Structural properties of colloidal complexes between condensed tannins and polysaccharide hyaluronan

Interactions of plant tannins with polysaccharide hyaluronan are studied by means of light scattering and small-angle X-ray scattering (SAXS). In this paper, we show that (1) the tannin-polysaccharide complexes remain stable in colloidal suspension; (2) the masses and structures of colloidal tannin-polysaccharide objects depend on the tannin degree of polymerization; and (3) the densities of tannin-polysaccharide aggregates are about 7 times lower than the density of a single solvated polysaccharide molecule. Short tannins and polysaccharides are aggregated in loose oligomeric structures whose sizes are comparable to a single polysaccharide molecule. Tannins longer than 10 nm and polysaccharides are aggregated in larger microgel-like particles whose sizes exceed 200 nm.     

Titration of tannin via alkaline phosphatase activity

An enzyme assay for tannin is described. It is based on the following steps: (i) bovine serum albumin (BSA) is absorbed onto polystyrene microplates; (ii) tannin is bound to the BSA-coated plates; (iii) alkaline phosphatase is then interacted with the free tannin-binding sites. The method takes advantage of the multiple hydroxyl groups of tannin which can associate more than one ligand, e.g., proteins. A pH-dependent dynamic equilibrium sets up between bound and unbound tannin and is controlled only by its initial concentration.     

Protection by plasma proteins against condensed tannin-mediated platelet activation

Condensed tannins isolated from cotton bracts are potent platelet agonists. They promote the secretion of 5-hydroxytryptamine from washed bovine platelets with an EC50 of 35 micrograms/ml. However, when platelet-rich plasma was used in lieu of washed platelets, the potency of tannin was decreased over 50-fold. Reconstitution experiments demonstrated that some component in the plasma was responsible for the diminished potency of tannin. Fractionation of platelet-poor plasma suggested that albumin may be the inhibitory plasma component. Confirmation of this hypothesis was obtained from the finding that purified serum albumin at physiologic concentrations inhibited the tannin-mediated release of platelet 5-HT by more than 70%. These data suggest that any activation of platelets which may occur in vivo by the tannins present in inhaled cotton dust would require higher tannin concentrations than would have been predicted from our previous studies using washed platelets.     

Automated measurement of the static light scattering of macromolecular solutions over a broad range of concentrations

A method and apparatus for automated measurement of the concentration dependence of static light scattering of protein solutions over a broad range of concentrations is described. The gradient of protein concentrations is created by successive dilutions of an initially concentrated solution contained within the scattering measurement cell, which is maintained at constant total volume. The method is validated by measurement of the concentration dependence of light scattering of bovine serum albumin, ovalbumin, and ovomucoid at concentrations up to 130 g/L. The experimentally obtained concentration dependence of scattering obtained from all three proteins is quantitatively consistent with the assumption that no significant self-association occurs over the measured range of concentrations.     

The factors affecting the compatibility of serum albumin and pectinate in aqueous medium

Compatibility is observed in aqueous solutions of serum albumin and pectin (degree of esterification 57%) at pH levels above the isoelectric point of protein. Both the variation in the pH values from 5 to 8 and the increase of ionic strength from 0·1 to 1·0 do not result in phase separation. These facts enable us to conclude that the affinity in this system is of a nonelectrostatic nature. The interaction of serum albumin and pectinate fractions with different degrees of esterification was studied by light scattering. The negative sign of A₂₄ (the second virial coefficient component of the mixture associated with the interaction between different polymer molecules) means that for any degree of esterification there is affinity between pectin and serum albumin. Information concerning excess thermodynamic functions was obtained from the temperature dependence of light scattering. Mixing microcalorimetry was used for precise measurement of enthalpy. The experimental results indicate that thermodynamic compatibility of serum albumin and pectin is controlled by increase of mixing entropy, which mainly stems from dehydration of biopolymer macromolecules during contact formation.     

Protein analysis with tetra-substituted sulfonated cobalt phthalocyanine by the technique of Rayleigh light scattering

This is the first report of cobalt-tetrasulfonatophthalocyanine (CoTSPc) as a probe of Rayleigh light scattering (RLS) to determine proteins at nanogram levels. A highly sensitive method has been developed for the determination of proteins by the light scattering technique on a common spectrofluorimeter, based on the fact that the weak RLS of CoTSPc can be greatly enhanced in the presence of proteins. Under optimum conditions, the linear ranges of the calibration curves were 0.10-34.3 microg x mL(-1) for both human serum albumin and bovine serum albumin, with detection limits of 15.5 and 13.9 ng x mL(-1), respectively. Moreover, there is almost no interference of any amino acids and metal ions. The method has been applied to the direct determination of total proteins in human serum samples, and the results were satisfactory with clinical data provided.     

Size and structure of antigen-antibody complexes. Electron microscopy and light scattering studies

Size parameters of model antigen-antibody (Ag-Ab) complexes formed by the interaction of bovine serum albumin (BSA) and pairs of monoclonal anti-BSA antibodies (mAb) were evaluated by quasielastic light scattering, classical light scattering, and electron microscopy (EM). Mean values for the hydrodynamic radius, radius of gyration, and molecular weight were determined by light scattering. Detailed information regarding the molecular weight distribution and the presence of cycles or open chains was obtained with EM. Average molecular weights were calculated from the EM data, and the Porod-Kratky wormlike chain theory was used to model the conformational behavior of the Ag-mAb complexes. Ag-mAb complexes prepared from three different mAb pairs displayed significantly different properties as assessed by each of the techniques employed. Observations and size parameter calculations from EM photomicrographs were consistent with the results from light scattering. The differences observed between the mab pairs would not have been predicted by idealized thermodynamic models. These results suggest that the geometric constraints imposed by the individual epitope environment and/or the relative epitope location are important in determining the average size of complexes and the ratio of linear to cyclic complexes.     

Scattering phenomena effects on growth of 308-nm laser-irradiated bacteria in suspension

In this study we analyzed the effect of 308-nm laser exposure on recovery of irradiated Staphylococcus epidermidis held in liquid after irradiation and before plating. Coexistence of bacterial growth inhibition and stimulation phenomena was observed. Under certain conditions, bacterial recovery was about fivefold higher in irradiated samples than in the controls. The available evidence suggests that the growth inhibition was due to the bactericidal activity of the 308-nm wavelength light, whereas the growth stimulation effect was associated with broadband radiation generated by scattering phenomena in the bacterial suspensions. Spectroscopic investigations revealed that the nutrient broth plays a decisive role in the scattering of laser radiation within the suspension.     

Ecological tannin assays

Summary Estimations of condensed tannin content are generally based on calibration standard curves from Quebracho condensed tannins. We generated calibration standard curves from eight Sonoran Desert species for comparison with estimates of tannin concentrations derived from the Quebracho standard curve. Estimates of leaf tannin concentrations of each of the eight species using each species standard curve differed significantly with the estimates given by the Quebracho standard curve. Standard curves constructed from tannins from different individuals of three of the species varied significantly between, but not within, species. The efficiency of precipitation of protein bovine serum albumin (BSA) by each different tannin varied up to a factor of fifty for tannins of different species. Ordering species from highest to lowest based on tannin concentrations or binding efficiencies gave two different ranks. We argue that concentration or efficiency alone do not describe adequately tannin ecological activity. Instead, we suggest combining tannin concentrations and binding efficiencies to measure the protein precipitating potential of a leaf. Leaf protein precipitating potential is a more ecologically realistic parameter, we feel, for between-species comparisons than tannin content or binding efficiencies alone.Operated for the U.S. Department of Energy by the University of California under Contract No. DE-AC03-76-SF00012. This article was supported by the Director of the Office of Energy Research, Office of Health and Environmental Research     

Particle size and shape of thermally denatured bovine serum albumin by light scattering

Solutions of bovine serum albumin in various stages of thermal denaturation, ranging from very early to fairly advanced, were prepared in a light scattering cell. The molecular weights and the molecular sizes of the subsequent aggregate particles were determined by the light scattering method. In the early stages of thermal denaturation, in the presence of sodium chloride, the aggregate particles were fairly stable, growing essentially in rods, relatively uniform in size. In the absence of sodium chloride, the aggregate particles extended fairly widely and were not uniform in size even in the early stages.     

Investigation of Ternary Complex Formations of Polyacrylic Acid with Bovine Serum Albumin in the Presence of Metal Ions by Fluorescence and Dynamic Light Scattering Measurements

In this paper, the complex formation of bovine serum albumin (BSA) and polyacrylic acid (PAA) in the presence metal ions at pH = 7 has been examined by using fluorescence and dynamic light scattering measurements. It has been observed that the most stable complexes of polyacrylic acid and bovine serum albumin have occurred in the presence of copper(II) ions. The other ions have the ability to form weak complexes between polyions and bovine serum albumin. To prior characterizing the interaction between bovine serum albumin and polyacrylic acid, the dynamic light scattering technique have been applied to determine the intensity-size distributions of the solutions of bovine serum albumin, polyacrylic acid, and ternary mixtures containing various molar ratios of bovine serum albumin to polyacrylic acid (the molar ratios of bovine serum albumin to polyacrylic acid has been taken equal to 0.5, 1.0, 1.5, 2.0 and 2.5) prepared at different molar ratios of copper(II) ions/acrylic acid unit. When the molar ratio of copper(II) ions to acrylic acid in the ternary mixtures has been lower than and equals to 0.3, two peaks have been observed in the curves of the intensity-size distributions due to contents of free bovine serum albumin and ternary complexes of polyacrylic acid-copper(II)-bovine serum albumin whereas when the molar ratio of copper(II) ions to acrylic acid equals to 0.4, the hydrodynamic diameter has pointed out only one peak. This result indicates that soluble and stable ternary complexes has occurred when the molar ratio of copper(II) ions to acrylic acid has been taken equal to 0.4.     

The Influence of pH Alteration and Pharmacological Modulators of Adenylate Cyclase System on Human Serum Albumin Conformation

Abstract

The report describes the results of a study the effect of pH and binding of six physiologically active compounds (isoproterenol, yohimbine, theophylline, propranolol, Clonidine and carbachol) on the molecular structure of human serum albumin (HSA) using dynamic light scattering. It was found that the albumin globule had the most compact configuration (Stokes diameter 59–62Å) at physiological pH 7.4. The changes in pH both increased to 8.0 and decreased to 5.4, resulting in the growth of globule size to 72–81Å. At acidic shift of pH an additional peak arose in the correlation spectra. This peak was caused by the light scattering on the structures with the Stokes diameters of 29—;37 Å, which conformed to the sizes of the albumin subdomains. The additional peak was not displayed at basic shift of pH.

The interaction with propranolol, Clonidine and carbachol, which hinder adenylate cyclase (AdC) signaling system of a cell, initiated structural rearrangements similar to acidic transitions. Isoproterenol, yohimbine and theophylline, which activate AdC, caused the conformational changes of HSA similar to basic transitions.     

Influence of the NaCl or CaCl2 concentration on the structure of heat-set bovine serum albumin gels at pH 7

The structure of heat-set systems of the globular protein bovine serum albumin (BSA) was investigated at pH 7 in different salt conditions (NaCl or CaCl(2)) using light scattering. Cross-correlation dynamic light scattering was used to correct for multiple scattering from turbid samples. After heat treatment, aggregates are formed whose size increases as the protein concentration increases. Beyond a critical concentration that decreases with increasing salt concentration, gels are formed. The heterogeneity and the reduced turbidity of the gels were found to increase with increasing salt concentration and to decrease with increasing protein concentration. The structure of the gels is determined by the strength of the repulsive electrostatic interactions between the aggregated proteins. The results obtained in NaCl are similar to those reported in previous studies for other globular proteins. CaCl(2) was found to be much more efficient in reducing electrostatic interactions than NaCl at the same ionic strength.     

单宁细胞形态与部分柿属种及品种相关性研究

采用软柿果肉直接压涂法,在光学显微镜下对204个柿属种及品种果实中的单宁细胞形态特征进行观察分析.结果显示:(1)在6个种柿属植物果实中,均含有单宁细胞,其外形大多属于短形和近圆形,但在数量、大小和颜色上存在差异,其中,油柿(Diospyros oleifera Cheng.)、君迁子(D.lotus Linn.)、柿(D.kaki Thunb.)、浙江柿(D.glaucifolia Metc.)的单宁细胞通常无色,而黑柿(D.nitida Mcrr.)为黄绿色,乌材(D.eriantha Champ.)为紫红色,乌柿(D.eathayensis Stheward.)为淡紫色;单宁细胞从大到小依次为油柿>君迁子>浙江柿>乌材>黑柿>乌柿.(2)单宁细胞在不同品种类型间差异明显,其中涩柿单宁细胞多为无色,单宁细胞比较宽大;甜柿品种均会出现褐变的单宁细胞,单宁细胞较小或瘦长;完全甜柿品种大多存在着凝固型褐变单宁细胞,仅少数凝聚呈球形,且单宁细胞分散存在于果肉中,果肉中的褐斑较细小;不完全甜柿在种子周围的褐斑处,可以看到大量的表面凹形且褐变的收缩型单宁细胞,且常以单宁细胞束的形态存在于果肉中,使果肉中的褐斑大而密;原产我国的完全甜柿中不存在凝聚型的单宁细胞,只有凝固型的单宁细胞.(3)聚类分析结果表明,单宁细胞的特征可以作为不同类型柿属种的分类依据.     

Treatment of mobile phase particulate matter in low-angle quasi-elastic light scattering

The method of quasi-elastic laser light scattering (QLS), particularly at low forward scattering angles, has been complicated by the transient presence of Mie or large Rayleigh scattering particles which contaminate the scattering volume. These large contaminating particles have substantial effects on photon correlation spectroscopy because the presence of these larger scatterers tends to decrease the value of the apparent diffusion coefficient of the particle of interest. A method is presented which yields more accurate diffusion constants by autocorrelation of selected photon count periods representative of minimal Mie or large Rayleigh particle contamination. This method was applied to the determination of the apparent diffusion constant for four proteins—ovalbumin, chymotrypsinogen-A, bovine serum albumin, and ribonuclease-A.     

Characterization of the soluble nanoparticles formed through Coulombic interaction of bovine serum albumin with anionic graft copolymers at low pH

A static light scattering (SLS) study of bovine serum albumin (BSA) mixtures with two anionic graft copolymers of poly(sodium acrylate-co-sodium 2-acrylamido-2-methyl-1-propanesulphonate)-graft-poly(N,N-dimethylacrylamide), with a high composition in poly(N,N-dimethylacrylamide) (PDMAM) side chains, revealed the formation of oppositely charged complexes, at pH lower than 4.9, the isoelectric point of BSA. The core-corona nanoparticles formed at pH = 3.00 were characterized. Their molecular weight and radius of gyration were determined by SLS, while their hydrodynamic radius was determined by dynamic light scattering. Small angle neutron scattering measurements were used to determine the radius of the insoluble complexes, comprising the core of the particles. The values obtained indicated that their size and aggregation number of the nanoparticles were smaller when the content of the graft copolymers in neutral PDMAM side chains was higher. Such particles should be interesting drug delivery candidates, if the gastrointestinal tract was to be used.     

Conformational studies of BSA using laser light scattering

The usefulness of laser light scattering as a technique for determining protein conformation has been investigated by studying the self-association and drug binding of bovine serum albumin (BSA). The diffusion coefficients of BSA monomers and dimers have been measured and the ratio of these two quantities indicates that in the dimer, the subunit separation is 2.2 times the monomeric hydrodynamic radius. The binding of salicylate to BSA causes an increase in its diffusion coefficient corresponding to a reduction in the frictional drag of the solvent on the protein molecules. It has been found that data obtained using laser light scattering may be interpreted confidently only when proper care has been taken in sample preparation and the scattered intensity autocorrelation function has been appropriately analyzed.     

Optical characteristics of flowing blood

The transmitted light strength (TS) through a thin blood layer changes with variation in blood flow, such as positive streaming transparency for low hematocrits and negative streaming transparency for high hematocrits. These phenomena are examined theoretically and experimentally. Maxwell’s equations are solved assuming that erythrocytes are oblate spheroids to investigate these phenomena due to flowing blood. The theoretical results reveal that the scattering and absorption cross sections for flowing blood are larger than those for stagnant blood. Experimental results indicate that the TS for both oxygenated and deoxygenated flowing blood, with a hematocrit of up to approximately 20%, was stronger than that for stagnant blood. The TS decreased for flowing blood with a hematocrit of approximately 20% or greater. Applying the theoretical scattering and absorption cross sections to the absorption and multiple scattering theory of Victor Twersky, the changes in the TS due to flowing blood are obtained theoretically. From the theoretical and experimental results, the positive streaming transparency phenomenon of flowing blood with a low hematocrit and the negative streaming transparency phenomenon with a high hematocrit are found to result from increased scattering and absorption cross sections because of the orientation of flowing erythrocytes.     

Lab-on-a-chip immunoassay for multiple antibodies using microsphere light scattering and quantum dot emission

Double detection of microsphere light scattering and quantum dot emission was demonstrated for lab-on-a-chip immunoassay without using stationary support. We conjugated quantum dots (QDs) onto microspheres to enable multiplex assays as well as to enhance the limit of detection (LOD). We named this configuration "nano-on-micro" or "NOM". Upon radiation with UV light (380nm), a stronger light scattering signal is observed with NOMs than QDs or microspheres alone. Additionally, NOMs are easier to handle than QDs. Since QDs also provide fluorescent emission, we are able to utilize an increase in light scattering for detecting antigen-antibody reaction and a decrease in QD emission to identify which antibody (or antigen) is present. Two types of NOM combinations were used. One batch of microspheres was coated with QDs emitting at 655 nm and mouse IgG (mIgG); the other with QDs emitting at 605 nm and bovine serum albumin (BSA). A mixture of these two NOMs was used to identify either anti-mIgG or anti-BSA. NOM particles and target solutions were mixed in a microfluidic device (using highly carboxylated microspheres as previously demonstrated by our group) and on-chip detection was performed using proximity optical fibers. Forward light scattering at 380 nm was collected. With the positive target, the scattering signal was increased. The LOD was as low as 50 ng ml(-1) (330 pM) with p<0.05. Fluorescent emission (655 or 605 nm) was simultaneously collected. With the positive target, the emission signal was attenuated. Therefore, we were able to detect two different antibodies simultaneously with two different detection protocols. We believe this NOM bioassay has the ability to screen for and detect multiple antibodies with minimal sample processing and handling (one-step lab-on-a-chip immunoassay).     

A new technique for mapping epitope specificities of monoclonal antibodies using quasi-elastic light scattering spectroscopy

Quasi-elastic light scattering (QLS) was used to determine relative epitope specificities of a group of anti-bovine serum albumin (BSA) monoclonal antibodies (MAb). QLS is a non-invasive technique which can determine the mean size and size distributions of macromolecular scatterers by analysis of the fluctuations in the intensity of laser light scattering. When two MAbs are mixed together with antigen, QLS detects the complex formation which results from the Ag-Ab reaction, and can easily distinguish between the large complexes formed by interaction of non-competitive MAbs and the smaller complexes formed by competitive MAbs. In this report, the competitive or non-competitive behavior of six anti-BSA MAbs were assessed by radioimmunoassay (RIA) and QLS analysis. The results obtained by QLS analysis confirmed the RIA findings indicating that the six MAbs examined can be categorized into three distinct, non-interacting groups. QLS analysis represents a simple, and extremely rapid technique for epitope mapping studies.