Biomedical subjects

H Cölfen

Publications and source records attributed to H Cölfen.

18 recordsLinked to original sources

PubMed2004

Time-resolved SAXS study of the effect of a double hydrophilic block-copolymer on the formation of CaCO3 from a supersaturated salt solution.

The effect of a double hydrophilic block-copolymer additive (made of polyaspartic acid and polyethyleneglycol, pAsp(10)-b-PEG(110)) on the initial formation of calcium carbonate from a supersaturated salt solution has been studied in situ by means of time-resolved synchrotron small-angle X-ray scattering (SAXS). A stopped-flow cell was used for rapidly mixing the 20 mM aqueous reactant solutions of calcium chloride and sodium carbonate. In reference measurements without polymer additive the very rapid formation of primary, overall spherical CaCO(3) particles with a radius of ca. 19 nm and a size polydispersity of ca. 26% was observed within the first 10 ms after mixing. A subsequent, very rapid aggregation of these primary particles was evidenced by a distinct upturn of the SAXS intensity at smallest angles. During the aggregation process the size of the primary particles remained unchanged. From an analysis of the absolute scattering intensity the mass density of these particles was determined to 1.9 g/cm(3). From this rather low density it is concluded that those precursor particles are amorphous, which has been confirmed by simultaneous wide-angle X-ray diffraction measurements. Upon adding 200 pm of the block-copolymer no influence on the size, the size polydispersity and morphology of the primary particles, nor on the kinetics of their formation and growth, was found. On the other hand, the subsequent aggregation and precipitation process is considerably slowed down by the additive and smaller aggregates result. The crystalline morphology of the sediment was studied in situ by WAXS ca. 50 min after mixing the reactants. Several diffraction rings could be detected, which indicate that a transformation of the metastable, amorphous precursor particles to randomly oriented vaterite nanocrystallites has taken place. In addition, a few isolated Bragg spots of high intensity were detected, which are attributed to individual, oriented calcite microcrystals that nucleated at the wall of the capillary.

Journal Article↗

PubMed2001

Formation of BaSO4 fibres with morphological complexity in aqueous polymer solutions.

BaSO4 fibres with morphological complexity were formed in aqueous solution with polyacrylate and partially monophosphonated poly(ethyleneoxide)-block-poly(methacrylic acid) additives by a simple precipitation reaction. For polyacrylate, formation of the fibrous deposits was strongly dependent on the level of supersaturation (S) and Ba2+:polymer molar ratio (R). At S = 60 to 80, and R = 3 to 14, highly anisotropic crystalline fibres consisting of bundles of BaSO4 nanofilaments were formed after several weeks, although the yield was low. The nanofilaments were also organized into cone-shaped aggregates at S = 80, and at lower R values these formed higher-order structures that consisted of multiple cone-on-cone assemblies with remarkable self-similarity. Increasing the supersaturation produced ovoid or cross-shaped dendritic particles for the range of molar ratios studied. In contrast, BaSO4 crystallisation in the presence of a partially phosphonated block copolymer gave a high yield of BaSO4 fibres up to 100 microm in length, and consisting of co-aligned bundles of 30 nm-diameter defect-free single-crystal nanofilaments with a uniform growth tip. A model for the defect-free growth of BaSO4 nanofilaments in aqueous polymer solutions based on amorphous precursor particles, vectorially directing forces and van der Waals attraction is proposed.

Journal Article↗

PubMed2001

A systematic examination of the morphogenesis of calcium carbonate in the presence of a double-hydrophilic block copolymer.

In this paper, a systematic study of the influence of various experimental parameters on the morphology and size of CaCO3 crystals after room-temperature crystallization from water in the presence of poly(ethylene glycol)-block-poly(methacrylic acid) (PEG-b-PMAA) is presented. The pH of the solution, the block copolymer concentration, and the ratio [polymer]/[CaCO3] turned out to be important parameters for the morphogenesis of CaCO3, whereas a moderate increase of the ionic strength (0.016 M) had no influence. Depending on the experimental conditions, the crystal morphologies can be tuned from calcite rhombohedra via rods, ellipsoids or dumbbells to spheres. A morphology map is presented which allows the prediction of the crystal morphology from a combination of pH, and CaCO3 and polymer concentration. Morphologies reported in literature for the same system but under different crystallization conditions agree well with the predictions from the morphology map. A closer examination of the growth of polycrystalline macroscopic CaCO3 spheres by TEM and time-resolved dynamic light scattering showed that CaCO3 macrocrystals are formed from strings of aggregated amorphous nanoparticles and then recrystallize as dumbbell-shaped or spherical calcite macrocrystal.

Calcium Carbonate↗

PubMed2000

Surfactant-encapsulated clusters (SECs): (DODA)20(NH4)[H3Mo57V6(NO)6O183(H2O)18], a case study.

We present a comprehensive study of the partially reduced polyoxomolybdate [H3-Mo57V6(NO)6O183(H2O)18]21-encapsulated in a shell of dimethyldioctadecylammonium (DODA) surfacmolecules. Treatment of an aqueous solution of (NH4)21[H3Mo57V6-(NO)6O183(H2O)18] . 65H2O (1a) with a trichloromethane solution of the surfactant leads to instant transfer of the encapsulated complex anion into the organic phase. Results from vibrational spectroscopy. analytical ultracentrifugation, small-angle X-ray scattering, transmission electron microscopy, elemental analysis, and Langmuir compression isotherms are consistent with a single polyoxometalate core encapsulated within a shell of 20 DODA molecules. The molar mass of the supramolecular assembly is 20249 gmol(-1) and the diameter is 3.5 nm. A material with the empirical formula (DODA)20(NH4)[H3-Mo57V6NO)6O183(H2O)18] (2) was isolated as a dark violet solid, which readily dissolves in organic solvents. Slow evaporation of solutions of 2 on solid substrates forces the hydrophobic particles to aggregate into a cubic lattice. Annealing these so-formed films at elevated temperature causes de-wetting with terrace formation similar to liquid crystals and block copolymers. Compound 2 forms a stable Langmuir monolayer at the air-water interface; Langmuir-Blodgett multilayers are readily prepared by repeated transfer of monolayers on solid substrates. The films were characterized by optical ellipsometry, Brewster angle microscopy, transmission electron microscopy, and X-ray reflectance.

Journal Article↗

PubMed2000

Crystal Design of Barium Sulfate using Double-Hydrophilic Block Copolymers.

Peach, peanut, fiber, and flower (see picture) crystal morphologies are achieved from the precipitation of simple minerals in the presence of specifically adsorbing polymers. These crystal design effects are illustrated using BaSO(4) and double-hydrophilic block copolymers, the latter featuring carboxylate, sulfonate, phosphonate, and aspartic acid groups.

Journal Article↗

PubMed2000

Interaction of kappa-carrageenan with nickel, cobalt, and iron hydroxides.

In a previous paper, it was shown that the functional polysaccharide kappa-carrageenan acts as an efficient stabilizer to prevent the precipitation of iron oxides and hydroxides up to very high pH values (Jones, et al. Colloid Polym. Sci. 2000, 278, 491-501). Here, this process was investigated for its application to synthesize stable cobalt and nickel oxide particles. Nickel hydroxide nanoparticles were well stabilized for long periods, while the hydroxide of cobalt(II), although initially stable at pH 13, progressively showed some complex structure rearrangement, invoked by alkaline oxidation of the primary particles. The final product of this oxidation/self-assembly process are spherical, fluffy superstructures of cobalt(III) oxide platelets with an overall diameter of 12 microns, in coexistence with 65-75% of free kappa-carrageenan.

Carrageenan↗

PubMed1999

Stabilization of Metal Nanoparticles in Aqueous Medium by Polyethyleneoxide-Polyethyleneimine Block Copolymers.

The branched polyethyleneoxide-polyethyleneimine (PEO)n-b-PEI block copolymers are synthesized and fractionated. The (PEO)n-b-PEI block copolymers are found to be able to form micelles in aqueous solution after addition of certain metal compounds (H2PtCl6, Na2PtCl4, RhCl3). The degree of copolymer polydispersity seems to play an important role in metal nanoparticle stabilization. Comparison of diblock and branched copolymers shows that as a rule branched block copolymers are the better stabilizing systems for metal nanoparticles. In the case of micelle formation due to interaction with metal compounds, fine control on colloid morphology can be provided, which leads to very uniform particles. Variation of different parameters (such as concentration, pH, type of reducing agent) affects the nanoparticle morphology as well. Copyright 1999 Academic Press.

Journal Article↗

PubMed1999

Morphological Characterization of the Molecular Superstructure of Polyphenylene Ethynylene Derivatives.

Two different polyphenylene ethynylene derivatives, one partly hydrophobic and one hydrophilic, were investigated with a combination of X-ray and light scattering techniques and hydrodynamic techniques, as well as scanning force microscopy and transmission electron microscopy to elucidate their molecular structure and aggregation behavior in tetrahydrofuran and water, respectively. It turns out that both polymers possess a rod-like molecular architecture which, however, is the basis of a cascade of aggregation steps. Both, electron microscopy and X-ray analysis support the concept of a primary back-to-back aggregation of polymer chains into cylindrically shaped aggregates with high anisometry. The thickness of these aggregates was between 4.0 and 4.5 nm. The aggregates of the hydrophobic polymer further associate into fibrils and these fibrils form clusters of globular shape, though with high internal anisometry. Copyright 1999 Academic Press.

Journal Article↗

PubMed1999

Hydrodynamic properties of human erythrocyte band 3 solubilized in reduced Triton X-100.

The oligomeric state and function of band 3, purified by sulfhydryl affinity chromatography in reduced Triton X-100, was investigated. Size exclusion high-performance liquid chromatography showed that a homogeneous population of band 3 dimers could be purified from whole erythrocyte membranes. The elution profile of band 3 purified from membranes that had been stripped of its cytoskeleton before solubilization was a broad single peak describing a heterogeneous population of oligomers with a mean Stokes radius of 100 A. Sedimentation velocity ultracentrifugation analysis confirmed particle heterogeneity and further showed monomer/dimer/tetramer equilibrium self-association. Whether the conversion of dimer to the form described by a Stokes radius of 100 A was initiated by removal of cytoskeletal components, alkali-induced changes in band 3 conformation, or alkali-induced loss of copurifying ligands remains unclear. After incubation at 20 degrees C for 24 h, both preparations of band 3 converted to a common form characterized by a mean Stokes radius of 114 A. This form of the protein, examined by equilibrium sedimentation ultracentrifugation, is able to self-associate reversibly, and the self-association can be described by a dimer/tetramer/hexamer model, although the presence of higher oligomers cannot be discounted. The ability of the different forms of the protein to bind stilbene disulfonates revealed that the dimer had the highest inhibitor binding affinity, and the form characterized by a mean Stokes radius of 114 A to have the lowest.

Anion Exchange Protein 1, Erythrocyte↗

PubMed1998

Ultracentrifugation studies on the transmembrane domain of the human erythrocyte anion transporter band 3 in the detergent C12E8.

The dilute solution behaviour of the transmembrane domain (TMD) of the human erythrocyte anion exchanger Band 3 was studied by analytical ultracentrifugation. Sedimentation velocity and equilibrium studies of the TMD solubilized with the detergent C12E8 demonstrate that the protein is a stable dimer in the concentration range 0.1 to 1 mg/ml. There is no evidence of a dissociation at low concentrations or of an association at higher concentrations. Hydrodynamic calculations applying a prolate ellipsoid of revolution and assuming a hydration of w = 0.35 result in an asymmetrical particle with an axial ratio (a/b) of approximately 3.5.

Algorithms↗

PubMed1997

An ultracentrifugal approach to quantitative characterization of the molecular assembly of a physiological electron-transfer complex: the interaction of electron-transferring flavoprotein with trimethylamine dehydrogenase.

The interaction between two physiological redox partners, trimethylamine dehydrogenase and electron-transferring flavoprotein, has been characterized quantitatively by analytical ultracentrifugation at 4 degrees C. Analysis of sedimentation-equilibrium distributions obtained at 15 000 rpm for mixtures in 10 mM potassium phosphate, pH 7.5, by means of the psi function [Wills, P. R., Jacobsen, M. P. & Winzor, D. J. (1996) Biopolymers 38, 119-130] has yielded an intrinsic dissociation constant of 3-7 microM for the interaction of electron-transferring flavoprotein with two equivalent and independent sites on the homodimeric enzyme. This investigation indicates the potential of sedimentation equilibrium for the quantitative characterization of interactions between dissimilar macromolecules.

Bacteria↗

PubMed1997

The ELLIPS suite of macromolecular conformation algorithms.

This paper describes a series of four programmes for the PC based on ellipsoidal representations of macromolecular shape in solution using Universal shape functions, ELLIPS1 is based on simple ellipsoid of revolution models (where two of the three axes of the ellipsoid are fixed equal to each other). If the user types in a value for a shape function from sedimentation or other types of hydrodynamic measurement, it will return a value for the axial ratio of the ellipsoid. ELLIPS2 is based on the more general triaxial ellipsoid with the removal of the restriction of two equal axes. The user enters the three semi-axial dimensions of the molecule or the equivalent two axial ratios and ELLIPS2 returns the value of all the hydrodynamic shape functions. It also works of course for ellipsoids of revolution. ELLIPS3 and ELLIPS4 do the reverse of ELLIPS2, that is they both provide a method for the unique evaluation of the triaxial dimensions or axial ratios of a macromolecule (and without having to guess a value for the so-called "hydration") after entering at least three pieces of hydrodynamic information: ELLIPS3 requires EITHER the intrinsic viscosity with the second virial coefficient (from sedimentation equilibrium, light scattering of osmometry) and the radius of gyration (from light or x-ray scattering) OR the intrinsic viscosity with the concentration dependence term for the sedimentation coefficient and the (harmonic mean) rotational relaxation time from fluorescence depolarisation measurements. ELLIPS4 evaluates the tri-axial shape of a macromolecule from electro-optic decay based Universal shape functions using another Universal shape function as a constraint in the extraction of the decay constants.

Algorithms↗

PubMed1997

Alteration of the quaternary structure of glutamate dehydrogenase from Clostridium symbiosum by a single mutation distant from the subunit interfaces.

X-ray crystallographic studies have previously shown that glutamate dehydrogenase from Clostridium symbiosum is a homohexamer. Mutation of the active-site aspartate-165 to histidine causes an alteration in the structural properties of the enzyme. The mutant enzyme, D165H exists predominantly as a single species of lower molecular mass than the wild-type enzyme as indicated by gel filtration and sedimentation velocity analysis. The latter technique gives an S20,w value for D165H of (6.07 +/- 0.01)S which compares with (11.08 +/- 0.01)S for the wild-type, indicative of alteration of the homohexameric quaternary structure of the native enzyme to a dimeric form, a result confirmed by sedimentation equilibrium experiments. Further support for this is provided by chemical modification by Ellman's reagent of cysteine-144 in the mutant, a residue which is buried at the dimer-dimer interface in the wild-type enzyme and is normally inaccessible to modification. The results suggest a possible structural route for communication between the active sites and subunit interfaces which may be important for relaying signals between subunits in allosteric regulation of the enzyme.

Chromatography, Gel↗

PubMed1996

Construction of a dimeric form of glutamate dehydrogenase from Clostridium symbiosum by site-directed mutagenesis.

By using site-directed mutagenesis, Phe-187, one of the amino-acid residues involved in hydrophobic interaction between the three identical dimers comprising the hexamer of Clostridium symbiosum glutamate dehydrogenase (GDH), has been replaced by an aspartic acid residue. Over-expression in Escherichia coli led to production of large amounts of a soluble protein which, though devoid of GDH activity, showed the expected subunit M(r) on SDS-PAGE, and cross-reacted with an anti-GDH antibody preparation in Western blots. The antibody was used to monitor purification of the inactive protein. F187D GDH showed altered mobility on non-denaturing electrophoresis, consistent with changed size and/or surface charge. Gel filtration on a calibrated column indicated an M(r) of 87000 +/- 3000. The mutant enzyme did not bind to the dye column routinely used in preparing wild-type GDH. Nevertheless suspicions of major misfolding were allayed by the results of chemical modification studies: as with wild-type GDH, NAD+ completely protected one-SH group against modification by DTNB, implying normal coenzyme binding. A significant difference, however, is that in the mutant enzyme both cysteine groups were modified by DTNB, rather than C320 only. The CD spectrum in the far-UV region indicated no major change in secondary structure in the mutant protein. The near-UV CD spectrum, however, was less intense and showed a pronounced Phe contribution, possibly reflecting the changed environment of Phe-199, which would be buried in the hexamer. Sedimentation velocity experiments gave corrected coefficients S20,W of 11.08 S and 5.29 S for the wild-type and mutant proteins. Sedimentation equilibrium gave weight average molar masses M(r,app) of 280000 +/- 5000 g/mol. consistent with the hexameric structure for the wild-type protein and 135000 +/- 3000 g/mol for F187D. The value for the mutant is intermediate between the values expected for a dimer (98000) and a trimer (147000). To investigate the basis of this, sedimentation equilibrium experiments were performed over a range of protein concentrations. M(r,app) showed a linear dependence on concentration and a value of 108 118 g/mol at infinite dilution. This indicates a rapid equilibrium between dimeric and hexameric forms of the mutant protein with an equilibrium constant of 0.13 l/g. An independent analysis of the radial absorption scans with Microcal Origin software indicated a threefold association constant of 0.11 l/g. Introduction of the F187D mutation thus appears to have been successful in producing a dimeric GDH species. Since this protein is inactive it is possible that activity requires subunit interaction around the 3-fold symmetry axis. On the other hand this mutation may disrupt the structure in a way that cannot be extrapolated to other dimers. This issue can only be resolved by making alternative dimeric mutants.

Aspartic Acid↗

PubMed1996

Hydrodynamic examination of the dimeric cytoplasmic domain of the human erythrocyte anion transporter, band 3.

Solution studies of the cytoplasmic domain (molecular mass approximately 40kDa) of band 3, the anion exchanger from human erythrocyte membranes, previously suggested a dimeric molecule on the basis of the relative techniques of calibrated gel filtration and calibrated preparative ultracentrifugation. This dimeric behavior is firmly established on an absolute basis by a combination of calibrated gel chromatography and absolute ultracentrifugation techniques. Sedimentation velocity in the analytical ultracentrifuge combined with calibrated gel chromatography give a molecular mass M of (77 +/- 4) kDa, a value confirmed by low-speed sedimentation equilibrium. Velocity sedimentation in the analytical ultracentrifuge gave a single sedimenting species with an s o 20,w of (3.74 +/- 0.07)S. Sedimentation equilibrium analysis was also used to establish the strength of the binding via the dissociation constant Kd, with a value from direct fitting of the concentration distribution curves of (2.8 +/- 0.5) microM, confirmed by a value of approximately 3 microM obtained from fitting a plot of molecular weight Mw,app versus cell loading concentration. Hydrodynamic calculations based on the classical translational frictional ratio showed that the protein was highly asymmetric, with an axial ratio of approximately 10:1, consistent with observations from electron microscopy.

Anion Exchange Protein 1, Erythrocyte↗

PubMed1995

Mycobacterium tuberculosis chaperonin 10 forms stable tetrameric and heptameric structures. Implications for its diverse biological activities.

The chaperonin activity of sequence-related chaperonin 10 proteins requires their aggregation into heptameric structures. We describe size-exclusion chromatography and ultracentrifugation studies that reveal that while Escherichia coli chaperonin 10 exists as a heptamer, the Mycobacterium tuberculosis chaperonin 10 is tetrameric in dilute solutions and in whole M. tuberculosis lysate. At high protein concentration and in the presence of saturating amounts of divalent ions, the protein is heptameric. Human chaperonin 10 is predominantly heptameric, although smaller oligomers were detected. These differences in structural assembly between species may explain differences in biological activity such as antigenicity. Using C-terminal and N-terminal fragments, sequence 1-25 was identified as indispensable for aggregation. CD spectroscopy studies revealed that (i) a minimum at 202-204 nm correlates with aggregation and characterizes not only the spectrum of the mycobacterial protein, but also those of E. coli and human chaperonin 10 proteins; (ii) the interactions between subunits are of the hydrophobic type; and (iii) the anti-parallel beta-pleated sheet is the main secondary structure element of subunits in both tetrameric and heptameric proteins.

Amino Acid Sequence↗

PubMed1995

Inversion formulae for ellipsoid of revolution macromolecular shape functions.

Explicit relations linking ellipsoid of revolution shape parameters with the axial ration (a/b) have been available for a long time. It is not possible to directly invert these functions to give a/b as a function of the shape parameter, so in the past graphical or tabular interpolation methods have been necessary. By the use of simple unconstrained polynomial expansions this restriction is no longer necessary. For the two salient hydration-dependent shape functions (P from the frictional ratio and v from the intrinsic viscosity) and the four salient hydration-independent shape functions (beta and R, both from sedimentation and viscosity measurements, and Lambda and Pi from combining viscosity measurements with respectively fluorescence depolarization or thermodynamic second virial coefficient measurements) the polynomial expansion coefficients (to no more than seventh order) are given for the ranges 1.1 < a/b < 2.0, 2 < 2/b < 10, and 10 < a/b < 100. The polynomial coefficients give an excellent fit to the real data values, except for four uninteresting cases (where the shape function is too insensitive for practical use).

Computer Simulation↗

PubMed1994

A modified experimental setup for sedimentation equilibrium experiments with gels. Part 2: Technical developments.

This part of the paper trilogy describes technical developments for an efficient experimental setup to investigate gels with equilibrium analytical ultracentrifugation. New 10-channel centerpieces for the Schlieren optics, a new programmable multiplexer, a modified Schlieren optical system, and a photo pickup with impulse transformer are introduced as major developments. Also, some new centerpieces suitable for equilibrium experiments with solutions using the Rayleigh interference and the uv-absorption optics are presented. These centerpieces allow the investigation of 10, 12, or even 26 samples per centerpiece. The problem to find suitable materials for cell centerpieces and windows in the case of adhering samples is discussed for the system gelatin/water. A phase volume calculation for circular sample channels as a correction for the case of broadened menisci is presented. The method described allows an accurate measurement of up to 70 samples simultaneously in an equilibrium experiment if the 8-hole rotor presented in part 1 of the trilogy is used. The number of samples is sufficient to characterize a gel/solvent system in the experimentally accessible range under identical conditions, which is not possible by means of any of the methods known before. All parts described are also applicable for the investigation of solutions.

Gelatin↗