Antithrombin activity of fucoidan. The interaction of fucoidan with heparin cofactor II, antithrombin III, and thrombin

Fucoidan, poly(L-fucopyranose) linked primarily alpha 1----2 with either a C3- or a C4-sulfate, is an effective anticoagulant in vitro and in vivo (Springer, G. F., Wurzel, H. A., McNeal, G. M., Jr., Ansell, N. J., and Doughty, M. F. (1957) Proc. Soc. Exp. Biol. Med. 94, 404-409). We have determined the antithrombin effects of fucoidan on the glycosaminoglycan-binding plasma proteinase inhibitors antithrombin III and heparin cofactor II. Fucoidan enhances the heparin cofactor II-thrombin reaction more than 3500-fold. The apparent second-order rate constant of thrombin inhibition by heparin cofactor II increases from 4 x 10(4) (in the absence of fucoidan) to 1.5 x 10(8) M-1 min-1 as the fucoidan concentration increases from 0.1 to 10 micrograms/ml and then decreases as fucoidan is increased above 10 micrograms/ml. The fucoidan reaction with heparin cofactor II-thrombin is kinetically equivalent to a "template model." Apparent fucoidan-heparin cofactor II and fucoidan-thrombin dissociation constants are 370 and 1 nM, respectively. The enhancement of thrombin inhibition by fucoidan, like heparin and dermatan sulfate, is eliminated by selective chemical modification of lysyl residues either of heparin cofactor II or of thrombin. The fucoidan-antithrombin III reactions with thrombin and factor Xa are accelerated maximally 285- and 35-fold at fucoidan concentrations of 30 and 500 micrograms/ml, respectively. Using human plasma and 125I-labeled thrombin in an ex vivo system, the heparin cofactor II-thrombin complex is formed preferentially over the antithrombin III-thrombin complex in the presence of 10 micrograms/ml fucoidan. Our results indicate that heparin cofactor II is activated by fucoidan in vitro and in an ex vivo plasma system and suggest that the major antithrombin activity of fucoidan in vivo is mediated by heparin cofactor II and not by antithrombin III.     

On the domain structure of antithrombin III. Tentative localization of the heparin binding region using 1H NMR spectroscopy

The denaturation of human and bovine antithrombin III by guanidine hydrochloride has been followed by 1H NMR spectroscopy. The same unfolding transition seen previously from circular dichroism studies [Villanueva, G. B., & Allen, N. (1983) J. Biol. Chem. 258, 14048-14053] at low denaturant concentration was detected here by discontinuous changes in the chemical shifts of the C(2) protons of two of the five histidines in human antithrombin III and of three of the six histidines in bovine antithrombin III. These two histidines in human antithrombin III are assigned to residue 1 and, more tentatively, to residue 65. Two of the three histidines similarly affected in the bovine protein appear to be homologous to residues in the human protein. This supports the proposal of similar structures for the two proteins. In the presence of heparin, the discontinuous titration behavior of these histidine resonances is shifted to higher denaturant concentration, reflecting the stabilization of the easily unfolded first domain of the protein by bound heparin. From the tentative assignment of one of these resonances to histidine-1, it is proposed that the heparin binding site of antithrombin III is located in the N-terminal region and that this region forms a separate domain from the rest of the protein. The pattern of disulfide linkages is such that this domain may well extend from residue 1 to at least residue 128. Thermal denaturation also leads to major perturbation of these two histidine resonances in human antithrombin III, though stable intermediates in the unfolding were not detected.     

Identification by 1H NMR spectroscopy of flexible C-terminal extensions in bovine lens alpha-crystallin.

Two-dimensional 1H NMR spectroscopy of bovine eye lens alpha-crystallin and its isolated alpha A and alpha B subunits reveals that these aggregates have short and very flexible C-terminal extensions of eight (alpha A) and ten (alpha B) amino acids which adopt little preferred conformation in solution. Total alpha-crystallin forms a tighter aggregate than the isolated alpha A and alpha B subunit aggregates. Our results are consistent with a micelle model for alpha-crystallin quaternary structure. The presence of terminal extensions is a general feature of those crystallins, alpha and beta, which form aggregates.     

Secondary structure determination of human beta-endorphin by 1H NMR spectroscopy

The 1H NMR spectra of human beta-endorphin indicate that the peptide exists in random-coil form in aqueous solution but becomes helical in mixed solvent. Thermal denaturation NMR experiments show that in water there is no transition between 24 and 75 degrees C, while a slow noncooperative thermal unfolding is observed in a 60% methanol-40% water mixed solvent in the same temperature range. These findings are consistent with circular dichroism studies by other workers concluding that beta-endorphin is a random coil in water but that it forms 50% alpha-helix or more in mixed solvents. The peptide in the mixed water-methanol solvent was further studied by correlated spectroscopy (COSY) and nuclear Overhauser effect spectroscopy (NOESY) experiments. These allow a complete set of assignments to be made and establish two distinct stretches over which the solvent induces formation of alpha-helices: the first occurs between Tyr-1 and Thr-12 and the second between Leu-14 and extending to Lys-28. There is evidence that the latter is capped by a turn occurring between Lys-28 and Glu-31. These helices form at the enkephalin receptor binding site, which is at the amino terminus, and at the morphine receptor binding site, located at the carboxyl terminus [Li, C. H. (1982) Cell (Cambridge, Mass.) 31, 504-505]. Our findings suggest that these two receptors may specifically recognize alpha-helices.     

1H NMR spectroscopic studies on the interactions between human plasma antithrombin III and defined low molecular weight heparin fragments.

The effects of length and composition upon the antithrombin-binding properties of heparin have been investigated for two series of structurally related heparin oligosaccharides. Each series consists of a tetrasaccharide, hexasaccharide, and octasaccharide heparin fragment composed of alternating hexuronic acid (either iduronate 2-sulfate or glucuronate) and glucosamine 6,N-disulfate residues. These two series represent dominant structural motifs in intact heparin and differ from each other by the presence of a glucuronic acid in one series in place of an iduronate 2-sulfate residue penultimate to the reducing end of the fragment. Perturbations to the 1H resonances in the NMR spectrum of antithrombin upon binding of the two series of heparin fragments are compared to those generated by intact heparin binding, as well as to the effects of binding of a synthetic high-affinity pentasaccharide. All of the heparin fragments examined appear to bind to antithrombin at the same site. Three of the heparin fragments (hexasaccharide-2, octasaccharide-2, and octasaccharide-1) produce almost identical perturbations in the antithrombin 1H NMR spectrum compared to binding of intact heparin, including perturbations of resonances from tryptophan 49. This indicates that neither the glucuronic acid nor the trisulfated glucosamine residue (structural elements known to be part of the high-affinity heparin motif) are necessary for the majority of the conformational changes induced upon heparin fragment binding to antithrombin. However, the low anticoagulant activity of these fragments indicates that the changes in protein conformation upon fragment binding, as manifested by these 1H resonance perturbations, are not sufficient for catalytic activation of the inhibitor.(ABSTRACT TRUNCATED AT 250 WORDS)     

FTIR, 1H NMR and EPR spectroscopy studies on the interaction of flavone apigenin with dipalmitoylphosphatidylcholine liposomes

Apigenin (5,7,4′-trihydroxyflavone) is a cancer chemopreventive agent and a member of the family of plant flavonoids. Apigenin interaction with liposomes formed with dipalmitoylphosphatidylcholine (DPPC) was investigated by means of FTIR spectroscopy, ¹H NMR and EPR techniques. Fluorescent microscopy and electron microscopy were applied to study the apigenin effects on colon myofibroblasts and human skin fibroblasts. The strong rigidifying effect of apigenin with respect to polar head groups was concluded on the basis of the action of the flavone on partition coefficient of Tempo spin label between the water and lipid phases. The ordering effect was also found in hydrophobic region at the depth monitored by 5-SASL and 16-SASL spin labels. The inclusion of apigenin to the membrane restricted the motional freedom of polar head groups lowering penetration of Pr^3 + ions to the membranes. The ¹H NMR technique supported also the restriction of motional freedom of the membrane in the hydrophobic region, especially in the zone of CH₂ groups of alkyl chains. FTIR analysis showed that apigenin incorporates into DPPC liposomes via hydrogen bonding between its own hydroxyl groups and lipid polar head groups in the COPOC segment. It is also very likely that hydroxyl groups of apigenin link with polar groups of DPPC by water bridges. Electron and fluorescence microscopic observations revealed changes in the internal membrane organization of the examined cells. In conclusion, the changes of the structural and dynamic properties of membranes can be crucial for processes involving tumor suppression signal transduction pathways and cell cycle regulation.     

1H NMR and NOE studies of the purple acid phosphatases from porcine uterus and bovine spleen.

The diiron active sites of the purple acid phosphatases from porcine uterus (also called uteroferrin, Uf) and bovine spleen (BSPAP) and their complexes with tungstate are compared by 1H NMR and NOE techniques. The paramagnetically shifted features of the 1H NMR spectrum of reduced BSPAP are similar to those of reduced Uf, while the spectra of the tungstate complexes are almost identical. These observations suggest that the two active sites are quite similar, in agreement with the greater than 90% sequence homology found in the two enzymes. Nuclear Overhauser effect (NOE) experiments on the His N-H resonances show that the Fe(III)-His residue is N epsilon-coordinated, while the Fe(II)-His is H delta-coordinated in both enzymes. On the basis of the above NMR and NOE results, our previously proposed model for the dinuclear iron active site of Uf [Scarrow, R. C., Pyrz, J. W., & Que, L., Jr. (1990) J. Am. Chem. Soc. 112, 657-665] is corroborated, refined, and found to represent the diiron center of BSPAP as well.     

Myosin II from rabbit skeletal muscle and Dictyostelium discoideum and its interaction with F-actin studied by (1)H NMR spectroscopy

Mg-F-actin occurs in two conformational states, I and M, where the N-terminal amino acids are either immobile or highly mobile. In the rigor or ADP complex of rabbit myosin S1 with Mg-F-actin the N-terminal acetyl group of actin stays in its highly mobile state. The same is true for the complexes with the myosin motor domain from Dictyostelium discoideum. This excludes a direct strong interaction of the N-terminal amino acids with myosin in the rigor state as suggested. An interaction of the N-terminus of F-actin with myosin is also not promoted by occupying its low-affinity binding site(s) with divalent ions. The N-terminal high-mobility region may be part of a structural system which has evolved for releasing inadequate stress applied to the actin filaments.     

Mestranol: the assignment of its 1H and 13C NMR spectra by means of two-dimensional NMR spectroscopy, and its photochemical decomposition

The 1H- and 13C-nmr spectra of mestranol were assigned with the help of a 2 D-J-resolved, a 2D spin echo J-correlated (SECSY) and a 2D 1H-13C hetero-shift correlation experiment. The analysis of the spectra facilitated the identification of some of the photodecomposition products of mestranol. It was shown that, upon irradiation with UV-B light in water-ethanol (1:1, v/v), products are formed by oxidation of rings B and C of the steroid.     

Backbone dynamics of the antifungal Psd1 pea defensin and its correlation with membrane interaction by NMR spectroscopy

Plant defensins are cysteine-rich cationic peptides, components of the innate immune system. The antifungal sensitivity of certain exemplars was correlated to the level of complex glycosphingolipids in the membrane of fungi strains. Psd1 is a 46 amino acid residue defensin isolated from pea seeds which exhibit antifungal activity. Its structure is characterized by the so-called cysteine-stabilized α/β motif linked by three loops as determined by two-dimensional NMR. In the present work we explored the measurement of heteronuclear Nuclear Overhauser Effects, R1 and R2 ¹⁵N relaxation ratios, and chemical shift to probe the backbone dynamics of Psd1 and its interaction with membrane mimetic systems with phosphatidylcholine (PC) or dodecylphosphocholine (DPC) with glucosylceramide (CMH) isolated from Fusarium solani. The calculated R2 values predicted a slow motion around the highly conserved among Gly12 residue and also in the region of the Turn3 His36-Trp38. The results showed that Psd1 interacts with vesicles of PC or PC:CMH in slightly different forms. The interaction was monitored by chemical shift perturbation and relaxation properties. Using this approach we could map the loops as the binding site of Psd1 with the membrane. The major binding epitope showed conformation exchange properties in the μs-ms timescale supporting the conformation selection as the binding mechanism. Moreover, the peptide corresponding to part of Loop1 (pepLoop1: Gly12 to Ser19) is also able to interact with DPC micelles acquiring a stable structure and in the presence of DPC:CMH the peptide changes to an extended conformation, exhibiting NOE mainly with the carbohydrate and ceramide parts of CMH.     

Examination, by 1H-n.m.r. spectroscopy, of the binding of a synthetic, high-affinity heparin pentasaccharide to human antithrombin III

Binding of a synthetic, high-affinity heparin pentasaccharide and of intact heparin to both native and elastase-modified human antithrombin III have been examined by 1H-n.m.r. spectroscopy. The pentasaccharide perturbs many protein resonances in the same way as does intact heparin. There are, however, differences that seem to arise both from fewer contacts in the heparin binding-site when the pentasaccharide binds and from dissimilar conformational changes in the protein. The resonance of the H-2 atom of the histidine, considered to be the N-terminal residue and to be located in the heparin binding-site, is strongly perturbed by heparin binding both to native and modified antithrombin. The pentasaccharide has little effect on this histidine in either protein. Resonances from two of the remaining four histidine units are sensitive to longer-range conformational changes, and show differences between binding of the two heparin species both in native and modified ATIII. It is concluded that the pentasaccharide only partly fills the heparin binding-site and does not produce a conformational change identical to that caused by intact heparin. This is particularly significant as regards the mechanism of action of heparin, because the synthetic pentasaccharide activates ATIII towards Factor Xa, but not towards thrombin.     

Cloning of the Full-Length cDNA of Porcine Antithrombin III and Comparison with its Human Homolog

The characterization of porcine antithrombin III (ATIII)—a highly powerful anticoagulant—is essential for using porcine liver in xenotransplantation applications. The objective of this study was to clarify the functions of porcine ATIII through comparison with human ATIII. We cloned porcine ATIII and compared its important functional sites with those of human ATIII. The full-length cDNA of porcine ATIII was cloned by screening a porcine liver cDNA library, and the ATIII activities of 23 pigs were determined. The full-length cDNA of porcine ATIII spanned 1498 bp and encoded 463 amino acids. Porcine ATIII shared 87.67% nucleotide identity and 89.06% amino acid identity with human ATIII. Complete identity was found at active center Arg393–Ser394, and remarkably high similarities were found at 2 critical heparin-binding sites (residues 41 through 49 and 114 through 156) and in some key residues involved in heparin binding. An ATIII assay found no significant difference between porcine and human plasma. The high level of similarity between porcine ATIII and human ATIII suggests that porcine ATIII will function in a manner similar to human ATIII in xenotransplantation.Abbreviation: ATIII, antithrombin IIIAntithrombin III (ATIII) is a single-chain glycoprotein found in mammalian plasma that inhibits thrombin and other serine proteinases involved in the blood coagulation cascade, such as factor IX, factor X,²⁰ and plasmin. ATIII is considered the most powerful serine proteinase inhibitor (serpin) and the most important contributor to the anticoagulation system.¹¹ Previous studies have provided detailed knowledge of human ATIII¹ and have identified 2 essential activities as prerequisites for its effective function: (1) recognizing and attacking target proteases and (2) interacting with its cofactor, heparin.²The pig has played an important role in biomedical research,^3,6,15,33 especially as a large animal model for surgical experiments and a promising candidate for xenotransplantation.^9,12Although knowledge of the physiologic features of the porcine coagulation system is important for its successful application, differences (if any) between human and porcine coagulation factors have not been studied in detail.¹⁶ Considering the unknown properties of porcine ATIII when it is secreted into human blood and interacts with human thrombin and heparin after liver or hepatocyte xenotransplantation, there is a clear need for elucidating the properties of porcine ATIII.Treatment with high doses of recombinant human ATIII prevents coagulopathy and protects renal xenografts from early injury in the pig-to-baboon model.⁸ Heparin-dependent inhibition of human factor Xa by porcine arterial endothelial cells is blocked completely by neutralizing ATIII but is unaffected by the antitissue factor pathway inhibitor antibody.¹⁸ These results suggest that human ATIII is an effective anticoagulant in the xenotransplantation model, but porcine tissue factor pathway inhibitor and human Xa are incompatible.¹⁸ However, porcine ATIII has not been evaluated with regard to xenotransplantation models. Consequently, in the present study, we cloned and characterized the full-length cDNA of porcine ATIII. We then compared sequence and function of porcine ATIII cDNA with those of human ATIII to gain insight into the molecular compatibility of porcine and human coagulation-related molecules.     

The three-dimensional structure of bovine calcium ion-bound osteocalcin using 1H NMR spectroscopy

Structural information on osteocalcin or other noncollagenous bone proteins is very limited. We have solved the three-dimensional structure of calcium bound osteocalcin using (1)H 2D NMR techniques and proposed a mechanism for mineral binding. The protons in the 49 amino acid sequence were assigned using standard two-dimensional homonuclear NMR experiments. Distance constraints, dihedral angle constraints, hydrogen bonds, and (1)H and (13)C chemical shifts were all used to calculate a family of 13 structures. The tertiary structure of the protein consisted of an unstructured N terminus and a C-terminal loop (residues 16-49) formed by long-range hydrophobic interactions. Elements of secondary structure within residues 16-49 include type III turns (residues 20-25) and two alpha-helical regions (residues 27-35 and 41-44). The three Gla residues project from the same face of the helical turns and are surface exposed. The genetic algorithm-molecular dynamics simulation approach was used to place three calcium atoms on the NMR-derived structure. One calcium atom was coordinated by three side chain oxygen atoms, two from Asp30, and one from Gla24. The second calcium atom was coordinated to four oxygen atoms, two from the side chain in Gla 24, and two from the side chain of Gla 21. The third calcium atom was coordinated to two oxygen atoms of the side chain of Gla17. The best correlation of the distances between the uncoordinated Gla oxygen atoms is with the intercalcium distance of 9.43 A in hydroxyapatite. The structure may provide further insight into the function of osteocalcin.     

Comparative studies of the interaction of human and bovine platelet factor 4 with heparin using histidine NMR resonances as spectroscopic probes

ThepK _a values of His-38 and His-50 of the heparin-binding protein, bovine platelet factor 4, are 5.6 and 6.5, respectively, as determined by¹H NMR spectroscopy. The¹H NMR resonance of His-38 of bovine platelet factor 4 which exhibits the lowerpK _a value is perturbed upon heparin binding to a greater degree than the resonance of His-50. Human platelet factor 4 contains the homologous residues His-23 and His-35. ThepK _a values of the two histidine residues of human platelet factor 4 are 5.3 and 6.4. The¹H NMR resonance of the histidine of human platelet factor 4 exhibiting the lowerpK _a value also is perturbed upon heparin binding to a greater degree than the histidine resonance exhibiting the higherpK _a , thereby suggesting comparable heparin-protein interactions in bovine and human platelet factor 4.     

Conformational studies of the N-terminal lipid-associating domain of human apolipoprotein C-I by CD and 1H NMR spectroscopy.

A peptide comprising the N-terminal 38 residues of human apolipoprotein C-I (apoC-I(1-38)) was synthesized using solid-phase methods and its solution conformation studied by CD and 1H NMR spectroscopy. The CD data indicate that apoC-I(1-38) has a similar helical content (55%) in the presence of saturating amounts of SDS or egg yolk lysophosphatidylcholine. A structural ensemble of SDS-bound apoC-I(1-38) was calculated from 464 NOE-based distance restraints using distance geometry methods. ApoC-I(1-38) adopts a helical structure between residues V4 and K30 and an extended C-terminus from Q31 when associated with SDS. The region K12-G15 undergoes slow conformational exchange as indicated by above-average amide resonance linewidths, large temperature coefficients, and fast exchange (< 2 h) of backbone amide protons with deuterium. The mobility of K12-G15 is reflected in the poorly defined dihedral angles of K12 and E13 in the calculated ensemble of structures. The average structure of apoC-I(1-38) is curved toward its hydrophobic face with bends of 125 degrees, centered at K12/E13, and 150 degrees, centered at K21. This curvature appears to be driven by the interaction of two hydrophobic clusters, one formed by residues L8, L11, F14, and L18, and the other by L25, I26, and I29, with the amphiphile SDS. Based on our present structural definition of apoC-I(1-38) and the previously obtained structure of the fragment apoC-I(35-53), we propose the secondary structure of intact apolipoprotein C-I.     

The asparagine-linked oligosaccharides on bovine fetuin. Structural analysis of N-glycanase-released oligosaccharides by 500-megahertz 1H NMR spectroscopy

The structures of the entire population of sialylated asparagine-linked oligosaccharides present on bovine fetuin were elucidated. Asparagine-linked oligosaccharides were released from fetuin with N-glycanase, radiolabeled by reduction with NaB[3H]4, and fractionated by anion-exchange high performance liquid chromatography (HPLC), ion-suppression amine adsorption HPLC, and concanavalin A affinity chromatography. The 3H-labeled oligosaccharide fractions obtained were analyzed by 500-MHz 1H nuclear magnetic resonance spectroscopy, revealing the presence of 23 distinct oligosaccharide structures. These oligosaccharides differed in extent of sialylation (3% mono-, 35% di-, 54% tri-, and 8% tetrasialylated), number of peripheral branches (17% di- and 83% tribranched), linkage (alpha 2,3 versus alpha 2,6) and location of sialic acid moieties, and linkage (beta 1,4 versus beta 1,3) of galactose residues. This represents the first time that the asparagine-linked oligosaccharides of fetuin have been successfully fractionated and characterized as sialylated species. The sialylated oligosaccharides derived from fetuin were also used to further define the specificities of the lectins leukoagglutinating phytohemagglutinin and Ricinus communis agglutinin I. The behavior of these oligosaccharides during lectin affinity HPLC further establishes the structural features which predominate in the interaction of oligosaccharides with leukoagglutinating phytohemagglutinin and R. communis agglutinin I.     

Site-specific N-glycosylation of human chorionic gonadotrophin--structural analysis of glycopeptides by one- and two-dimensional 1H NMR spectroscopy.

Glycopeptides representing individual N-glycosylation sites of the heterodimeric glycoprotein hormone human chorionic gonadotrophin (hCG) were obtained from subunits hCG alpha (N-glycosylated at Asn-52 and Asn-78) and hCG beta (N-glycosylated at Asn-13 and Asn-30) by digestion with trypsin and chymotrypsin, respectively. Following purification by reverse-phase HPLC and identification by amino acid sequencing, the glycopeptides were analysed by one- and two-dimensional 1H NMR spectroscopy. The results are summarized as follows: (i) oligosaccharides attached to Asn-52 of hCG alpha comprised monosialylated 'monoantenary' NeuAc alpha 2-3Gal beta 1-4GlcNAc beta 1-2Man alpha 1-3[Man alpha 1-6]Man beta 1-4GlcNAc beta 1-4GlcNAc (N1-4'), disialylated diantennary NeuAc alpha 2-3Gal beta 1-4GlcNAc beta 1-2Man alpha 1-3[NeuAc alpha 2-3-Gal beta 1-4GlcNAc beta 1-2Man alpha 1-6]Man beta 1-4GlcNAc beta 1-4GlcNAc (N2), and the monosialylated hybrid-type structures NeuAc alpha 2-3Gal beta 1-4GlcNAc beta 1-2Man alpha 1-3[Man alpha 1-3Man alpha 1-6]Man beta 1-4GlcNAc beta 1-4GlcNAc (N1-A) and NeuAc alpha 2-3Gal-beta 1-4GlcNAc beta 1-2Man alpha 1-3[Man alpha 1-3(Man alpha 1-6)Man alpha 1-6]Man beta 1-4GlcNAc beta 1-4GlcNAc (N1-AB) in a ratio approaching 5:2:2:1; (ii) Asn-78 of hCG alpha carried N2 and N1-4' almost exclusively (ratio approximately 3:2); (iii) both N-glycosylation sites of hCG beta contained predominantly component N2, partially (approximately 25%) and completely alpha 1-6-fucosylated at the N-acetylglucosamine linked to Asn-13 and Asn-30, respectively. The distinct site-specific distribution of the oligosaccharide structures among individual N-glycosylation sites of hCG appears to reflect primarily the influence of the surrounding protein structure on the substrate accessibility of the Golgi processing enzymes alpha-mannosidase II, GlcNAc transferase II and alpha 1,6-fucosyltransferase.     

Sequence-specific interaction of Hoechst 33258 with the minor groove of an adenine-tract DNA duplex studied in solution by 1H NMR spectroscopy.

The interaction of Hoechst 33258 with the minor groove of the adenine-tract DNA duplex d(CTTTTGCAAAAG)2 has been studied in both D2O and H2O solutions by 1D and 2D 1H NMR spectroscopy. Thirty-one nuclear Overhauser effects between drug and nucleotide protons within the minor groove of the duplex, together with ring-current induced perturbations to the chemical shifts of basepair and deoxyribose protons, define the position and orientation of the bound dye molecules. Two drug molecules bind cooperatively and in symmetry related orientations at the centre of the 5'-TTTT and 5'-AAAA sequences with the binding interactions spanning only the four A-T basepairs. The positively charged N-methylpiperazine moieties point towards the centre of the duplex while the phenol groups are disposed towards the 3'-ends of the sequence. Resonance averaging is apparent for both the D2/D6 and D3/D5 phenol protons and D2"'/D6"' and D3"'/D5"' of the N-methylpiperazine ring and is consistent with these groups being involved in rapid rotation or ring-flipping motions in the bound state. Interstrand NOEs between adenine H2s and deoxyribose H1' are consistent with a high degree of propeller twisting of the A-T basepairs at the binding site of the aromatic benzimidazole and phenol rings of Hoechst. The data imply that the minor groove is particularly narrow with many contacts between the complementary curved surfaces of the drug and DNA indicating that strong van der Waals interactions, involving the floor and the walls of the minor groove, stabilize the complex. In our model the NH groups of the benzimidazole rings are positioned to make a pair of bifurcated hydrogen bonds with the adenine N3 and thymine O2 on the floor of the minor groove.     

Desulfation of sulfated galactans with chlorotrimethylsilane. Characterization of beta-carrageenan by 1H NMR spectroscopy

A desulfation method using chlorotrimethylsilane for treatment of pyridinium salts of sulfated galactans was developed. It proved to be appropriate for desulfation of polysaccharides of both agar and carrageenan families. In order to evaluate its efficiency in presence of the maximum content of 3,6-anhydrogalactose, it was applied to commercial kappa-carrageenan, leading to obtention of a product mainly composed by beta-carrageenan. Best experimental conditions for achieving desulfation of kappa-carrageenan--in terms of low sulfate content, high recovery and low degradation of the product--were found. In addition, the complete assignment of the 1H NMR spectrum of beta-carrageenan was achieved by means of 1D and 2D NMR techniques.