Deconvolution can be used in electrooptic studies to correct for non-ideal electric excitation pulses only when the electric dipole moment of the studied molecules is predominantly induced.

The electric field pulses used for most measurements of transient electrooptic properties such as birefringence and dichroism, are rectangular and assumed to be ideal, but in practice do all such pulses have non-zero rise and fall times. Claims have been made that this non-ideality may be taken into account by employing standard deconvolution techniques. We find that this approach yields exact results in the zero electric field limit when the electric dipole moment of the studied macromolecules is predominantly induced. However, for finite electric field strengths and/or macromolecules with partly or fully permanent electric dipole moments, we find that the deconvolution method yields erroneous estimates of the electrooptic relaxation times. When the decay time of the electric pulse and the electrooptic decay time are equal, and the system is operated in the Kerr domain, this systematic error for macromolecules with purely permanent electric dipole moment equals 37%. In a research field where the uncertainty of the reported relaxation times normally is assumed to be only a few percent this is an error that may seriously mislead unsuspecting users. We find that this systematic error can readily be avoided by employing standard numerical integration of a set of coupled first-order differential equations instead of the standard deconvolution techniques.

Exploring the (1 --> 3)-beta-D-glucan conformational phase diagrams to optimize the linear to macrocycle conversion of the triple-helical polysaccharide scleroglucan

The immunologically important (1 --> 6) comb-like branched (1 --> 3)-beta-D-glucans scleroglucan, schizophyllan, lentinan, and others, exist mainly as linear triple-helical structures in aqueous solution. Partial interconversion from linear to circular topology has been reported to take place following conformational transition of the triple-helical structure and subsequent regeneration of the triplex conformation. We here report on experimental data indicating that complete strand separation of the triple-helical structure is required for this interconversion. NaOH or dimethylsulfoxide was used to induce dissociation of the triplex at combinations of concentrations and temperatures shown by calorimetry to yield a conformational transition of the triplex structures. For the alkaline treatment at 55 degrees C, it is found that up to about 30% of the material readily can be converted to the cyclic topology. This fraction increased to about 60% when the subsequent annealing of the scleroglucan in aqueous solution at pH 7 was carried out at 100 degrees C. Further increase of the annealing temperature yielded a smaller relative amount of cyclic species. The data indicate that the lower molecular weight fraction of the molecular weight distributions can be converted selectively to the macrocyclic topology by conditions that do not yield complete strand separation of the whole sample. These findings add to previous reports by providing more details about how the conditions required for the linear triplex to macrocycle interconversion relate to the conformational properties of the triple-helical structure. Copyright 1999 John Wiley & Sons, Inc.

Transient electric birefringence of human erythroid spectrin dimers and tetramers at ionic strengths of 4 mM and 53 mM.

In conventional electrooptic studies the sample ionic strength must for technical reasons be kept below about 3 mM, which is only 2% of the ionic strength at physiological conditions. In particular for flexible polyelectrolytic macromolecules it can in general not be ruled out that both the conformational average and dynamics at ionic strength 3 mM and below may differ significantly from what it is at physiological conditions. Here we report on the first electrooptic study of human erythroid spectrin dimers and tetramers at ionic strengths higher than 3 mM. All measurements in this study were carried out at both ionic strength 4 mM (2.5 mM HEPES + 1 mM NaCl) and 53 mM (2.5 mM HEPES + 50 mM NaCl). Spectrin tetramers were studied only at 4 degrees C whereas the dimers were studied at both 4 degrees C and 37 degrees C. At 4 degrees C there is a striking quantitative similarity between the transient electric bire-fringence (TEB) of spectrin dimers and tetramers. Also, the TEB of spectrin dimers at 37 degrees C was very similar to the results at 4 degrees C. The contour length and the molecular weight of spectrin dimers and tetramers are known. The dominating TEB relaxation time is in all cases only a fraction of what is predicted theoretically if the spectrin dimers and tetramers are assumed to be stiff and extended molecules. In sum, the new TEB data constitute strong electrooptic evidence confirming that spectrin dimers and tetramers have a highly flexible structure, and demonstrate for the first time that a major part of the intrachain dynamics of the spectrin is quite insensitive to an increase of the ionic strength from 4 mM to 53 mM. Use of the reversing electric field pulse technique for all conditions studied yields TEB data suggesting that the orientation of both spectrin dimers and tetramers in an electric field is dominated by a permanent rather than an induced electric dipole moment.

Electric birefringence of recombinant spectrin segments 14, 14-15, 14-16, and 14-17 from Drosophila alpha-spectrin.

Members of the spectrin protein family can be found in many different cells and organisms. In all cases studied, the major functional role of these proteins is believed to be structural rather than enzymatic. All spectrin proteins are highly elongated and consist mainly of homologous repeats that constitute rigid segments connected in tandem. It is commonly believed that the details of the spectrin function depend critically on the flexibility of the links between the segments. Here we report on a work addressing this question by studying the transient electric birefringence of recombinant spectrin fragments consisting of segments 14, 14-15, 14-16, and 14-17, respectively, from Drosophila alpha-spectrin. Transient electric birefringence depends sharply on both molecular length and flexibility. We found that the birefringence relaxation time of segment 14 measured at 4 degrees C, but scaled to what is expected at 20 degrees C, equals 16 ns (+/-15%) at pH 7.5 and ionic strength 6 mM. This is consistent with this single segment being rigid, 5 nm long and having an axial ratio equal to about two. Under the same conditions, segments 14-15, 14-16 and 14-17 show relaxation times of 45, 39 and 164 ns (all +/-20%), respectively, scaled to what is expected at 20 degrees C. When the temperature is increased to 37 degrees C the main relaxation time for each of these multisegment fragments, scaled to what is expected at 20 degrees C, increased to 46, 80, and 229 ns (all +/-20%), respectively. When the ionic strength and the Debye shielding is low, the dynamics of these short fragments even at physiological temperature is nearly the same as for fully extended weakly bending rods with the same lengths and axial ratios. When the ionic strength is increased to 85 mM, the main relaxation time for each of these multisegment fragments is reduced 20-50% which suggests that at physiological salt and temperature conditions the links in 2-4-segment-long fragments exhibit significant thermally induced flexing. Provided that the recombinant spectrin fragments can serve as a model for native spectrin, this implies that, at physiological conditions, the overall conformational dynamics of a native spectrin protein containing 20-40 segments equals that of a flexible polymer.

Electrooptic analysis of macromolecule dipole moments using asymmetric reversing electric pulses.

The use of symmetric reversing electric field pulses in electrooptic studies of rigid macromolecules in order to determine the ratio between the permanent and the induced dipole moments is well established. Application of this method to studies of small macromolecules requires a field reversal time of only a few nanoseconds. No high current pulse generator capable of producing symmetric kV pulses with such a short reversal time is available for studies of small macromolecules in physiological salt solutions, but it has long been known how to make such reversing pulses that are asymmetric. In order to take advantage of the opportunity offered by the latter fact, we here present a theoretical analysis in the thermal domain of the electrooptic properties of solutions containing rigid macromolecules with axial symmetry when exposed to asymmetric reversing electric field pulses. The analytical expressions needed for quantitative determination of the ratio between the permanent and the induced electric dipole moments of rigid macromolecules using electrooptic data obtained employing reversing electric pulses with given asymmetry are presented. The feasibility of this new approach is demonstrated by including experimental electric birefringence data for a 12 kDa protein (segment 14 of alpha-spectrin from Drosophila brains) in near physiological salt solutions obtained using a coaxial cable pulser producing 2 microseconds long pulses with a reversal time of about 15 ns.

An antitumor, branched (1-->3)-beta-D-glucan from a water extract of fruiting bodies of Cryptoporus volvatus.

A water-soluble, (1-->6)-branched (1-->3)-beta-D-glucan (H-3-B) was isolated from a hot-water extract of the fruiting bodies of the fungus, Cryptoporus volvatus (Basidiomycetes). Enzymatic analysis using exo-(1-->3)-beta-D-glucanase and methylation analysis indicated that this polysaccharide has a main chain composed of beta-(1-->3)-linked D-glucopyranosyl residues, and single, beta-(1-->6)-linked D-glucopyranosyl residues attached as side chains to, on average, every fourth sugar residue of the main chain. This structure was confirmed by 13C NMR spectra of the glucan in Me2SO-d6. The weight-average molecular weight (Mw) of H-3-B was determined to be 44.0 x 10(4) by gel permeation chromatography equipped with a low-angle laser-light-scattering photometer. The electron microscopic observations showed that H-3-B and its sonicated sample (S-H-3-B, Mw = 13.7 x 10(4)) can be described as linear worm-like chains. The mass per unit length for native and sonicated H-3-B was determined to be 1750 and 1780 g mol-1 nm-1, respectively, from the contour lengths obtained by electron microscopy and the molecular weights. These values are in good agreement with that expected for the triple stranded structure. A sample denatured in 0.1 M NaOH and subsequently renatured by neutralization showed a mixture of linear and cyclic structures, and larger aggregates with less well-defined morphology. The H-3-B and S-H-3-B had antitumor activity against the Sarcoma 180 tumor.

Conformation, order-disorder conformational transitions and gelation of non-crystalline polysaccharides studied using electron microscopy.

Direct imaging of polysaccharides using transmission electron microscopy (EM) is an important alternative to physical characterization of non-crystalline polysaccharides in solution. The polymer nature of stiff-chain polysaccharides is quite apparent from direct visualization of the electron micrographs, despite the fact that commonly employed preparation techniques reduce the resolution limit to about 1-2 nm. Electron microscopy has recently been used to study polysaccharides with emphasis both on quantitative properties like contour length, end-to-end distance and chain stiffness, and on qualitative structural features such as cyclization at the macromolecular level. The structural richness observed for polysaccharides of the beta-D0glucan family after a denaturation-renaturation treatment of the specimen, in particular, illustrates the unique potential of EM as a tool for obtaining conformational information about carbohydrate macromolecules. Examples of the latter also include the recent discoveries of cyclic beta-D-glucan and l-carrageenan structures. The EM technique provides information that is not only complementary to what can be obtained using other physical techniques, but also offers important insight otherwise masked by the averaging implicit in most physical techniques used to study aqueous polysaccharide solutions.

Physicochemical properties of (1-->6)-branched (1-->3)-beta-D-glucans. 1. Physical dimensions estimated from hydrodynamic and electron microscopic data.

The physical dimensions of several (1-->6) branched (1-->3)-beta-D-glucan samples obtained from different organisms and their derivatives have been studied by electron microscopy, light scattering measurements, viscometry, and gel permeation chromatography. The electron micrographs indicate that in most samples these biopolymers are adequately described as linear worm-like coils. A sample reconstituted from alkaline media appeared as a blend of the linear, circular, and aggregated polymer morphologies. The average mass per unit length, ML = Mw/Lw for the macroscopically linear samples, was estimated to be 2100 +/- 200 g mol-1 nm-1. The parameter ML was determined from the contour lengths obtained by electron microscopy and the molecular weight by light scattering measurements. The observed ML was consistent with the triple-helical structure reported from x-ray diffraction studies and observed degree of side-chain substitution. From the molecular snapshots shown in the electron micrographs, the persistence lengths of these beta-D-glucans were determined to be 140 +/- 30 nm. The experimentally determined intrinsic viscosities were consistent with these estimates of ML and persistence length. Comparison of the molecular weight distributions obtained from gel permeation chromatography and those deduced from the electron micrographs indicates that number and weight average contour lengths are more reliable than z and z + 1 averages.

Macrocyclization of polysaccharides visualized by electron microscopy.

Topological features of the polysaccharides schizophyllan, l-carrageenan and gellan gum were studied using electron microscopy. Electron micrographs of schizophyllan not subjected to any thermal or solvent composition history destabilizing the triple helix, show stiff, linear chains consistent with the structure being triple helical and with contour length proportional to the molecular weight in solution. A blend of linear, cyclic and hairpin topologies and higher molecular weight clusters were observed after renaturation, i.e. return to conditions favouring the triple helical structure, from solvent conditions dissociating the triple helix. Electron micrographs of l-carrageenan in salt-free solution reveal linear extended structures. Addition of 0.15 M LiI to the solution before preparation for electron microscopy, i.e. salt conditions that favour ordering but not gelation, yields a large fraction of cyclic structures with circumference of different lengths. Likewise, adding KCl to aqueous gellan gum changes their appearance from dispersed polymers to suprastrands with several associated chains. Macrocyclic species can also be observed in gellan gum after the addition of a gel-promoting salt. The tendency to form macrocyclic structures in competition with intermolecular aggregates is determined by the three factors: (1) chain stiffness relative to overall length; (2) parallel or antiparallel alignment of interacting chain segments; and (3) polymer concentration. The present study indicates that electron microscopy provides information about the topology adopted by polysaccharides.

Frequency dependence of the shear moduli of spectrin studied using a multiple lumped resonator viscoelastometer.

The frequency dependence (119-7860 Hz) of the storage and loss shear moduli, G' and G'', of human erythrocyte spectrin dimer crude solutions at 22.5 degrees C has been measured using a Birnboim-Schrag multiple lumped resonator viscoelastometer. The measurements were carried out on solutions of ionic strength 1 mM containing 1.1-3.7 mg ml-1 spectrin. This corresponds to the terminal zone for G' and G''. Analysis of the data using the standard theory of hybrid relaxation spectra yields a relaxation time of 22.5 +/- 1 microseconds. The pure spectrin dimer relaxation time is estimated to be 16 +/- 3 microseconds. This result suggests that at an ionic strength of 1 mM, the spectrin dimers are extended and that the main relaxation process is simple end-over-end rotation.

The molecular size and shape of xanthan, xylinan, bronchial mucin, alginate, and amylose as revealed by electron microscopy.

Electron microscopy of some selected, vacuum-dried and rotary-shadowed, polyelectrolytic polysaccharides and glycoproteins adsorbed to mica indicates that this technique can yield reliable information about polymer conformation for chains with persistence lengths q exceeding about 10 nm. Statistical analyses of the local polymer tangent-direction yield q = 150 nm for double-stranded xanthan, q = 60 nm for single-stranded xanthan, q = 45 nm for xylinan, q = 16 nm for alginate (90% beta-D-mannuronic acid), and q = 15 nm for human-bronchial mucin. These values are all in adequate agreement with values of q obtained by using other techniques. Amylose, on the other hand, appears as non-randomly aligned chains. The observed contour lengths of amylose indicate a mass per unit length of 1440 dalton/nm, consistent with a pseudo-helical conformation.

The molecular basis of erythrocyte shape.

Recent discoveries about the molecular organization and physical properties of the mammalian erythrocyte membrane and its associated structural proteins can now be used to explain, and may eventually be used to predict, the shape of the erythrocyte. Such explanations are possible because the relatively few structural proteins of the erythrocyte are regularly distributed over the entire cytoplasmic surface of the cell membrane and because the well-understood topological associations of these proteins seem to be stable in comparison with the time required for the cell to change shape. These simplifications make the erythrocyte the first nonmuscle cell for which it will be possible to extend our knowledge of molecular interactions to the next hierarchical level of organization that deals with shape and shape transformations.

The human erythrocyte membrane skeleton may be an ionic gel. III. Micropipette aspiration of unswollen erythrocytes.

We have carried out a theoretical analysis of micropipette aspiration of unswollen erythrocytes using the protein-gel-lipid-bilayer membrane model and taking into account that the modulus of area compression of the membrane skeleton may depend on the environmental conditions. Our analysis shows that the aspiration pressure needed to obtain a certain membrane projection length is strongly dependent on the ratio between the membrane skeleton modulus of area compression and the elastic shear modulus. Our analysis therefore predicts that micropipette aspiration of unswollen erythrocytes may be a sensitive method for detection of changes in this ratio. The analysis thus also shows that micropipette aspiration of unswollen erythrocytes can not be used to determine the membrane shear modulus unless something is known about the membrane skeleton modulus of area compression.

The human erythrocyte membrane skeleton may be an ionic gel. I. Membrane mechanochemical properties.

Biochemical and biophysical observations indicate that the erythrocyte membrane skeleton is composed of a swollen network of long, flexible and ionizable macromolecules located at the cytoplasmic surface of the fluid membrane lipid bilayer. We have analyzed the mechanochemical properties of the erythrocyte membrane assuming that the membrane skeleton constitutes an ionic gel (swollen ionic elastomer). Using recently established statistical thermodynamic theory for such gels, our analysis yields mathematical expressions for the mechanochemical properties of erythrocyte membranes that incorporate membrane molecular parameters to an extent not achieved previously. The erythrocyte membrane elastic shear modulus and maximum elastic extension ratio predicted by our membrane model are in quantitative agreement with reported values for these parameters. The gel theory predicts further that the membrane skeleton modulus of area compression, KG, may be small as well as large relative to the membrane elastic shear modulus, G, depending on the environmental conditions. Our analysis shows that the ratio between these two parameters affects both the geometry and the stability of the favoured cell shapes.

The human erythrocyte membrane skeleton may be an ionic gel. II. Numerical analyses of cell shapes and shape transformations.

In the first paper in this series (Stokke et al. Eur Biophys J 1986, 13:203-218) we developed the general theory of the mechanochemical properties and the elastic free energy of the protein gel--lipid bilayer membrane model. Here we report on an extensive numerical analysis of the human erythrocyte shapes and shape transformations predicted by this new cell membrane model. We have calculated the total elastic free energy of deformation of four different cell shape classes: disc-shaped cells, cup-shaped cells, crenated cells, and cells with membrane invaginations. We find that which of these shape classes is favoured depends strongly on the spectrin gel osmotic tension, IIGu, and the surface tensions, IIEu and IIPu, of the extracellular and protoplasmic halves of the membrane lipid bilayer, respectively. For constant ratio IIEu/IIPu greater than O large negative or positive values of IIGu favour respectively the crenated and invaginated cell shape classes. For small absolute values of IIGu, IIEu, and IIPu, biconcave or cup-shaped cells are the stable ones. Our numerical analysis shows that the higher the membrane skeleton compressibility is, the smaller are the values of IIGu needed to induce cell shape transformation. We find that the stable and metastable shapes of discocytes and stomatocytes generally depend both on the shape of the stressfree membrane skeleton and the membrane skeleton compressibility.

Spectrin, human erythrocyte shapes, and mechanochemical properties.

Physical studies of human erythrocyte spectrin indicate that isolated spectrin dimers and tetramers in solution are worm-like coils with a persistence length of approximately 20 nm. This finding, the known polyelectrolytic nature of spectrin, and other structural information about spectrin and the membrane skeleton molecular organization have lead us to the hypothesis that the human erythrocyte membrane skeleton constitutes a two-dimensional ionic gel (swollen ionic elastomer). This concept is incorporated in what we refer to as the protein gel-lipid bilayer membrane model. The model accounts quantitatively for red elastic shear modulus and the maximum elastic extension ratio reported for the human erythrocytes membrane. Gel theory further predicts that depending on the environmental conditions, the membrane skeleton modulus of area compression may be small or large relative to the membrane elastic shear modulus. Our analyses show that the ratio between these two parameters affects both the geometry and the stability of the favored cell shapes and that the higher the membrane skeleton compressibility the smaller the values of the gel tension needed to induce cell shape transformations. The main virtue of the protein gel-lipid bilayer membrane model is that it offers a novel theoretical and molecular basis for the various mechanical properties of the membrane skeleton such as the membrane skeleton modulus of area compression and osmotic tension, and the effects of these properties on local membrane skeleton density, cell shape, and shape transformations.

A computerized low-shear pendulum viscoelastometer, stress-relaxation, shear creep, and dynamic elastic moduli measurements of soft biogels.

A computerized low-shear pendulum viscoelastometer that is well suited for measurements of the viscoelastic properties of soft biogels is described. The instrument has three modes of operation: (i) measurement of dynamic elastic moduli, (ii) stress relaxation, and (iii) shear creep-creep recovery. The main mechanical parts of the instrument include a specimen cuvette and a pendulum with a thin metal plate extending into the specimen cuvette. The cuvette and pendulum can undergo independent angular movements relative to a horizontal axis located about 12 cm above the specimen cuvette. The absolute angular position of the cuvette is under on-line computer control through a stepping motor driven micrometer. The angular position of the pendulum relative to the cuvette is measured using an inductive position detector. The necessary software for automatic operation, data acquisition and processing has been developed for all the three modes of operation. The instrument is well suited for measurements of dynamic elastic moduli in the range 0.02-150 dyn/cm2. Calibration data obtained using 100% ethanol are presented. The instrument suitability for measurements of stress relaxation and shear creep-creep recovery is illustrated by presenting the results of such measurements on a 5.5-mg/ml erythrocyte spectrin gel in isotonic solution.