NMR methods to monitor the enzymatic depolymerization of heparin

Heparin and the related glycosaminoglycan, heparan sulfate, are polydisperse linear polysaccharides that mediate numerous biological processes due to their interaction with proteins. Because of the structural complexity and heterogeneity of heparin and heparan sulfate, digestion to produce smaller oligosaccharides is commonly performed prior to separation and analysis. Current techniques used to monitor the extent of heparin depolymerization include UV absorption to follow product formation and size exclusion or strong anion exchange chromatography to monitor the size distribution of the components in the digest solution. In this study, we used 1H nuclear magnetic resonance (NMR) survey spectra and NMR diffusion experiments in conjunction with UV absorption measurements to monitor heparin depolymerization using the enzyme heparinase I. Diffusion NMR does not require the physical separation of the components in the reaction mixture and instead can be used to monitor the reaction solution directly in the NMR tube. Using diffusion NMR, the enzymatic reaction can be stopped at the desired time point, maximizing the abundance of larger oligosaccharides for protein-binding studies or completion of the reaction if the goal of the study is exhaustive digestion for characterization of the disaccharide composition. In this study, porcine intestinal mucosa heparin was depolymerized using the enzyme heparinase I. The unsaturated bond formed by enzymatic cleavage serves as a UV chromophore that can be used to monitor the progress of the depolymerization and for the detection and quantification of oligosaccharides in subsequent separations. The double bond also introduces a unique multiplet with peaks at 5.973, 5.981, 5.990, and 5.998 ppm in the 1H-NMR spectrum downfield of the anomeric region. This multiplet is produced by the proton of the C-4 double bond of the non-reducing end uronic acid at the cleavage site. Changes in this resonance were used to monitor the progression of the enzymatic digestion and compared to the profile obtained from UV absorbance measurements. In addition, in situ NMR diffusion measurements were explored for their ability to profile the different-sized components generated over the course of the digestion

Actinide covalency measured by pulsed electron paramagnetic resonance spectroscopy

Our knowledge of actinide chemical bonds lags far behind our understanding of the bonding regimes of any other series of elements. This is a major issue given the technological as well as fundamental importance of f-block elements. Some key chemical differences between actinides and lanthanides—and between different actinides—can be ascribed to minor differences in covalency, that is, the degree to which electrons are shared between the f-block element and coordinated ligands. Yet there are almost no direct measures of such covalency for actinides. Here we report the first pulsed electron paramagnetic resonance spectra of actinide compounds. We apply the hyperfine sublevel correlation technique to quantify the electron-spin density at ligand nuclei (via the weak hyperfine interactions) in molecular thorium(III) and uranium(III) species and therefore the extent of covalency. Such information will be important in developing our understanding of the chemical bonding, and therefore the reactivity, of actinides

Detection of the 1H and 15N NMR resonances of sulfamate groups in aqueous solution: A new tool for heparin and heparan sulfate characterization

Sulfamate (NHSO3-) groups are critically important structural elements of the glycosaminoglycans heparin and heparan sulfate (HS). Experimental conditions are presented for detection of the sulfamate 1H NMR resonances in aqueous solution. NMR spectra reported for N-sulfoglucosamine (GlcNS) and the synthetic pentasaccharide drug fondaparinux demonstrate the broad utility of the sulfamate group 1H chemical shifts to reflect differences in molecular structure. The sulfamate protons also provide an efficient route for detection of 15N chemical shifts through proton-nitrogen correlations measured with the heteronuclear single quantum coherence (HSQC) experiment. The HSQC spectra of GlcNS, fondaparinux, and the low-molecular weight heparin enoxaparin illustrate the power of the 1H and 15N chemical shifts of the sulfamate NH groups for the structural characterization of heparin and HS. © 2011 American Chemical Society

Getting to know the nitrogen next door: HNMBC measurements of amino sugars

Long-range 1H-15N correlations detected by the heteronuclear multiple-bond correlation (HMBC) experiment are explored for the characterization of amino sugars. The gradient-enhanced HMBC, IMPACT-HMBC, and a modified pulse sequence with the 1J-filters removed, IMPACT-HNMBC, are compared for sensitivity and resolution. 15N chemical shifts and long-range proton correlations are reported using the IMPACT-HNMBC experiment for N-acetyl-glucosamine, N-acetyl-galactosamine, and for a series of glucosamine analogs with an N-sulfo substitution, unmodified amino group, and 6-O-sulfonation. As is common with sugars, for all the compounds examined both anomeric forms are present in solution. For each compound studied, the 15N chemical shifts of the α anomer are downfield of the β form. For the N-acetylated sugars, the β anomer has a unique long-range 15N correlation to the anomeric proton not observed for the α anomer. Though N-sulfonation results in a significant change in the 15N chemical shift of the glucosamine analogs, 6-O sulfo substitution has no significant effect on the local environment of the amino nitrogen. For N-acetylated sugars in D2O solution, peaks in the 15N projection of the HMBC spectrum appear as triplets as a result of J-modulation due to 2H-15N coupling. © 2011 Published by Elsevier Inc

Heparin characterization:challenges and solutions

Although heparin is an important and widely prescribed pharmaceutical anticoagulant, its high degree of sequence microheterogeneity and size polydispersity make molecular-level characterization challenging. Unlike nucleic acids and proteins that are biosynthesized through template-driven assembly processes, heparin and the related glycosaminoglycan heparan sulfate are actively remodeled during biosynthesis through a series of enzymatic reactions that lead to variable levels of O- and N-sulfonation and uronic acid epimers. As summarized in this review, heparin sequence information is determined through a bottom-up approach that relies on depolymerization reactions, size- and charge-based separations, and sensitive mass spectrometric and nuclear magnetic resonance experiments to determine the structural identity of component oligosaccharides. The structure-elucidation process, along with its challenges and opportunities for future analytical improvements, is reviewed and illustrated for a heparin-derived hexasaccharide. </jats:p

The efficient structure elucidation of minor components in heparin digests using microcoil NMR

The structural complexity and microheterogeneity of the glycosaminoglycans heparin and heparan sulfate make their characterization a daunting task. The methodology described herein utilizes a combination of enzymatic digestion, size-exclusion chromatography, strong anion-exchange HPLC, reverse-phase ion-pair ultrahigh performance liquid chromatography-mass spectrometry, and microcoil NMR for the efficient sequencing of heparin-derived tetrasaccharides. The high mass sensitivity of microcoil NMR makes this technique well suited for the characterization of mass-limited samples removing a bottleneck in the analysis workflow and permitting structural characterization of minor components isolated from a heparin enzymatic digestion. Complete characterization of one tetrasulfonated, five pentasulfonated isomers and two hexasulfonated tetrasaccharide sequences is described. To our knowledge, two of the identified minor tetrasaccharides are unique, and have not been previously reported: IdoA(2S)-GlcNS(6S)-IdoA(2S)-GlcNS(6S) and ΔUA(2S)-GlcNS(6S)-IdoA-GlcNS(6S) . © 2011 Elsevier Ltd. All rights reserved

Assessment of Barotrauma from Rapid Decompression of Depth-Acclimated Juvenile Chinook Salmon Bearing Radiotelemetry Transmitters

This study investigated the mortality of and injury to juvenile Chinook salmon Oncorhynchus tshawytscha exposed to simulated pressure changes associated with passage through a large Kaplan hydropower turbine. Mortality and injury varied depending on whether a fish was carrying a transmitter, the method of transmitter implantation, the depth of acclimation, and the size of the fish. Juvenile Chinook salmon implanted with radio transmitters were more likely than those without to die or sustain injuries during simulated turbine passage. Gastric transmitter implantation resulted in higher rates of injury and mortality than surgical implantation. Mortality and injury increased with increasing pressure of acclimation. Injuries were more common in subyearling fish than in yearling fish. Gas emboli in the gills and internal hemorrhaging were the major causes of mortality. Rupture of the swim bladder and emphysema in the fins were also common. This research makes clear that the exposure of juvenile Chinook salmon bearing radiotelemetry transmitters to simulated turbine pressures with a nadir of 8-19 kPa can result in barotrauma, leading to immediate or delayed mortality. The study also identified sublethal barotrauma injuries that may increase susceptibility to predation. These findings have significant implications for many studies that use telemetry devices to estimate the survival and behavior of juvenile salmon as they pass through large Kaplan turbines typical of those within the Columbia River hydropower system. Our results indicate that estimates of turbine passage survival for juvenile Chinook salmon obtained with radiotelemetry devices may be negatively biase