Unveiling the binding interaction of zinc (II) complexes of homologous Schiff-base ligands on the surface of BSA protein: A combined experimental and theoretical approach

Four new zinc (II) complexes [Zn (HL¹H)Br₂] (1), [Zn (HL¹H)Cl₂] (2), [Zn₂(HL²)Br₃] (3), and [Zn (HL²)Cl] (4) have been synthesized by adopting template synthetic strategy and utilizing two homologous Schiff base ligands (H₂L¹ = 4-bromo-2-{[2-(2-hydroxyethylamino)-ethylimino]-methyl}-6-methoxyphenol, H₂L² = 4-bromo-2-{[3-(2-hydroxyethylamino)propylimino]methyl}-6-methoxyphenol), differing in one -CH₂- unit in the ligating backbone, by adopting template synthetic strategy. All the complexes have been characterized by single crystal X-ray diffraction analysis as well as by other routine physicochemical techniques. Ligand mediated structural variations have been observed and rationalized by density functional theoretical (DFT) calculations. Interaction of the complexes 1–4 with Bovine Serum Albumin protein (BSA) has been studied by different spectroscopic techniques. A complete thermodynamic profile (ΔH^o, ΔS^o and ΔG^o) was evaluated initially from the change in absorption and fluorescence spectra upon addition of BSA to the complexes. Appreciable binding constant values in the range ~ 0.94–4.51 × 10⁴ M⁻¹ indicate efficient binding tendency of the complexes to BSA with the sequence 1 ≅ 2 > 3 ≅ 4. Circular dichroism (CD), isothermal calorimetric titration experiments, molecular docking and molecular dynamics have been performed to gain deep insight into the binding regions of complex 1 to BSA. Experimental evidences suggest an interaction of zinc complexes at the surface of BSA protein and this particular binding has been exploited to determine unknown concentration of BSA protein. For this purpose complex 1 was explored as a BSA protein quantification tool.     

The effect of glucose on bovine serum albumin denatured aggregation kinetics at high concentration

The effect of glucose (0–15 mass%) on the kinetics of bovine serum albumin (BSA) denatured aggregation at high concentration in aqueous solution has been studied by differential scanning calorimetry. The observed denatured aggregation process was irreversible and could be characterized by a denaturation temperature (T _m), apparent activation energy (E _a), the approximate order of reaction, and pre-exponential factor (A). As the glucose concentration increased from 0 to 15 mass%, T _m increased, E _a also increased from 514.59409±6.61489 to 548.48611±7.81302 kJ mol⁻¹, and A/s⁻¹ increased from 1.24239E79 to 5.59975E83. The stabilization increased with an increasing concentration of glucose, which was attributed to its ability to alter protein denatured aggregation kinetics. The kinetic analysis was carried out using a composite procedure involving the iso-conversional method and the master plots method. The iso-conversional method indicated that denatured aggregation of BSA in the presence and absence of glucose should conform to single reaction model. The master plots method suggested that the simple order reaction model best describe the process. This study shows the combination of iso-conversional method and the master plots method can be used to quantitatively model the denatured aggregation mechanism of the BSA in the presence and absence of glucose.     

Bovine serum albumin adsorption onto 316L stainless steel and alumina: a comparative study using depletion,protein radiolabeling,quartz crystal microbalance and atomic force microscopy

The importance of protein adsorption on biomaterials is widely recognized, but the dependence of the adsorption results on the chosen technique has not been much addressed. The objective of this work is to compare adsorption data obtained using several techniques under experimental conditions as closely as possible. Two case studies were investigated: adsorption of bovine serum albumin (BSA) onto 316L stainless steel (SS) and onto alumina. Both materials were used as powders and plates, whose characterization was done through zeta potential (ZP) measurements. The experimental techniques were depletion, protein radiolabeling, quartz crystal microbalance with dissipation (QCM‐D) and atomic force microscopy (AFM). The adsorption isotherms obtained with depletion and QCM‐D techniques, although quantitatively different, present some similarities in shape. Both techniques suggest the existence of a compact end‐on monolayer of protein on the SS surface, while on the alumina surface a less dense side‐on monolayer is formed at lower BSA concentration, followed by a second layer at higher concentration. AFM topographical characterization of the protein films adsorbed on both materials confirms those findings. Further use of AFM in determining the thickness of the film adsorbed on SS yielded values in good agreement with the QDM‐D results. Different surface charges measured on powders and plates do not seem to affect adsorption. Protein radiolabeling seems to be the least reliable technique because it yields, for both materials, adsorption values higher than those from the other techniques. In the case of SS, the difference amounts to one order of magnitude. Copyright © 2008 John Wiley & Sons, Ltd.     

Development of an enzyme-linked immunosorbent assay (ELISA)-like fluorescence assay to investigate the interactions of glycosaminoglycans to cells

Sulfated glycosaminoglycans were labeled with biotin to study their interaction with cells in culture. Thus, heparin, heparan sulfate, chondroitin 4-sulfate, chondroitin 6-sulfate and dermatan sulfate were labeled using biotin-hydrazide, under different conditions. The structural characteristics of the biotinylated products were determined by chemical (molar ratios of hexosamine, uronic acid, sulfate and biotin) and enzymatic methods (susceptibility to degradation by chondroitinases and heparitinases). The binding of biotinylated glycosaminoglycans was investigated both in endothelial and smooth muscle cells in culture, using a novel time resolved fluorometric method based on interaction of europium-labeled streptavidin with the biotin covalently linked to the compounds. The interactions of glycosaminoglycans were saturable and number of binding sites could be obtained for each individual compound. The apparent dissociation constant varied among the different glycosaminoglycans and between the two cell lines. The interactions of the biotinylated glycosaminoglycans with the cells were also evaluated using confocal microscopy. We propose a convenient and reliable method for the preparation of biotinylated glycosaminoglycans, as well as a sensitive non-competitive fluorescence-based assay for studies of the interactions and binding of these compounds to cells in culture.     

Affinity of a new copper(II) complex to DNA/BSA and antioxidant/radical scavenging activities: crystal structure of [Cu(4,7-diphenyl-1,10-phenanthroline)(leucine)(NO3)(H2O)]

A new copper(II) complex, [Cu(Bphen)(Leu)(NO₃)(H₂O)] (Bphen = 4,7-diphenyl-1,10-phenanthroline, leu = L-leucine), has been synthesized and characterized by IR spectroscopy, CHN analysis, and single-crystal X-ray diffraction techniques. The CT-DNA binding properties of the complex have been investigated by both absorption and emission spectroscopy. The binding parameters for the fluorescence Scatchard plot were also determined. Further, the interaction of the complex with bovine serum albumin (BSA) has been investigated using absorption and emission spectroscopy. The thermodynamic parameters, free energy change (ΔG), enthalpy change (ΔH), and entropy change (ΔS), were calculated by the van’t Hoff equation and discussed. The distance between BSA and the complex has been obtained according to fluorescence resonance energy transfer. Conformational changes of BSA have been observed from synchronous fluorescence. Antioxidant and radical scavenging activities of the complex were determined by various in vitro assays such as 1,1-diphenyl-2-picryl-hydrazyl free radicals (DPPH˙), 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) radicals (ABTS˙⁺), and reducing ability determination by H₂O₂ scavenging methods.     

Synthesis,structural characterization,in vitro cytotoxicities,and BSA interaction of di-organotin(IV) complexes derived from salicylaldehyde nicotinoyl hydrazone

Two organotin(IV) compounds were synthesized from salicylaldehyde nicotinoyl hydrazone and the corresponding dialkyltin(IV) precursor. Their structures were determined by IR, elemental analysis, NMR, and single crystal X-ray diffraction analysis. Compound 1 exhibits a distorted trigonal bipyramidal geometry around tin, which is coordinated to the Schiff-base ligand in an enolic tridentate fashion. For 2, structural analysis reveals that it is a centro-symmetric trimer, in which the central Sn adopts a six-coordinate octahedral geometry and the other two Sn ions adopt five-coordinate trigonal bipyramidal geometry. In vitro cytotoxicities of the compounds against three human cisplatin-resistant tumor cell lines (A549, HeLa, and MCF-7) were assessed by MTT assay. Further, the interaction of 1 and 2 with bovine serum albumin (BSA) has been explored by the titration method with fluorescence quenching spectra and synchronous ?uorescence spectra. Studies reveal that di-n-butyltin(IV) complex 1 with significant antiproliferative effects in the cells shows stronger BSA interaction.     

Spectrophotometric determination of total protein in serum using a novel near-infrared cyanine dye, 5,5′-dicarboxy-1,1′-disulfobutyl-3,3,3′,3′-tetramethylindotricarbocyanine

The application of near-infrared (NIR) dyes (λ _em > 750 nm) to the analysis of biological samples shows much promise, because the long emission wavelengths of such dyes allow interferences from biomolecule matrices to be minimized. In this paper, a novel NIR dye, 5,5′-dicarboxy-1,1′-disulfobutyl-3,3,3′,3′-tetramethylindotricarbocyanine (DCDSTCY) has been developed for the spectrophotometric determination of total protein in serum. Under acidic conditions, the binding of DCDSTCY to proteins caused a new peak at 878 nm, the height of which was proportional to the concentration of protein. The linear range of the method was found to be 0.04–0.5 μg mL⁻¹ for bovine serum albumin (BSA) and human serum albumin (HSA), and detection limits of 5 ng mL⁻¹ were obtained for these substances. The maximum binding number of BSA with DCDSTCY was measured to be 133. The method proposed here has been applied to the quantitation of total protein in serum, and recoveries of 96.6–104% were achieved. Figure Near-infrared probe for protein determination     

Hapten heterology for a specific and sensitive indirect enzyme-linked immunosorbent assay for organophosphorus insecticide fenthion

Five haptens with different spacer-arm attachment sites on the structure of the organophosphorus insecticide fenthion were designed and synthesized. All of the haptens were conjugated with ovalbumin (OVA) for the coating antigen, and three haptens containing all or most of the structure of fenthion were conjugated with bovine serum albumin (BSA) for the immunogen. Six polyclonal antisera were raised against the three BSA conjugates, and 30 antibody/coating conjugate combinations were selected for studies of assay sensitivity and specificity for fenthion. The study revealed the best combination with high sensitivity (I₅₀ of 0.08 ng mL⁻¹) and high assay specificity, which indicated that when structural difference between the analyte and an immunizing hapten is less than that between a coating hapten and the immunizing hapten, a high sensitive enzyme-linked immunosorbent assay (ELISA) in the heterologous system may stand a good chance to be developed. The immunity results showed that heterology in the hapten spacer-arm attachment site of the immunogen could achieve a remarkable improvement in the quantity, sensitivity, and/or specificity of antibody, and that the moiety of an analyte, which is the same as the moiety near/on the immunizing spacer-arm hapten attachment site, contributes greatly to the interaction of antibody and hapten.     

Squaraine Dyes: Interaction with Bovine Serum Albumin to Investigate Supramolecular Adducts with Aggregation‐Induced Emission (AIE) Properties

Bovine serum albumin (BSA)–squaraine supramolecular adducts with aggregation‐induced emission (AIE) properties were prepared and characterized by spectroscopic methods. While squaraine dyes showed a very low fluorescence quantum yield in water, a great enhancement in the fluorescence of the aggregated BSA adducts was achieved due to the abnormal aggregation‐induced emission properties of squaraines. The adducts formation was studied from a kinetic point of view showing unexpected structure–properties relationships.     

固态纳米孔对蛋白质易位的实验研究

蛋白质因其多样性和功能性,是生物体内一类非常重要的分子.通常蛋白质的表征需要借助荧光或者酶的标记.而纳米孔技术,得益于免标记、单分子检测等优势,为蛋白质的表征提供了新方向.我们使用固态纳米孔完成了单个蛋白质分子及蛋白质-蛋白质结合物的检测.可以发现,外部电压和电解质溶液的酸碱度会直接影响蛋白质分子表面带电量,从而加快或延迟其在孔内的易位时间.抗原、抗体本质上都是蛋白质,两者之间具有高度特异性.通过比较抗体溶液在添加特异性抗原前后的易位事件,实现了单个蛋白质分子和蛋白质-蛋白质结合物的区分.未来,纳米孔技术有望应用于多蛋白质分子的辨识、蛋白质分子相互作用机制等方面的研究.