of the complex diffusion coefficient (D_ML), (ii) determination at low pH of the total metal concentration initially present in the sample, (iii) a metal titration at the desired pH. The free and bound metal concentrations are determined for each point of the titration and modeled with the non-ideal competitive adsorption (NICA-Donnan) model in order to obtain the binding parameters. In this methodology, it is recommended to determine the hydrodynamic transport parameter, α, for each set of hydrodynamic conditions used in the voltammetric measurements.The methodology was tested using two fractions of natural organic matter (NOM) isolated from the Loire river, namely the hydrophobic organic matter (HPO) and the transphilic organic matter (TPI), and a well characterized fulvic acid (Laurentian fulvic acid, LFA). The complex diffusion coefficients obtained at pH 5 were 0.4 ± 0.2 for Pb and Cu/HPO, 1.8 ± 0.2 for Pb/TPI and (0.612 ± 0.009) × 10⁻¹⁰ m² s⁻¹ for Pb/LFA. NICA-Donnan parameters for lead binding were obtained for the HPO and TPI fractions. The new lead/LFA results were successfully predicted using parameters derived in our previous work.     

Chemistry and biology of glycosaminoglycans in blood coagulation

Glycosaminoglycans (GAGs) are widely distributed in animal tissues where they are usually associated with proteins. Six types are commonly recognized: heparin (Hep), heparan sulfate (HS), dermatan sulfate (DS), chondroitin sulfate (Ch-S), keratan sulfate (KS) and hyaluronic acid (Hyal). They are structurally related with a carbohydrate backbone consisting of alternating hexuronic acid (L-iduronic acid and/or D-glucuronic acid) or galactose units and hexosamine (D-glucosamine or D-galactosamine) residues. All GAGs, except Hyal, show sulfate groups along their chains. Certain sulfate glycoaminoglycans have the ability to interfere with blood coagulation, as demonstrated by the extensive clinical use of Hep as an anticoagulant agent. HS and DS show a good anticoagulant activity, although weaker than that of Hep. In contrast, Ch-S has a low ability to inhibit plasma serine proteases, and KS and Hyal are devoid of any effect on coagulation cascade. The interaction between blood coagulation serine proteases and GAGs can be found to have two principle mechanisms: the specific “lock and key” binding and the nonspecific cooperative electrostatic association. This different ability of GAGs to interact with coagulation cascade proteins depends on the molecular weight, the ratio of iduronic/glucoronic acid and the sulfation degree. Many attempts have been made to improve or induce anticoagulant activity of natural GAGs-by chemical modification. Increasing sulfation degree of DS and Ch-S is followed by their biological activity increasing. Hyal, which is devoid of any anticoagulant effect, acquires a good ability to inactivate plasma serine proteases, i.e. thrombin and Factor Xa, when it is sulfated. This ability increases by increasing the number of sulfate groups per disaccharide unit, although the mechanism of action is different from that of Hep, but seems to be independent of its molecular weight.     

Molecular recognition and fluorescence sensing of monophosphorylated peptides in aqueous solution by bis(zinc(II)-dipicolylamine)-based artificial receptors

The phosphorylation of proteins represents a ubiquitous mechanism for the cellular signal control of many different processes, and thus selective recognition and sensing of phosphorylated peptides and proteins in aqueous solution should be regarded as important targets in the research field of molecular recognition. We now describe the design of fluorescent chemosensors bearing two zinc ions coordinated to distinct dipicolylamine (Dpa) sites. Fluorescence titration experiments show the selective and strong binding toward phosphate derivatives in aqueous solution. On the basis of (1)H NMR and (31)P NMR studies, and the single-crystal X-ray structural analysis, it is clear that two Zn(Dpa) units of the binuclear receptors cooperatively act to bind a phosphate site of these derivatives. Good agreement of the binding affinity estimated by isothermal titration calorimetry with fluorescence titration measurements revealed that these two receptors can fluorometrically sense several phosphorylated peptides that have consensus sequences modified with natural kinases. These chemosensors display the following significant features: (i) clear distinction between phosphorylated and nonphosphorylated peptides, (ii) sequence-dependent recognition, and (iii) strong binding to a negatively charged phosphorylated peptide, all of which can be mainly ascribed to coordination chemistry and electrostatic interactions between the receptors and the corresponding peptides. Detailed titration experiments clarified that the phosphate anion-assisted coordination of the second Zn(II) to the binuclear receptors is crucial for the fluorescence intensification upon binding to the phosphorylated derivatives. In addition, it is demonstrated that the binuclear receptors can be useful for the convenient fluorescent detection of a natural phosphatase (PTP1B) catalyzed dephosphorylation.     

Recognition of guanine-guanine mismatches by the dimeric form of 2-amino-1,8-naphthyridine.

Dimeric 2-amino-1,8-naphthyridine selectively binds to a G-G mismatch with high affinity (K(d) = 53 nM). We have investigated a binding mechanism of naphthyridine dimer 2 to a G-G mismatch by spectroscopic studies, thermodynamic analysis, and structure-activity studies for the thermal stabilization of the mismatch. 1H NMR spectra of a complex of 2 with 9-mer duplex d(CATCGGATG)2 containing a G-G mismatch showed that all hydrogens in two naphthyridine rings of 2 were observed upfield compared to those of 2 in a free state. The 2D-NOESY experiments showed that each naphthyridine of 2 binds to a guanine in the G-G mismatch within the pi-stack. In CD spectra, a large conformational change of the G-G mismatch-containing duplex was observed upon complex formation with 2. Isothermal calorimetry titration of 2 binding to the G-G mismatch showed that the stoichiometry for the binding is about 1:1 and that the binding is enthalpy-controlled. It is clarified by structure-activity studies that show (i) the linker connecting two naphthyridine rings was essential for the stabilization of the G-G mismatch, (ii) the binding efficiency was very sensitive to the linker structure, and (iii) the binding of two naphthyridines to each one of two Gs in the G-G mismatch is essential for a strong stabilization. These results strongly supported the intercalation of both naphthyridine rings of 2 into DNA base pairs and the formation of a hydrogen bonded complex with the G-G mismatch.     

Variations in content and structure of glycosaminoglycans of the vitreous gel from different mammalian species

The vitreous of all species is composed of essentially the same type of extracellular matrix macromolecules organized to a transparent gel. In this study, the composition and fi ne chemical structure of the glycosaminoglycans (GAGs) in the vitreous gel from sheep and goat were determined and compared with those of human and pig vitreous gels. The results showed that, in all examined species; hyaluronan (HA) was the predominant GAG, whereas chondroitin sulphate (CS) was the minor one. In the vitreous gel of the most relative species, i.e. sheep and goat, higher amounts of both of HA and CS were estimated as compared with pig and human tissues. The distribution of hydrodynamic sizes of HA and CS was significantly differed among different species. All HA preparations consisted of molecules with great variability in hydrodynamic sizes. The relative proportions of the large HA molecules (size >1.8 x 10(6) kDa) were significantly higher in sheep and goat as compared with human and pig vitreous gel. The length of CS chains was also of larger size in sheep and goat (50 and 58 kDa, respectively) than the respective chains in human and pig vitreous gel (38 and 28 kDa, respectively). The sulphation patterns of CS preparations were determined following enzymic treatments, HPLC and capillary electrophoretic analyses. The human vitreous-derived CS chains showed quite different sulphation profile than that of CS isolated from other species, since 4-sulphated disaccharides were identified as the dominant moiety. In conclusion, significant compositional and structural variations between the vitreous matrixes of different species at the GAG level were identified. The functional significance of these species-dependent variations is discussed.     

Non-trivial solution chemistry between amido-pyridylcalix[4]arenes and some metal salts

Mercury ion complexation reactions were carried out between 3 and various mercury(II) salts. (1)H NMR studies showed that the role of solvent, the anion chosen and the initial reaction conditions were critical and that the formation of a "simple" mercury(II) complex was non-trivial. The mercury(II) ion can cause either (i) the formation of an ion-pair system, which have a characteristic doubling of all signals in the (1)H NMR spectrum, (ii) a cleavage reaction to occur resulting in the reformation of the calix[4]arene diester compound 2, but only when the reaction is heated and (iii) "simple" mercury binding to the pyridine rings when the binding studies are carried out using NMR titration techniques. The electrochemistry results, on the same systems, show that the initial reaction involves the removal of the phenoxide protons followed by the resulting catalysis of the mercury species. This proton removal is not observed in the NMR spectra of any of the mercury reactions. It was also found that 3 could bind silver and zinc salts and was not selective for mercury(II) as was previously described.     

用蛋白质作探针共振瑞利散射和共振非线性散射法测定硫酸软骨素A

在pH值为2.5~4.0的BR缓冲溶液介质中,牛血清白蛋白(BSA)、糜蛋白酶(Chy)和α-淀粉酶(α-Amy)等蛋白质与酸性多糖硫酸软骨素A(CS)形成结合物。 此时将会使共振瑞利散射(RRS)和二级散射(SOS)、倍频散射(FDS)等共振非线性散射的强度显著增大。 在蛋白质过量时,3种散射增强(ΔIRRS、ΔISOS和ΔIFDS)均在一定范围内与CS的浓度成正比,方法具有高灵敏度。 当用Chy、BSA和α-Amy作探针时,3种散射法对于CS的检出限分别在1.4~5.8 μg/L、2.0~13.2 μg/L和1.8~9.6 μg/L。 其中以Chy-CS体系的RRS法最灵敏(检出限1.4 μg/L),可用于痕量CS的测定。 研究了反应体系的RRS、SOS和FDS的光谱特征、适宜的反应条件和影响因素,并以Chy-CS体系为例考察了共存物质的影响,方法有良好的选择性,将其用于滴眼液中CS的测定,取得了较好的结果。     

Supramolecular complex induced by the binding of sodium dodecyl sulfate to PAMAM dendrimers

Isothermal titration calorimetry (ITC) and dynamic light scattering (DLS) were employed to study the spontaneous supramolecular complexation of amine terminated PAMAM dendrimer (G3[EDA] PAMAM-NH2) induced by the binding of an anionic surfactant, sodium dodecyl sulfate (SDS). At pHor=10, the electrostatic binding ceased because the deprotonated PAMAM dendrimer was uncharged, and hence the surfactant-induced supramolecular assembly could not be formed.     

An isothermal titration calorimetry study of phytate binding to lysozyme

Isothermal titration calorimetry (ITC) was used to detect phytate binding to the protein lysozyme. This binding interaction was driven by electrostatic interaction between the positively charged protein and negatively charged phytate. When two phytate molecules bind to the protein, the charge on the protein is neutralised and no further binding occurs. The stoichiometry of binding provided evidence of phytate–lysozyme complex formation that was temperature dependent, being most extensive at lower temperatures. The initial stage of phytate binding to lysozyme was less exothermic than later injections and had a stoichiometry of 0.5 at 313 K, which was interpreted as phytate crosslinking two lysozyme molecules with corresponding water displacement. ITC could make a valuable in vitro assay to understanding binding interactions and complex formation that normally occur in the stomach of monogastric animals and the relevance of drinking water temperature on the extent of phytate–protein interaction. Interpretation of ITC data in terms of cooperativity is also discussed.     

Multiple steps and critical behaviors of the binding of calcium to alginate

Previous research on the binding and gelation of calcium/alginate in aqueous solution were mostly conducted in the (semi-)concentrated regime, and it did not provide details of the binding process and the formation of egg-box junctions. In the present investigation, the binding of calcium to alginate, of low and high molecular weight and different guluronate/mannuronate ratios, was investigated in dilute solutions using isothermal titration calorimetry (ITC), Ca2+-selective potentiometry, and viscometry techniques. The results reveal three distinct and successive steps in the binding of calcium to alginate with increased concentration of Ca ions. They were assigned to (i) interaction of Ca2+ with a single guluronate unit forming monocomplexes; (ii) propagation and formation of egg-box dimers via pairing of these monocomplexes; and (iii) lateral association of the egg-box dimers, generating multimers. The third step has different association modes depending on the molecular weight of alginate. The boundaries between these steps are reasonably critical, and they closely correlate with the Ca/guluronate stoichiometry expected for egg-box dimers and multimers with 2/1 helical chains. The formation of egg-box dimers and their subsequent association are thermodynamically equivalent processes and can be fitted by a model of independent binding sites. The binding of Ca to alginates of different guluronate contents is controlled by a balance between enthalpy and entropy.     

Thermodynamics of folding, stabilization, and binding in an engineered protein--protein complex

We analyzed the thermodynamics of a complex protein-protein binding interaction using the (engineered) Z(SPA)(-)(1) affibody and it's Z domain binding partner as a model. Free Z(SPA)(-)(1) exists in an equilibrium between a molten-globule-like (MG) state and a completely unfolded state, wheras a well-ordered structure is observed in the Z:Z(SPA)(-)(1) complex. The thermodynamics of the MG state unfolding equilibrium can be separated from the thermodynamics of binding and stabilization by combined analysis of isothermal titration calorimetry data and a separate van't Hoff analysis of thermal unfolding. We find that (i) the unfolding equilibrium of free Z(SPA)(-)(1) has only a small influence on effective binding affinity, that (ii) the Z:Z(SPA)(-)(1) interface is inconspicuous and structure-based energetics calculations suggest that it should be capable of supporting strong binding, but that (iii) the conformational stabilization of the MG state to a well-ordered structure in the Z:Z(SPA)(-)(1) complex is associated with a large change in conformational entropy that opposes binding.     

Solid-phase synthesis, characterization and DNA binding properties of the first chloro(polypyridyl)ruthenium conjugated peptide complex

A general method for the synthesis of chloro(polypyridyl)ruthenium conjugated peptide complexes via a solid-phase strategy is described. The method is applied to synthesize two positional isomers of the complex [Ru(terpy)(4-CO2H-4'-Mebpy-Gly-L-His-L-LysCONH2)Cl](PF6). Even though the separation of the isomers was only partially achieved chromatographically, the isomers were unambiguously assigned by NMR spectroscopy. The interactions of the complex [Ru(terpy)(4-CO2H-4'-Mebpy-Gly-L-His-L-LysCONH2)Cl](PF6) with CT-DNA and plasmid DNA, have been studied with various spectroscopic techniques, showing that (i) the complexes coordinatively bind to DNA preferring the bases guanine and cytosine over the bases thymine and adenine after hydrolysis of the coordinated chloride, (ii) electrostatic interactions between the complex cation and the polyanionic DNA chain assist this binding (iii) only in the case of one isomer the peptide does interact further with DNA as evidenced from 31P NMR spectroscopy, (iv) DNA unwinding occurs in all cases with high binding ratio (Ru/base) values (r > 0.3).     

Theoretical analysis of factors affecting the formation and stability of multilayered colloidal dispersions

A mathematical analysis of the major factors influencing the formation and stability of colloidal dispersions containing spherical particles surrounded by multilayered polymeric interfacial membranes formed by the layer-by-layer electrostatic deposition technique is carried out. The mathematical model assumes that (i) the colloidal dispersion initially consists of a mixture of electrically charged monodisperse spherical particles and oppositely charged polymer molecules, (ii) the adsorption of polymer molecules to the particle surfaces is diffusion-limited, and (iii) the dominant particle-particle collision mechanism is Brownian motion. This approach was used to produce stability maps that highlight conditions under which bridging flocculation, multilayer formation, or depletion flocculation occurs. The stability maps are derived from calculations of the critical polymer concentrations required to (i) saturate the particle surfaces (C(Sat)), (ii) ensure that polymer adsorption is faster than particle collisions (C(Ads)), and (iii) promote depletion flocculation (C(Dep)). In addition, the influence of interfacial properties on the stability of multilayer colloidal dispersions was assessed by calculating the colloidal interactions between the coated particles (i.e., van der Waals, electrostatic, steric, and depletion). These calculations indicated that the major factors are the interfacial charge and composition rather than the interfacial thickness. This article provides useful insights into the factors affecting the formation of stable multilayer colloidal dispersions.