Crystallization of macromolecular heparin (Short Communication)

X-ray fibre-diffraction photographs were obtained from oriented films of the sodium salt of macromolecular heparin (molecular weight approx. 10⁶) prepared from rat skin. Two distinct molecular chain conformations corresponding to two different crystal lattices were observed as a function of relative humidity. The first conformation, obtained at 78% relative humidity, has a layer-line spacing of 1.73nm, which can be interpreted as an approximate twofold helix. On increasing the relative humidity to 84% a second phase with a layer-line repeat of 1.65nm is obtained with the reflexions indexing on a triclinic unit cell similar to that obtained previously (Nieduszynski & Atkins, 1973) for pig mucosal heparin.     

Structural changes in the thin filament during activation studied by X-ray diffraction of highly stretched skeletal muscle

The actin layer-lines were recorded from a frog semitendinosus muscle stretched to a sarcomere length greater than 4.4 microM. On activation of the muscle, the equator, the second layer-line at 1/18 nm-1 and the 5.9 nm layer-line increased in integrated intensity. On the other hand, the integrated intensity of the first layer-line at 1/36 nm-1 decreased markedly on activation. This decrease was not fully attributable to shifts of tropomyosin strands and therefore suggested a structural change in the actin subunit. The decrease may account for the apparent lack of an intensity increase of this layer-line on activation at normal muscle lengths where attachment of myosin heads to actin increases the intensities of other layer-lines.     

Structure of polar pili from Pseudomonas aeruginosa strains K and O

The polar pili of Pseudomonas aeruginosa strains K and O are hollow cylinders with 52 Å outer diameter and 12 Å inner diameter. There is a girdle of low electron density (interpreted as due to a local concentration of hydrophobic amino acid side-chains) centred at 31 Å diameter. Similar X-ray diffraction patterns are obtained from oriented fibres of the two types of pili, to a resolution of 7 Å in the equatorial direction and 4 Å in the meridional direction. The two types of pilin protein subunits have a similar molecular weight, and their sequences contain a number of homologous regions. They form a helical array with 4.06 to 4.08 units per turn of a basic helix that has a pitch of 40.8 Å for strain K pili and 41.3 Å for strain O pili at 75% relative humidity. A method is described for distinguishing between very similar diffraction patterns.There is strong intensity at 10 Å near the equator and at 5 Å near the meridian on the diffraction patterns. This intensity distribution is characteristic of α-helical rods running roughly in the direction of the fibre axis. The orientation of these rods was established by the fit between the transform of an α-helical polyalanine model and the strong near-equatorial layer-line.     

Structure of the filamentous bacteriophage,Pf3, by X-ray fiber diffraction

Fiber diffraction data have been obtained for the filamentous bacteriophage Pf3. The virus crystallizes on a hexagonal net with lattice constants a = b = 5.61 nm and c = 7.50 nm at 0% relative humidity and a = 6 = 6.20 nm and c = 7.99 nm at 98% relative humidity. The X-ray diffraction pattern resembles those of two other bacteriophages, Pf 1 and Xf, and therefore belongs to class II. The data are consistent with a 27₅ helix symmetry with an axial rise of 0.277 (dry) to 0.296 (wet) nm.     

The conformation of the mucopolysaccharides. Hyaluronates

X-ray-diffraction patterns of hyaluronate fibres from a variety of sources were obtained. Sodium hyaluronate gives well-defined patterns which index on a hexagonal unit cell with dimensions a=1.17+/-nm and a fibre repeat-distance of 2.85+/-0.03nm. A further form of sodium hyaluronate is produced by annealing at 60 degrees C in 75% relative humidity. This stable state indexes on a hexagonal unit cell of unchanged fibre repeat-distance but with a=1.87nm. The chain conformation is a threefold helix. Analysis of these diffraction patterns led to two tentative structures for sodium hyaluronate, involving different packing of the polysaccharide chains. The significance of side-chain interaction is discussed. Hyaluronic acid produces an X-ray pattern different from that obtained with the sodium salt. The fibre repeat-distance is 1.96+/-0.02nm and the unit cell appears to be monoclinic. The chain conformation is a twofold helix and conformational change between free acid and monovalent salt is discussed. These findings, together with model-building experiments, are interpreted as indicating a highly ordered structure, and the physical properties of hyaluronate solutions with regard to molecular shape and polyelectrolyte behaviour are rationalized.     

Solid-phase synthesis of fluorescent heparin

A method for linking heparin via its reducing terminus to a fluorescent ligand, 3-aminotyramine, is described. The procedure is such that all of the isolated product is labeled and each polysaccharide chain contains a single label. The derivatized heparin has a fluorescence excitation maximum at 330 nm and an emission maximum at 406 nm. Application of the procedure to heparin fractions with defined molecular-weight ranges yielded products in which the relative emission intensities for equimolar solutions were essentially the same. These preparations should be very useful in defining the molecular weight of fractionated heparin and for studies on heparin-protein interactions.     

Arrangement of myosin heads in relaxed thick filaments from frog skeletal muscle

The distribution of myosin heads on the surface of frog skeletal muscle thick filaments has been determined by computer processing of electron micrographs of isolated filaments stained with tannic acid and uranyl acetate. The heads are arranged in three strands but not in a strictly helical manner and so the structure has cylindrical symmetry. This accounts for the "forbidden" meridional reflections seen in diffraction patterns. Each layer-line therefore represents the sum of terms of Bessel orders 0, +/- 3, +/- 6, +/- 9 and so on. These terms interact so that, unlike a helical object without terms from overlapping Bessel orders, as the azimuth is changed, the amplitude on a layer-line at a particular radius varies substantially and its phase does not alter linearly. Consequently, a three-dimensional reconstruction cannot be produced from a single view. We have therefore used tilt series of three individual filaments to decompose the data on layer-lines 0 to 6 into terms of Bessel orders up to +/- 9 using a least-squares procedure. These data had a least-squares residual of 0.32 and enabled a three-dimensional reconstruction to be obtained at a nominal resolution of 6 nm. This showed, at a radius of about 10 nm, three strands of projecting morphological units with three units spaced along each strand every 42.9 nm axially. We have identified these units with pairs of myosin heads. Successive units along a strand are perturbed axially, azimuthally and radially from the positions expected if the structure was perfectly helical. This may simply be a consequence of steric restrictions in packing the heads on the thick filament surface, but could also reflect an underlying non-helical arrangement of myosin tails, which would be consistent with the thick filament shaft being constructed from three subfilaments in which the tails were arranged regularly. There was also material at a radius of about 6 nm spaced 42.9 nm axially, which we tentatively identified with accessory proteins. The filament shaft had a pronounced pattern of axial staining.     

Isolation and characterization of a heparin with high anticoagulant activity from the clam Tapes phylippinarum: evidence for the presence of a high content of antithrombin III binding site

Heparin with high anticoagulant activity (activated partial thromboplastin time of 347 +/- 56.4 and anti-Xa activity of 317 +/- 48.3) was isolated from the marine clam species Tapes phylippinarum in an amount of approximately 2.1 mg/g dry animals. Agarose-gel electrophoresis showed a high content of the slow-moving heparin component (22 +/- 6.8%) and 78 +/- 5.4% of the fast-moving species. An average molecular mass of 13,600 was calculated by PAGE analysis, whereas a number average molecular weight Mn value of 10,700, a weight average molecular weight Mw of 14,900, and a dispersity index Mn/Mw of 1.386 were obtained by high-performance size-exclusion chromatography. Structural analysis of clam heparin, performed by depolymerizing heparin samples with heparinase (EC 4.2.2.7) and then separating the resulting unsaturated oligosaccharides by strong anion exchange-HPLC revealed the presence of large amounts (more than 130% than standard pharmaceutical heparin obtained from bovine intestine) of the oligosaccharide sequence bearing part of the ATIII-binding region, DeltaUA2S (1-->4)-alpha-D-GlcN2S6S (1-->4)-alpha-L-IdoA (1-->4)-alpha-D-GlcNAc6S (1-->4)-beta-D-GlcA (1-->4)-alpha-D-GlcN2S3S6S in the T. phylippinarum heparin, in comparison with bovine mucosal heparin and a sample of porcine mucosal heparin previously published. Furthermore, as expected from the oligosaccharide compositional analysis, due to the presence of a great mol % (80.6%) of the trisulfated disaccharide DeltaUA2S(1-->4)-alpha-D-GlcN2S6S, mollusc heparin is a more sulfated polysaccharide than bovine mucosal heparin (73.5%) and a sample of porcine mucosal (72.8%) heparin previously reported. To our knowledge, this is the first article describing a clam heparin having the ATIII binding site mainly identical to that of human and porcine intestinal mucosal heparins and bovine intestinal mucosal heparin but different from that found in beef lung heparin.     

The solution structure of heparan sulfate differs from that of heparin: implications for function

The highly sulfated polysaccharides heparin and heparan sulfate (HS) play key roles in the regulation of physiological and pathophysiological processes. Despite its importance, no molecular structures of free HS have been reported up to now. By combining analytical ultracentrifugation, small angle x-ray scattering, and constrained scattering modeling recently used for heparin, we have analyzed the solution structures for eight purified HS fragments degree of polymerization 6-18 (dp6-dp18) and dp24, corresponding to the predominantly unsulfated GlcA-GlcNAc domains of heparan sulfate. Unlike heparin, the sedimentation coefficient s(20,)(w) of HS dp6-dp24 showed a small rotor speed dependence, where similar s(20,)(w) values of 0.82-1.26 S (absorbance optics) and 1.05-1.34 S (interference optics) were determined. The corresponding x-ray scattering measurements of HS dp6-dp24 gave radius of gyration (R(G)) values from 1.03 to 2.82 nm, cross-sectional radius of gyration (R(XS)) values from 0.31 to 0.65 nm, and maximum lengths (L) from 3.0 to 10.0 nm. These data showed that HS has a longer and more bent structure than heparin. Constrained scattering modeling starting from 5000-8000 conformationally randomized HS structures gave best fit dp6-dp16 molecular structures that were longer and more bent than their equivalents in heparin. No fits were obtained for HS dp18 or dp24, indicating their higher flexibility. We conclude that HS displays an extended bent conformation that is significantly distinct from that for heparin. The difference is attributed to the different predominant monosaccharide sequence and reduced sulfation of HS, indicating that HS may interact differently with proteins compared with heparin.     

X-ray-diffraction patterns from chondroitin 4-sulphate, dermatan sulphate and heparan sulphate

Ordered conformations from the sodium salts of chondroitin 4-sulphate, dermatan sulphate and heparan sulphate were observed by X-ray diffraction. Chondroitin 4-sulphate shows similar threefold helical character to that previously reported for chondroitin 6-sulphate and hyaluronates. Dermatan sulphate forms an eightfold helix with an axial rise per disaccharide of 0.93nm, which favours the l-iduronic acid moiety in the normal C1 chair form. The layer-line spacing and axial projection in heparan sulphate of 1.86nm favours a tetrasaccharide repeat with glycosidic linkages alternating beta-d-(1-->4) and alpha-d-(1-->4).     

Wheat-gluten-based natural polymer nanoparticle composites

A series of wheat-gluten-based nanocomposites were produced by dispersing Cloisite-30B nanoclay particles into plasticized wheat gluten systems under thermal processing conditions. The exfoliation of the nanoparticles as confirmed by wide-angle X-ray diffraction and transmission electron microscopy has resulted in significant enhancement of the mechanical properties for both deamidated proteins and vital gluten systems under 50% relative humidity (RH). Such strength improvement was also pronounced for wheat gluten (WG) systems under a high humidity condition (RH = 85%). A similar level of further strength enhancement was obtained for the WG systems that had been strengthened by blending with poly(vinyl alcohol) (PVA) and cross-linking with glyoxal. Although the nanoclay modifier, a quaternary ammonium, caused an additional plasticization to the materials, the interactions between the gluten matrix and the nanoparticles were predominant in all of these nanocomposites. A solid-state NMR study indicated that the polymer matrix in all of these nanocomposites displayed a wide distribution of chain mobilities at a molecular level (less than 1 nm). The interactions between the nanoparticles and the natural polymer matrix resulted in motional restriction for all components in the mobile phases including lipid, plasticizers, and plasticized components, although no significant influence from the nanoparticles was obtained in the mobility of the rigid phases (unplasticized components). On a scale of 20-30 nm, the deamidated protein systems tended to be homogeneous. The small domain size of the matrix resulted in modifications of the spin-lattice relaxation of these systems via spin diffusion. The residual starch seemed to remain in a relatively larger domain size in WG systems. The nanoparticles could enhance the miscibility between the starch and the other components in the WG nanocomposite, but such miscibility enhancement did not occur in the WG/PVA blend and the cross-linked system. These polymer matrixes were still heterogeneous on a scale of 20-30 nm.     

Structural transitions of deoxyribonucleic acid in aqueous electrolyte solutions. I. Reference spectra of conformational limits.

The circular dichroism properties of calf thymus DNA have been examined at 27 degrees over the wavelength range of 215-300 nm in aqueous solutions of NaCl, KCl, LiCl, CsCl, and NH4Cl at pH 7. The concentrations of these electrolytes were varied from 0.01 to ca. 5-10 m. The spectral changes induced by changes in concentration of NaCl and KCl and all but the highest concentrations of NH4Cl as well as lower concentrations of Cstcl and LiCl could be represented by a common two-state transition involving the conversion of the typical conservative spectrum commonly seen in dilute solutions of these salts to a nonconservative spectrum similar to that obtained by Tunis-Schneider and Maestre ((1970), J. Mol. Biol. 52, 521) for the C form of DNA. At higher concentrations of CsCl, LiCl, and NH4Cl, an additional component, resembling an A type spectrum, was required to account for the observed CD changes with changing concentration of electrolyte. Relying on the published spectra of the B, the C, and the A forms of DNA by Tunis-Schneider and Maestre for identification and approximate values of the molecular ellipticities of these forms, we have analyzed these spectral transitions by two least mean squares methods in order to obtain accurate reference spectra of aqueous "B", C, and "A" conformations of calf thymus DNA. The results obtained suggest that although the C form in solution is identical with that obtained in film, the aqueous B conformational limit is not identical with the crystallographic Watson-Crick structure. In addition, the A form generated in solution under our experimental conditions appears to be more similar to that assumed by low molecular weight Escherichia coli DNA at 75% relative humidity rather than calf thymus DNA at the same relative humidity.     

Controlled reduction of the humidity induces a shortcut recovery reaction in the photocycle of photoactive yellow protein

The photocycle of the blue-light photoreceptor protein Photoactive Yellow Protein (PYP) was studied at reduced relative humidity (RH). Photocycle kinetics and spectra were measured in thin films of PYP in which the relative humidity was set at values between 29 and 98% RH with saturated solutions of various salts. We show that in this range, approximately 200 water molecules per PYP molecule are released from the film. As humidity decreased, photocycle transition rates changed, until at low humidity (RH < 50%) an authentic photocycle was no longer observed and the absorption spectrum of the dark, equilibrium state of PYP started to shift to 355 nm, that is, to a form resembling that of pB(dark). At moderately reduced humidity (i.e., >50% RH), an authentic photocycle is still observed, although its characteristics differ from those in solution. As humidity decreases, the rate of ground state recovery increases, while the rate of depletion of the first red-shifted intermediate pR dramatically decreases. The latter observation contrasts all so-far known modulations of the rate of the transition of the red-shifted- to the blue-shifted intermediates of PYP, which is consistently accelerated by all other modulations of the mesoscopic context of the protein. Under these same conditions, the long-lived, blue-shifted intermediate was formed not only with slower kinetics than in solution but also to a smaller extent. Global analysis of these data indicates that in this low humidity environment the photocycle can take a different route than in solution, that is, part of pG recovers directly from pR. These experiments on wild-type PYP, in combination with observations on a variant of PYP obtained by site-directed mutagenesis (the E46Q mutant protein), further document the context dependence of the photocycle transitions of PYP and are relevant for the interpretation of results obtained in both spectroscopic and diffraction studies with crystalline PYP.     

Binding and endocytosis of heparin by human endothelial cells in culture

Binding of heparin and low molecular weight heparin fragments (CY 222, Mr range 1500-8000) to human vascular endothelial cells was studied. Primary culture of human umbilical vein endothelial cells and either 125I or 3H-labeled heparin or [125I]CY 222 were used. Slow, saturable and specific binding was found. No other tested glycosaminoglycan, excepting a highly sulfated heparan fraction, was able to compete for heparin binding. Two groups of binding sites for [3H]heparin could be distinguished: one with high affinity (Kd = 0.12 microM) and another with lower affinity (Kd = 1.37 microM) and a relative large capacity of binding (1.16 X 10(7) molecules/cell) was calculated. The Kd for unlabeled heparin, as calculated from competition experiments, was 0.23 microM. Much lower affinity was calculated for unlabeled low molecular weight heparin fragments CY 222 (Kd = 4.3 microM) from competition experiments with [125I]CY 222. The binding reversibility was only partial for unfractionated heparin. Even by chasing with unlabeled compound, a fraction of 25-30% was not dissociable from endothelial cells. This fraction was much lower if incubation was carried out at 4 degrees C. The addition of basic proteins (histones) to the incubation medium greatly enhanced the undissociable binding at 37 degrees C, but not at 4 degrees C. The undissociable fraction of heparin was not available to degradation by purified microbial heparinase. These results suggest that a fraction of bound heparin is internalized by the vascular endothelium.     

X-ray equatorial analysis of crab striated muscle in the relaxed and rigor states

The structure of muscle projected along the fiber axis was studied by equatorial X-ray diffraction. The clectron-density distributions in axial projection of muscle were derived by the Fourier syntheses to a resolution of 7 nm in the relaxed and rigor states. The structure of the thick filament backbone (diameter about 21.5 nm) has a nearly smooth cylindrical surface and a low electron-density core (diameter about 7 nm) in the center. In the relaxed state, the center of gravity of the myoXXXin heads is situated at a radius of 19.6 nm from the center of the thick filament, lying just between the surface of the thick filament backbone and the surface of the thin filament (diameter about 8.4 nm). From the electron-density distributions in two slates. the amount of mass transfer from the thick filament to the thin filament was estimated. It was in accordance with that predicted from the structure derived bv the X-ray layer-line analyses.     

Physical polymer matrices based on affinity interactions between peptides and polysaccharides

A rapidly forming polymer matrix with affinity-based controlled release properties was developed based upon interactions between heparin-binding peptides and heparin. Dynamic mechanical testing of 10% (w/v) compositions consisting of a 3:1 molar ratio of poly(ethylene glycol)-co-peptide (approximately 18,000 g/mol) to heparin (approximately 18,000 g/mol) revealed a viscoelastic profile similar to that of concentrated, large molecular weight polymer solutions and melts. In addition, the biopolymer mixtures recovered quickly following thermal denaturation and mechanical insult. These gel-like materials were able to sequester exogenous heparin-binding peptides and could release these peptides over several days at rates dependent on relative heparin affinity. The initial release rates ranged from 3.3% per hour for a peptide with low heparin affinity to 0.025% per hour for a peptide with strong heparin affinity. By altering the affinity of peptides to heparin, a series of peptides can be developed to yield a range of release profiles useful for controlled in vivo delivery of therapeutics.     

Prevention of deep vein thrombosis after hip replacement: randomised comparison between unfractionated heparin and low molecular weight heparin.

OBJECTIVE--To evaluate the efficacy and safety of two subcutaneous prophylactic regimens for postoperative deep vein thrombosis after total hip replacement. DESIGN--Prospective open randomised multicentre trial. SETTING--28 European departments of orthopaedic surgery. INTERVENTION--All patients had bilateral phlebography 10 days after surgery. 31 patients receiving low molecular weight heparin and 29 receiving unfractionated heparin were excluded from the efficacy analysis for various reasons. PATIENTS--349 patients undergoing total hip replacement between September 1988 and May 1989. 174 patients received subcutaneously a low molecular weight heparin (Fraxiparine) with anti-factor Xa activity of 41 IU/kg/day for three days, then 62 IU/kg/day from day 4 to day 10. 175 patients received subcutaneous unfractionated heparin at intervals of eight hours; doses were adjusted to maintain the activated thromboplastin time at two to five seconds above control values. MAIN OUTCOME MEASURE--Total incidence of deep vein thrombosis and incidence of proximal deep vein thrombosis on bilateral phlebography. RESULTS--The total incidence of deep vein thrombosis was 16% in patients receiving unfractionated heparin and 12.6% in patients receiving low molecular weight heparin (p = 0.45), and the incidence of thrombosis of the proximal veins was 13.1% and 2.9% respectively (p less than 0.001). Four patients receiving unfractionated heparin and one receiving low molecular weight heparin developed pulmonary embolism. The incidence of bleeding complications was low and comparable in the two groups. CONCLUSION--Low molecular weight heparin is at least as effective as unfractionated heparin in preventing deep vein thrombosis and is more effective at preventing thrombosis of the proximal veins in patients undergoing hip replacement. Low molecular weight heparin is not more likely to cause bleeding complications and is simpler to give than unfractionated heparin.     

The Solution Structure of Heparan Sulfate Differs from That of Heparin: IMPLICATIONS FOR FUNCTION*

The highly sulfated polysaccharides heparin and heparan sulfate (HS) play key roles in the regulation of physiological and pathophysiological processes. Despite its importance, no molecular structures of free HS have been reported up to now. By combining analytical ultracentrifugation, small angle x-ray scattering, and constrained scattering modeling recently used for heparin, we have analyzed the solution structures for eight purified HS fragments dp6–dp24 corresponding to the predominantly unsulfated GlcA-GlcNAc domains of heparan sulfate. Unlike heparin, the sedimentation coefficient s_20,w of HS dp6–dp24 showed a small rotor speed dependence, where similar s_20,w values of 0.82–1.26 S (absorbance optics) and 1.05–1.34 S (interference optics) were determined. The corresponding x-ray scattering measurements of HS dp6–dp24 gave radii of gyration R_G values from 1.03 to 2.82 nm, cross-sectional radii of gyration R_XS values from 0.31 to 0.65 nm, and maximum lengths L from 3.0 to 10.0 nm. These data showed that HS has a longer and more bent structure than heparin. Constrained scattering modeling starting from 5,000 to 12,000 conformationally randomized HS structures gave best fit dp6–dp24 molecular structures that were longer and more bent than their equivalents in heparin. Alternative fits were obtained for HS dp18 and dp24, indicating their higher bending and flexibility. We conclude that HS displays bent conformations that are significantly distinct from that for heparin. The difference is attributed to the different predominant monosaccharide sequence and reduced sulfation of HS, indicating that HS may interact differently with proteins compared with heparin.     

Isolation and characterization of a high molecular weight glycoprotein from human blood platelets.

A high molecular weight glycoprotein consisting of three disulfide-linked 142,000 molecular weight chains has been isolated from human blood platelets. The glycoprotein, designated thrombospondin, is released by platelets in response to thrombin treatment and is proteolyzed when left in the presence of platelets after liberation. It is relatively insensitive to degradation by thrombin. Thrombospondin is a filamentous protein of dimensions approximately 7 X 70 nm and contains 1.9% neutral sugars, 1.4% amino sugars, 0.7% sialic acid, and no hexuronic acid. Amino acid analysis reveals that the level of cysteine is approximately 260 residues per molecule. Thrombospondin binds to immobilized heparin but is released by 0.45 M sodium chloride. A single band is obtained by isoelectric focusing, indicating a pI of 4.7 as well as a relatively high degree of purity. Degradation of the intact molecule with trypsin yields a stable core particle of molecular weight 210,000 comprised of three 70,000 chains.