Novel protocols for acquiring quantitative (13)C NMR spectra of lignins have been developed using the internal reference compounds 1,3,5-trioxane and pentafluorobenzene. Trioxane offers a convenient internal standard for collecting inverse gated proton decoupled (13)C NMR spectra for lignins, whereas pentafluorobenzene can be used to provide information on the amount of methine carbon using the DEPT experiment. In each case, the internal reference compounds provide single, un-overlapped sharp signals in the middle of the spectral region, permitting facile integration. These integrals could be used to determine the amounts of different structural features of lignins, expressed in absolute units of millimoles per gram. The optimum parameters for these experiments were validated for a variety of spectrometer platforms, and standard errors were determined for different spectral areas using lignin model compounds and "standard" lignins. In addition, the data derived for the International Round Robin "standard" lignins showed good agreement with the data from quantitative (31)P NMR spectroscopy and published data, obtained by independent laboratories using independent methods of analysis.
Comprehensive 1H NMR assignments of the heme cavity proton resonances of sperm whale
metmyoglobin cyanide have provided the dipolar shifts for nonligated residues which, together with the crystal
coordinates of carbonyl myoglobin, allow accurate determination of both the anisotropies and orientation of
the paramagnetic susceptibility tensor, χ, in the molecular framework. The resulting axial, Δχax = 2.48 ×
10-8 m3/mol, and rhombic anisotropy, Δχrh = −0.58 × 10-8 m3/mol, values at 25 °C determined from the
most complete set of dipolar shifts are determined to 2% and 6% uncertainty, respectively, and agree well
with theoretical estimates (Horrocks, W. D., Jr. and Greenberg, E. S. Mol. Phys. 1974, 27, 993−999).
Numerically and spatially restricted input data sets lead to larger uncertainties in Δχax and Δχrh, but do not
systematically bias the orientation of the tensor. Determination of the anisotropies and orientation over the
temperature range 5−50 °C shows that the susceptibility tensor orientation is minimally influenced, with both
anisotropies well-behaved, and with Δχax exhibiting a temperature behavior close to that predicted for the
system. The quantitative determination of the magnetic anisotropies over temperature allows the quantitative
separation of contact and dipolar shifts for the iron ligands. The heme contact shifts reflect the expected dominant
π spin density at pyrrole positions, but the meso-protons exhibit low-field contact shifts indicative of unpaired
spin in a σ orbital. Such delocalized σ spin density could arise from either deformation of the heme from
planarity or the loss of σ/π separation for the d
xz
, d
yz
orbitals when the major magnetic axis is tilted strongly
from the heme normal as is experimentally observed. The observed anomalous temperature dependencies of
the heme methyl and axial His ring contact shifts, as well as that of the rhombic anisotropy, are all consistent
with thermal population of the excited orbital state. The limitations for quanitatively determining the excited
orbital state energy separation from the available NMR data are discussed.
Organic molecular hole-transport materials (HTMs) are appealing for the scalable manufacture of perovskite solar cells (PSCs) because they are easier to reproducibly prepare in high purity than polymeric and inorganic HTMs.
Shear rate can affect protein adsorption and platelet aggregation by regulating both the collision frequency and the capture efficiency (alpha). These effects were evaluated in well defined shear field in a micro-couette for shear rate G = 10 - 1000 s-1. The rate of protein binding was independent of G, shown for adsorption of albumin to latex beads and PAC1 to activated platelets. The initial aggregation rate for ADP-activated platelets in citrated platelet-rich plasma followed second order kinetics at the initial platelet concentrations between 20,000 and 60,000/microliters. alpha values, which dropped nearly fivefold for a 10-fold increase in G, were approximately proportional to G-1, contrary to a minor drop predicted by the theory that includes protein cross-bridging. Varying ADP concentration did not change alpha of maximally activated platelet subpopulations, suggesting that aggregation between unactivated and activated platelets is negligible. Directly blocking the unoccupied but activated GPIIb-IIIa receptors without affecting pre-bound Fg on "RGD"-activated, fixed platelets (AFP) by GRGDSP or Ro 43-5054 eliminated aggregation, suggesting that cross-bridging of GPIIb-IIIa on adjacent platelets by fibrinogen mediates aggregation. Alpha for AFP remained maximal (approximately 0.24) over 25-75% Fg occupancy, otherwise decreasing rapidly, with a half-maximum occurring at around 2% occupancy, suggesting that very few bound Fg were required to cause significant aggregation.
Dynamic and quantitative studies of the binding of fibrinogen (Fg) to its receptor, GPIIb-IIIa, on activated platelets, leading to platelet aggregation, are best studied with fluorescently-labelled Fg by flow cytometry. Due to conflicting reports on the functionality of FITC-labelled Fg, we have developed a reproducible and 'mild' labelling of fibrinogen with FITC-celite at pH 7.4-8.5 for direct and dynamic studies of specific Fg binding to activated platelets evaluated for native platelet-rich plasma, for washed platelets, and for activated, fixed platelets. We have demonstrated the equivalence of FITC-labelled and unlabelled Fg for binding to activated GPIIb-IIIa receptors, and in the rate and extent of mediating platelet aggregation. We found that FITC-Fg labelled at pH > or = 9 had reduced to absent specific binding to activated platelets, whether using soluble FITC or FITC-celite. The FITC-labelled Fg must be diluted 3-fold with unlabelled Fg when evaluating maximal Fg binding to activated platelets in order to prevent autoquenching of the FITC-Fg which leads to underestimation of Fg levels. The dissociation constant (KD) of Fg on stable preparations of activated, fixed platelets, determined with FITC-Fg binding to platelets by flow cytometry, was in the range reported for 125I-labelled Fg, 70-255 nm with Bmax = 10000-25000 Fg per platelet (n = 20). The FITC-Fg was used to monitor Fg binding to activated platelets directly by plasma, as well as to evaluate platelet subpopulations which maximally bind Fg according to the concentration of ADP used as activator. It is expected that this 'mildly' labelled FITC-Fg will stimulate further studies of platelet activation directly in native anticoagulated blood/plasma, for both basic and clinical research.
To define the abundance and comprehend the reactivity of dibenzodioxocins in lignin, model compound studies, specific degradation experiments on milled wood lignin, and molecular modeling calculations have been performed. Quantitative (31)P NMR measurements of the increase of biphenolic hydroxyl groups formed after a series of alkaline degradations in the presence of hydrosulfide anions (kraft conditions) showed the presence of 3.7 dibenzodioxocin rings/100 C9 units in milled wood lignin. The DFRC degradation protocol (Derivatization Followed by Reductive Cleavage) was chosen as an independent means to estimate their abundance. Initial experiments with a dibenzodioxocin model compound, trans-6,7-dihydro-7-(4-hydroxy-3-methoxyphenyl)-4,9-dimethoxy-2,11-dipropyldibenzo[e,g][1,4]dioxocin-6-ylmethanol, showed that it is not cleaved under DFRC conditions, but rather it isomerizes into a cyclic oxepine structure. Steric effects precluded this isomerization from occurring when DFRC was applied to milled wood lignin. Instead, monoacetylated biphenolic moieties were released and quantified by (31)P NMR, at 4.3 dibenzodioxocin rings/100 C9 units. The dibenzodioxocin content in residual lignins isolated from kraft pulps delignified to various degrees showed that during pulp delignification, the initial rate of dibenzodioxocin removal was considerably greater than the cleavage rate of arylglycerol-beta-aryl ether bonds. The activation energy for the degradation of dibenzodioxocins under kraft conditions in milled wood lignin was 96 +/- 9 kJ/mol, similar to that of arylglycerol-beta-aryl ether bond cleavage.
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