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Several oxygen- 17-enriched silicates were studied using dynamic-angle spinning (DAS) NMR spectroscopy at two magnetic field strengths. The DAS method averages wnd-order quadrupolar interactions by reorienting a sample about a hedependent axis, thereby yielding high-resolution spectra for half-odd integer spin quadrupolar nuclei such as oxygen-17. A narrow spectral line is observed for each distinct oxygen site in a powdered sample at the sum of the isotropic chemical shift and the field-dependent isotropic second-order quadrupolar shift. Using equations for the total shift observed at two field strengths, the chemical shift is uniquely determined together with a product of the quadrupolar coupling constant (CQ = $qQ/h) and the quadrupolar asymmetry parameter (7). For one silicate, we demonstrate a computer program that uses the isotropic shifts and quadrupolar products as constraints and provides simulations of overlapped magic-angle spinning line shapes. In this way the quadrupolar parameters, CQ and q, are determined separately for each crystallographic site. The silicates studied include the discrete orthosilicates lamite (Ca2Si04) and forsterite (Mg2Si04), as well as diopside (CaMgSi206), wollastonite (CaSi03), and clinocnstatite ( MgSi03), which are minerals composed of chains of silicon-oxygen tetrahedra.

The complete primary structures of the major Asn-linked oligosaccharides from the type II variant surface glycoproteins (VSGs), MITat 1.2 and MITat 1.7, and the type III VSG, MITat 1.5, were determined using a combination of exo- and endoglycosidase digestions, methylation analysis, acetolysis, and 500 MHz 1H NMR spectroscopy. Each variant contained classical branched oligomannose-type and biantennary complex oligosaccharides, a proportion of the latter substituted with terminal alpha(1-3)-linked galactose residues, the first report of the presence of this epitope in Trypanosoma brucei. In addition both the type II variants contained relatively large amounts of the unusual small oligomannose-type oligosaccharides, Man4GlcNAc2 and Man3GlcNAc2, and a diverse array of novel branched poly-N-acetyllactosamine oligosaccharides, similar but not identical to those from mammalian glycoproteins. These latter structures were also partially substituted with terminal alpha(1-3)-linked galactose residues. Glycosylation in the type II variants showed site specificity in that the poly-N-acetyllactosamine and Man(9-5)GlcNAc2 oligosaccharides were located exclusively at Asn-glycosylation site 1 very close to the C terminus, whereas the Man(4-3)GlcNAc2 and biantennary complex oligosaccharides were located exclusively at site 2. This is the first report of the presence of poly-N-acetyllactosamine oligosaccharides in protozoa.

The oligomannose series of oligosaccharides from bovine thyroglobulin (BTG) and the variant surface glycoprotein (VSG) of Trypanosoma brucei have been isolated and sequenced by 1H NMR. The structure of Man9GlcNAc2, the parent molecule of the series, is shown below. Structural isomerism occurs within this series through the removal of residues D1, D2, D3, and C. Using spin-spin coupling and chemical shift data the rotamer distributions about the dihedral angle omega for the Man alpha 1-6Man beta and Man alpha 1-6Man alpha linkages were determined for each member of the series. It is shown that the dihedral angle omega of the Man alpha 1-6Man beta linkage exhibits low flexibility with a preference for the omega = 180 degrees conformation when residue D2 is present and high flexibility when this residue is absent. Flexibility of omega for the Man alpha 1-6Man alpha is largely independent of primary sequence and is intermediate between the two Man alpha 1-6Man beta extremes, again with a preference for the omega = 180 degrees conformation. [see text] There are, however, data which indicate that removal of residue D3 may confer additional flexibility upon the dihedral angle omega of the Man alpha 1-6Man alpha linkage. Molecular graphics modelling, together with chemical and enzymatic modification studies, suggest that the origin of the observed primary sequence dependence of the Man alpha 1-6Man beta linkage arises from steric factors. On the basis of these observations taken together with previous work, it is postulated that recognition of individual oligomannose conformations may play a role in the control of N-linked oligosaccharide biosynthesis.

The complete primary structures of the major Asn-linked oligosaccharides from the type II variant surface glycoproteins (VSGs), MITat 1.2 and MITat 1.7, and the type III VSG, MITat 1.5, were determined using a combination of exo- and endoglycosidase digestions, methylation analysis, acetolysis, and 500 MHz 1H NMR spectroscopy. Each variant contained classical branched oligomannose-type and biantennary complex oligosaccharides, a proportion of the latter substituted with terminal alpha(1-3)-linked galactose residues, the first report of the presence of this epitope in Trypanosoma brucei. In addition both the type II variants contained relatively large amounts of the unusual small oligomannose-type oligosaccharides, Man4GlcNAc2 and Man3GlcNAc2, and a diverse array of novel branched poly-N-acetyllactosamine oligosaccharides, similar but not identical to those from mammalian glycoproteins. These latter structures were also partially substituted with terminal alpha(1-3)-linked galactose residues. Glycosylation in the type II variants showed site specificity in that the poly-N-acetyllactosamine and Man(9-5)GlcNAc2 oligosaccharides were located exclusively at Asn-glycosylation site 1 very close to the C terminus, whereas the Man(4-3)GlcNAc2 and biantennary complex oligosaccharides were located exclusively at site 2. This is the first report of the presence of poly-N-acetyllactosamine oligosaccharides in protozoa.

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.

NMR imaging has been applied to elastomeric materials of industrial and military interest The T2 spin-spin relaxation times of common elastomers, particularly after filling and curing, are sufficiently short that spin-echo sequences at submillisecond echo times cannot produce T2-independent images. The sensitivity to T2 makes spin echo imaging a good probe of elastomer blend composition, as demonstrated for a series of filled, cured cis-polybutadiene, styrene-butadiene rubber blends. The technique can distinguish good and bad carbon black dispersion in actual tire tread samples. The configuration of non- metallic tire cord, voids, rubber layer boundaries, apparent migration of additives, and other inhomogeneities can be detected in end-product tire samples. Arrowhead patterns, arising from magnetic susceptibility differences for defects in carbon-black-filled elastomers, were attributed to graphitized 'coke' particles from the carbon black. NMR images were obtained for porous glass disks of different porosities as models of materials such as oil cores. The mottled appearance often seen for such images is attributed largely to insufficient signal-to-noise ratio, and not pore structures. Comparison of spin- echo and gradient-echo images demonstrates the defect-magnification effect of the gradient-echo sequence seen previously for elastomers. The advantages of volume imaging, isotropic voxels in thin slices, and higher resolution are demonstrated for porous materials. defects, composites, spin echo, lithium-7, fluorine-19, carbon nuclear magnetic resonance, imaging, elastomers, tires, black, Interfaces, curing, filler, NMR imaging, relaxation coke.

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.