Characterization Of Macromolecules Research Articles

Persistence Length and Encounter Frequency Determination from Fluorescence Studies of Pyrene-Labeled Poly(oligo(ethylene glycol) methyl ether methacrylate)s

A series of nine pyrene-labeled poly(oligo(ethylene glycol) methyl ether methacrylate)s (Py-PEGnMAs) with n equal to 0–5, 9, 16, and 19 were prepared by random radical copolymerization of 1-pyrenebutyl methacrylate and nine different EGnMA monomers. The process of pyrene excimer formation (PEF) in the Py-PEGnMA samples was characterized in acetone, THF, toluene, N,N-dimethylformamide (DMF), dioxane, and dimethyl sulfoxide (DMSO). The fluorescence decays of all Py-PEGnMA samples were acquired in the six solvents and analyzed with the fluorescence blob model (FBM) to yield the number Nblob of structural units (SU) in the subvolume of the polymer coil probed by an excited pyrene and referred to as a blob, and the rate constant kblob describing the encounters between two SU bearing an excited and a ground-state pyrenyl label located inside a same blob. Nblob and kblob remained constant with pyrene content for a same series of Py-PEGnMA samples. After averaging Nblob and kblob over all pyrene contents for a same Py-PEGnMA series, ⟨Nblob⟩ and the product ⟨kblob × Nblob⟩ were found to decrease with increasing side chain length reflecting a progressive stiffening and decrease in the internal dynamics of the polymethacrylate backbone, respectively. ⟨Nblob⟩ and ⟨kblob × Nblob⟩ could be parametrized as a function of the molecular weight of an SU and the solvent viscosity. The parametrized form of ⟨Nblob⟩ was applied to determine the persistence length (lp) of the PEGnMA samples using the Kratky–Porod equation. lp was found to increase linearly with the square of the side chain length of the PEGnMA samples, as expected theoretically. The parametrized form of ⟨kblob × Nblob⟩ was used as a calibration curve against which the internal dynamics of several polypeptides and poly(methyl acrylate) could be compared in DMSO. This study illustrates the ability of PEF measurements to determine the persistence length and quantify the internal dynamics of polymers in solution, two important parameters in the characterization of macromolecules.

High-Resolution Native Mass Spectrometry.

Native mass spectrometry (MS) involves the analysis and characterization of macromolecules, predominantly intact proteins and protein complexes, whereby as much as possible the native structural features of the analytes are retained. As such, native MS enables the study of secondary, tertiary, and even quaternary structure of proteins and other biomolecules. Native MS represents a relatively recent addition to the analytical toolbox of mass spectrometry and has over the past decade experienced immense growth, especially in enhancing sensitivity and resolving power but also in ease of use. With the advent of dedicated mass analyzers, sample preparation and separation approaches, targeted fragmentation techniques, and software solutions, the number of practitioners and novel applications has risen in both academia and industry. This review focuses on recent developments, particularly in high-resolution native MS, describing applications in the structural analysis of protein assemblies, proteoform profiling of—among others—biopharmaceuticals and plasma proteins, and quantitative and qualitative analysis of protein–ligand interactions, with the latter covering lipid, drug, and carbohydrate molecules, to name a few.

A review of modern approaches to the hydrodynamic characterisation of polydisperse macromolecular systems in biotechnology

This short review considers the range of modern techniques for the hydrodynamic characterisation of macromolecules – particularly large glycosylated systems used in the food, biopharma and healthcare industries. The range or polydispersity of molecular weights and conformations presents special challenges compared to proteins. The review is aimed, without going into any great theoretical or methodological depth, to help the Industrial Biotechnologist choose the appropriate methodology or combination of methodologies for providing the detail he/she needs for particular applications.

Synthesis and characterisation of macromolecules containing multiple tetrazole functionalities

The synthesis of tetra-tetrazole macromolecules, containing various aromatic cores including benzene, pyridine and pyrazine directly attached to the tetrazole moieties, is described. This variation allowed for the generation of ligands with greater potential for metal ion complexation. Metal ion complexation reactions of the tetra-tetrazole macromolecules with the chelating pyridyl-tetrazole arms result in the formation of metal complexes where the metal ion was bound at the pendant arms rather than at the central core.

Probing mechanical properties of fully hydrated gels and biological tissues

A longstanding challenge in accurate mechanical characterization of engineered and biological tissues is maintenance of both stable sample hydration and high instrument signal resolution. Here, we describe the modification of an instrumented indenter to accommodate nanomechanical characterization of biological and synthetic tissues in liquid media, and demonstrate accurate acquisition of force–displacement data that can be used to extract viscoelastoplastic properties of hydrated gels and tissues. We demonstrate the validity of this approach via elastoplastic analysis of relatively stiff, water-insensitive materials of elastic moduli E>1000 kPa (borosilicate glass and polypropylene), and then consider the viscoelastic response and representative mechanical properties of compliant, synthetic polymer hydrogels (polyacrylamide-based hydrogels of varying mol%-bis crosslinker) and biological tissues (porcine skin and liver) of E<500 kPa. Indentation responses obtained via loading/unloading hystereses and contact creep loading were highly repeatable, and the inferred E were in good agreement with available macroscopic data for all samples. As expected, increased chemical crosslinking of polyacrylamide increased stiffness ( E⩽40 kPa) and decreased creep compliance. E of porcine liver (760 kPa) and skin (222 kPa) were also within the range of macroscopic measurements reported for a limited subset of species and disease states. These data show that instrumented indentation of fully immersed samples can be reliably applied for materials spanning several orders of magnitude in stiffness ( E=kPa–GPa). These capabilities are particularly important to materials design and characterization of macromolecules, cells, explanted tissues, and synthetic extracellular matrices as a function of spatial position, degree of hydration, or hydrolytic/enzymatic/corrosion reaction times.

Macromolecular Size-and-Shape Distributions by Sedimentation Velocity Analytical Ultracentrifugation

Sedimentation velocity analytical ultracentrifugation is an important tool in the characterization of macromolecules and nanoparticles in solution. The sedimentation coefficient distribution c( s) of Lamm equation solutions is based on the approximation of a single, weight-average frictional coefficient of all particles, determined from the experimental data, which scales the diffusion coefficient to the sedimentation coefficient consistent with the traditional s ∼ M 2/3 power law. It provides a high hydrodynamic resolution, where diffusional broadening of the sedimentation boundaries is deconvoluted from the sedimentation coefficient distribution. The approximation of a single weight-average frictional ratio is favored by several experimental factors, and usually gives good results for chemically not too dissimilar macromolecules, such as mixtures of folded proteins. In this communication, we examine an extension to a two-dimensional distribution of sedimentation coefficient and frictional ratio, c( s, f r), which is representative of a more general set of size-and-shape distributions, including mass-Stokes radius distributions, c( M, R S), and sedimentation coefficient-molar mass distributions c( s, M). We show that this can be used to determine average molar masses of macromolecules and characterize macromolecular distributions, without the approximation of any scaling relationship between hydrodynamic and thermodynamic parameters.

Micro-thermal field-flow fractionation in the characterization of macromolecules and particles: Effect of the steric exclusion mechanism

Abstract The influence of the steric exclusion mechanism on the dependence of the retention on the thickness of the micro-TFFF channel was studied theoretically. A good agreement of theory with experimental data was found. In order to preserve the use of micro-TFFF within an extended range of molar masses and particle sizes, the thickness of the channel cannot be reduced without taking into account a possible deterioration of the separation due to the contribution of steric exclusion to the separation mechanism.

Characterization of Macromolecules by Electrospray lonization Mass Spectrometry

Application of electrospray ionization (ESI) mass spectrometry (MS) to the analysis of oligomers and polymers is reviewed through several representative examples. The benefits and limitations of the technique are discussed considering the molecular weight and complexity (polydispersity) of the analyte. Recent results demonstrating the advantage of using hyphenated techniques such as size exclusion chromatography (SEC) coupled with ESI-MS are highlighted.

Osmolarity effects on red blood cell elution in sedimentation field-flow fractionation.

Field-flow fractionation (FFF) is an analytical technique particularly suitable for the separation, isolation, and characterization of macromolecules and micrometer- or submicrometer-sized particles. This chromatographic-like methodology can modulate the retention of micron-sized species according to an elution mode described to date as "steric hyperlayer". In such a model, differences in sample species size, density, or other physical parameters make particle selective elution possible depending on the configuration and the operating conditions of the FFF system. Elution characteristics of micron-sized particles of biological origin, such as cells, can be modified using media and carrier phases of different osmolarities. In these media, a cells average size, density, and shape are modified. Therefore, systematic studies of a single reference cell population, red blood cells (RBCs), are performed with 2 sedimentation FFF systems using either gravity (GrFFF) or a centrifugational field (SdFFF). However, in all cases, normal erythrocyte in isotonic suspension elutes as a single peak when fractionated in these systems. With carrier phases of different osmolarities, FFF elution characteristics of RBCs are modified. Retention modifications are qualitatively consistent with the "steric-hyperlayer" model. Such systematic studies confirm the key role of size, density, and shape in the elution mode of RBCs in sedimentation FFF for living, micronsized biological species. Using polymers as an analogy, the RBC population is described as highly "polydisperse". However, this definition must be reconsidered depending on the parameters under concern, leading to a matricial concept: multipolydispersity. It is observed that multipolydispersity modifications of a given RBC population are qualitatively correlated to the eluted sample band width.

13th Bratislava International Conference on Polymers: Separation and Characterization of Macromolecules, 4–9 July 1999, Bratislava, Slovakia

13th Bratislava International Conference on Polymers: Separation and Characterization of Macromolecules, 4–9 July 1999, Bratislava, Slovakia

Mechanisms of the formation and disposition of reactive metabolites that can cause acute liver injury.

Acetaminophen and pulegone are just two examples for many agents that can form reactive metabolites that can cause acute liver injury. Two other classic organic compounds that have been extensively studied are carbon tetrachloride (for a recent review see Ref. 159, and for other discussions see Refs. 8 and 9) and bromobenzene (for review see Ref. 160). Different kinds of protein adducts of reactive metabolites of bromobenzene have been partially characterized [161], and specific antibodies to these adducts are now being used to isolate and identify the proteins that are modified (162). In contrast, carbon tetrachloride and other agents, such as the herbicide diquat, may form radicals that bind to and/or oxidize lipids and proteins in causing liver injury (163, 164). Therefore, the recent development [165] of antibodies to detect oxidative damage to proteins will be important in the identification and characterization of macromolecules that do not form adducts with reactive metabolites but are damaged oxidatively. Thus, some major challenges in the coming years are to identify hepatocellular macromolecules that are modified by reactive metabolites, and then approach the more difficult task of integrating this information into a time course and sequence of events leading to lethal hepatocellular injury.

Influence of Mannaproteins from Yeast on the Aroma Intensity of a Model Wine

The influence of mannaproteins released from yeast cell walls during alcoholic fermentation on the volatility of aroma substances was investigated in a model wine. After the characterization of macromolecules (substrates), two techniques have been used to study the interactions with aroma compounds: headspace analysis and an equilibrium dialysis method. The assumed effects of these macromolecules from yeasts on the fixation of volatile compounds were demonstrated. The physico-chemical interactions between aroma substances and mannaproteins depended on the nature of volatile compounds. Protein concentration in substrates was an important factor in their binding capacity. The retention of aroma compounds (β-ionone, ethyl hexanoate) was attributed to mannaproteins having a high proportion of proteins.

Immunological and biochemical analysis of test matrix proteins in living and fossil foraminifers

The characterization of macromolecules from shell matrix proteins in living organisms is the fundamental first step in establishing molecular phylogenies. This is particularly important if ancient macromolecules will be used for the phylogenies. Approximately 500 individuals of Orbulina universa were picked from plankton tows taken in the Gulf of Mexico during spring 1990. Proteins were extracted from the tests of the individuals, and the molecular weights of the proteins were determined and compared to 2,000–4,000-year old samples. Gel electrophoresis of the soluble matrix (SM) from living O. universa showed at least ten protein bands, while electrophoresis of core-top O. universa showed two protein bands. Dot-immunobinding assays of proteins from living and fossil samples of O. universa, incubated against monoclonal antibodies raised against core-top O. universa SM, demonstrated a reactivity with the two proteins in common. Less reactivity was obtained with living samples of the benthic foraminifers Androsina lucasi and Archaias angulatus and core-top samples of the planktonic foraminifer Neogloboquadrina dutertrei. A partial protein sequence from one shell matrix protein approximately 65kD in size, from core-top Orbulina universa, showed a domain of polyaspartic acid at the NH2 terminus. This is consistent with data obtained for other matrix proteins found in invertebrates and vertebrates. Sequence data provide insight into the role that the protein may play in the biomineralization of the test and will aid in modelling degradation. □Foraminifera, test proteins, monoclonal antibodies, Orbulina, Androsina, Archaias, Neogloboquadrina, biomineralization, Dot-immunobinding assay, protein sequence.

Characterisation of Macromolecules Used as Pharmaceutical Excipients: Excipients and the Regulatory Authorities

AbstractThe sources of information on regulatory requirements for licensing (marketing authorisation) of pharmaceuticals in the European Community, including aspects applying to macromolecular excipients, are identified. The pharmaceutical, preclinical and clinical implications are discussed in outline.

Characterization of Macromolecules Used as Pharmaceutical Excipients予告

Characterization of Macromolecules Used as Pharmaceutical Excipients予告

Structure of agarose gels: Mean pore radius

To use an agarose gel's sieving for the biophysical characterization of macromolecules and supramolecular complexes, the effective gel's pore radius (PE) is determined as a function of the agarose percentage (A). In previous studies, performed by use of agarose gel electrophoresis, the sieving of spheres of known radius (R) was used, together with two different (unproven) theories of sieving, to obtain PE. The PE values obtained were self-consistent and independent of R. The PE vs. A relationship did not agree with that predicted by a model that represents the gel as straight, randomly-oriented fibers (random fiber model). To test the accuracy of both the empirical PE vs. A relationship and the previously assumed model of gel structure, in the present study thin sections of agarose gels have been examined by use of electron microscopy.Gels of the agarose previously used to quantify sieving (SeaKem LE, Marine Colloids) were cast in the buffer previously used: 0.025 M sodium phosphate, pH 7.4, 0.001M MgCl2. Pieces of gel were fixed with osmium tetroxide, dehydrated and embedded in Epon. Dark gold (120 nm) sections were examined with a JEM-100CX transmission electron microscope. Electron micrographs were captured with a DataTranslation QuickCapture video digitizer and were processed on a Macintosh II computer using the program, Image.4

Has immunoblotting replaced electroimmunoprecipitation? : Examples from the analysis of autoantigens and transglutaminase-induced polymers of the human erythrocyte membrane

The virtues and drawbacks of immunoblotting and electroimmunoprecipitation in the characterization of macromolecules in crude mixtures are presented. Interactions between autoantibodies and human erythrocyte membrane proteins were studied by means of crossed-affinoimmunoelectrophoresis with autologous immunoglobulins incorporated into the first dimension gel and by immunoblotting of sodium dodecyl sulphate-polyacrylamide gel electrophoresis separated erythrocyte membrane proteins with autologous immunoglobulins as primary antibodies. Substrates for transglutaminase in calcium-activated human erythrocyte membranes were examined by immunoelectrophoretic and immunoblotting methods. The experiments concerning autoantibodies complemented each other and showed that epitopes on Band 3 protein, spectrin and ankyrin are recognized by circulating immunoglobulin autoantibodies in normal individuals. The polymer experiments showed the presence of spectrin, ankyrin, Band 3, Band 4.1, glucose transporter, actin and haemoglobin epitopes in the polymer ( M r 3 · 10 6-5 · 10 6). It is concluded that the two techniques complement each other. The most evident advantage of immunoblotting is its sensitivity and applicability while electroimmunoprecipitation in some instances allows an easier identification of distinct protein species and still has a rôle for quantification and certain monitoring purposes.

1] Characterization of macromolecules by sedimentation equilibrium in the air-turbine ultracentrifuge

Publisher Summary The development of sedimentation equilibrium in the air-driven preparative ultracentrifuge has provided an independent, nonempirical method to characterize partially purified macromolecules within heterogeneous preparations, based on the equilibrium distribution of their biochemical or other specific activities as a function of radial distance in a centrifugal field. Since this method is based on a theoretical thermodynamic description of the system in sedimentation-diffusion equilibrium and is independent of molecular weight standards and zonal transport, the difficulties outlined in this chapter are effectively avoided. In addition, the method has opened this area of investigation to the extensive existing literature concerning the use of sedimentation equilibrium to determine the molecular weights and interactions of proteins in solution, which had been previously restricted to highly purified protein preparations detected optically in the analytical ultracentrifuge. This chapter illustrates that as the time required to approach to within a specific deviation from true sedimentation equilibrium is proportional to the square of this radial distance, 17 short distances are necessary for achievement of experimental sedimentation equilibrium within a reasonable time. Furthermore, the nonsectorial geometry of the tube walls initially creates convective transport of solute 18 which contributes to achievement of experimental sedimentation equilibrium within 24 hr.

AUTOMATED CELL SORTING WITH FLOW SYSTEMS

The major successes of biochemistry and molular biology have derived from the isolation and characterization of macromolecules from complex solutions and structures. The analogous requirement in the field of cellular biology is for the preparation of pure populations of cells from organs and tissue culture. This ob jec­ tive is of primary importance in basic research of cellular differentiation and regula­ tion, as well as in applied areas such as clinical diagnosis and treatment. The same considerations apply to subcellular structures that extend from organelles or chromosomes to the ultimate structural limit of individual molecules, thereby bridging the gap with solution biochemistry. Cell separation is not a new problem nor has it been neglected in the past. Numerous methods exist for the bulk isolation of cells, including, for example, sedimentation, electrophoresis, or phase partitioning (134). In many instances, however, these methods lack sufficient selectivity. The techniques of automated cell analysis and sorting discussed here deal with the problem of heterogeneity by examining individual cells dispersed in suspension. Thus, the distributions of physical, structural, and functional properties of the population can be assessed and coupled in a logical manner with the sorting of desired cells for morphological examination or biologicallbioch emical studies. The flow systems designed for this purpose are capable of high speed (currently up to lOLI05 cells/sec) and a selectiv­ ity limited only by the nature of the reagents and physical signals used to differenti­ ate between the cellular subpopulations. In many instances, the sorting procedure can be carried out under conditions that insure preservation of viability and sterility. In fact, by eliminating debris and dead cells, overall viability can actually be increased. The duality of analysis and sorting cannot be overstressed. Although many flow instruments exist for analytical purposes alone and are of great utility. the

Modification by sucrose of the catalytic activity and physical properties of glutamic dehydrogenase

Sucrose (and a variety of glycols) caused inhibition of the glutamate specific catalytic activity of glutamate dehydrogenase and stimulation of the monocarboxylic acid activity. In addition, sucrose prevented the concentration dependent aggregation of the enzyme and produced a sharp increase in stability toward heat denaturation. These studies suggest the potential usefulness of sucrose in studying protein structure transitions, but demand caution in the use of such media for separation and characterization of macromolecules unless possible effects on structure are taken into account.