pH Dependence Of Chemical Shifts Research Articles

Extracellular pH Mapping as Therapeutic Readout of Drug Delivery in Glioblastoma.

An acidic extracellular space is a hallmark of the tumor microenvironment. Acidosis has been postulated to promote the aggressive and invasive characteristics of tumors and also inhibit the therapeutic response, particularly in the context of novel immunotherapies. Therefore, methods to quantitatively measure the extracellular pH (pHe) are needed. Here we describe a magnetic resonance spectroscopic imaging (MRSI) technique termed Biosensor Imaging of Redundant Deviation in Shifts (BIRDS), which uses the pH-dependent chemical shifts of nonexchangeable protons of lanthanide-based contrast agents to generate quantitative spatial pHe maps. We assess this method in the context of evaluating the acidic pHe and therapeutic response in glioblastoma in rodents, where normalization of the pHe upon therapy can serve as a quantitative readout of successful drug delivery to the tumor.

Quantum mechanical reference spectrum simulation for precursors and degradation products of chemicals relevant to the Chemical Weapons Convention.

A selection of acidic, alkaline and neutral degradation products relevant to the Chemical Weapons Convention was studied in wide range of pH conditions to determine their spin systems as well as spectral parameters. The pH dependence of chemical shifts and J couplings was parameterized using Henderson-Hasselbalch-based functions using dichloromethane as additional shift reference in TSP-d4 referenced spectra. The resulting parameters allowed calculation of precise chemical shifts and J coupling constants in arbitrary pH conditions. The validity of the obtained spin system definitions and parameters as a source of quantum mechanically simulated reference data in chemical verification analysis is demonstrated.

Volumetric mapping of intra- and extracellular pH in the human brain using 31 P MRSI at 7T.

In vivo 31 P MRSI enables noninvasive mapping of absolute pH values via the pH-dependent chemical shifts of inorganic phosphates (Pi ). A particular challenge is the quantification of extracellular Pi with low SNR in vivo. The purpose of this study was to demonstrate feasibility of assessing both intra- and extracellular pH across the whole human brain via volumetric 31 P MRSI at 7T. 3D 31 P MRSI data sets of the brain were acquired from three healthy volunteers and three glioma patients. Low-rank denoising was applied to enhance the SNR of 31 P MRSI data sets that enables detection of extracellular Pi at high spatial resolutions. A robust two-compartment quantification model for intra- and extracellular Pi signals was implemented. In particular low-rank denoising enabled volumetric mapping of intra- and extracellular pH in the human brain with voxel sizes of 5.7 mL. The average intra- and extracellular pH measured in white matter of healthy volunteers were 7.00 ± 0.00 and 7.33 ± 0.03, respectively. In tumor tissue of glioma patients, both the average intra- and extracellular pH increased to 7.12 ± 0.01 and 7.44 ± 0.01, respectively, compared to normal appearing tissue. Mapping of pH values via 31 P MRSI at 7T using the proposed two-compartment quantification model improves reliability of pH values obtained in vivo, and has the potential to provide novel insights into the pH heterogeneity of various tissues.

Nontarget analysis of Murchison soluble organic matter by high-field NMR spectroscopy and FTICR mass spectrometry.

High-field NMR spectra of Murchison meteorite methanolic extracts revealed primarily aliphatic extraterrestrial organic matter (EOM) with near statistical branching of commonly C(3-5) units separated by heteroatoms and aromatic units. The ratios of CCH, OCH and C(sp2)H units were 89 : 8 : 3, whereas carbon-based aliphatic chain termination was in the order methyl > -COOH > -CH(CH3)COOH. Aliphatic methine carbon was abundant, but its weak NMR signatures were primarily deduced from JRES (J-resolved) NMR spectra. Carbon NMR spectra were dominated by methylene and methyl carbon; strong apodization revealed methine carbon, of which about 20% was aromatic. Extrapolation provided 5-7% aromatic carbon present in Murchison soluble EOM. Compositional heterogeneity in Murchison methanolic extracts was visible in NMR and Fourier transform ion cyclotron (FTICR) mass spectra obtained from a few cubic millimeters of solid Murchison meteorite; increasing sample size enhanced uniformity of NMR spectra. Intrinsic chemical diversity and pH-dependent chemical shift variance contributed to the disparity of NMR spectra. FTICR mass spectra provided distinct clustering of CHO/CHOS and CHNO/CHNOS molecular series and confirmed the prevalence of aliphatic/alicyclic (73%) over single aromatic (21%) and polyaromatic (6%) molecular compositions, suggesting extensive aliphatic substitution of aromatic units as proposed by NMR. Murchison soluble EOM molecules feature a center with enhanced aromatic and heteroatom content, which provides rather diffuse and weak NMR signatures resulting from a huge overall chemical diversity. The periphery of Murchison EOM molecules comprises flexible branched aliphatic chains and aliphatic carboxylic acids. These project on narrow ranges of chemical shift, facilitating observation in one-dimensional and two-dimensional NMR spectra. The conformational entropy provided by these flexible surface moieties facilitates the solubility of EOM.

PH imaging of mouse kidneys in vivo using a frequency-dependent paraCEST agent.

This study explored the feasibility of using a pH responsive paramagnetic chemical exchange saturation transfer (paraCEST) agent to image the pH gradient in kidneys of healthy mice. CEST signals were acquired on an Agilent 9.4 Tesla small animal MRI system using a steady-state gradient echo pulse sequence after a bolus injection of agent. The magnetic field inhomogeneity across each kidney was corrected using the WASSR method and pH maps were calculated by measuring the frequency of water exchange signal arising from the agent. Dynamic CEST studies demonstrated that the agent was readily detectable in kidneys only between 4 to 12 min postinjection. The CEST images showed a higher signal intensity in the pelvis and calyx regions and lower signal intensity in the medulla and cortex regions. The pH maps reflected tissue pH values spanning from 6.0 to 7.5 in kidneys of healthy mice. This study demonstrated that pH maps of the kidney can be imaged in vivo by measuring the pH-dependent chemical shift of a single water exchange CEST peak without prior knowledge of the agent concentration in vivo. The results demonstrate the potential of using a simple frequency-dependent paraCEST agent for mapping tissue pH in vivo. Magn Reson Med 75:2432-2441, 2016. © 2015 Wiley Periodicals, Inc.

PH-dependent random coil 1H, 13C, and 15N chemical shifts of the ionizable amino acids: a guide for protein pK a measurements

The pK a values and charge states of ionizable residues in polypeptides and proteins are frequently determined via NMR-monitored pH titrations. To aid the interpretation of the resulting titration data, we have measured the pH-dependent chemical shifts of nearly all the (1)H, (13)C, and (15)N nuclei in the seven common ionizable amino acids (X=Asp, Glu, His, Cys, Tyr, Lys, and Arg) within the context of a blocked tripeptide, acetyl-Gly-X-Gly-amide. Alanine amide and N-acetyl alanine were used as models of the N- and C-termini, respectively. Together, this study provides an essentially complete set of pH-dependent intra-residue and nearest-neighbor reference chemical shifts to help guide protein pK a measurements. These data should also facilitate pH-dependent corrections in algorithms used to predict the chemical shifts of random coil polypeptides. In parallel, deuterium isotope shifts for the side chain (15)N nuclei of His, Lys, and Arg in their positively-charged and neutral states were also measured. Along with previously published results for Asp, Glu, Cys, and Tyr, these deuterium isotope shifts can provide complementary experimental evidence for defining the ionization states of protein residues.

Determination of pK a of N-alkyl-N,N-dimethylamine-N-oxides using 1H NMR and 13C NMR spectroscopy

AbstractThe pK a values of N-alkyl-N,N-dimethylamine-N-oxides were determined from the pH dependences of chemical shifts in 1H NMR and 13C NMR spectra. The dependences were measured at concentrations below and above the critical micelle concentration of the amine oxides. Above the critical micelle concentration, the plots of the peak positions vs. pH were either sigmoid (the groups close to the nitrogen) or “peak type” (farther groups). The sigmoidal behaviour was a direct result of the acid-base reaction; on the other hand, the “peak type” behaviour was probably an indirect consequence of the pH variation, mediated with the change in micelle size.

Combining Hydrophilic Interaction Chromatography (HILIC) and Isotope Tagging for Off-Line LC-NMR Applications in Metabolite Analysis.

The complementary use of liquid chromatography (LC) and nuclear magnetic resonance (NMR) has shown high utility in a variety of fields. While the significant benefit of spectral simplification can be achieved for the analysis of complex samples, other limitations remain. For example, 1H LC-NMR suffers from pH dependent chemical shift variations, especially during urine analysis, owing to the high physiological variation of urine pH. Additionally, large solvent signals from the mobile phase in LC can obscure lower intensity signals and severely limit the number of metabolites detected. These limitations, along with sample dilution, hinder the ability to make reliable chemical shift assignments. Recently, stable isotopic labeling has been used to detect quantitatively specific classes of metabolites of interest in biofluids. Here we present a strategy that explores the combined use of two-dimensional hydrophilic interaction chromatography (HILIC) and isotope tagged NMR for the unambiguous identification of carboxyl containing metabolites present in human urine. The ability to separate structurally related compounds chromatographically, in off-line mode, followed by detection using 1H-15N 2D HSQC (two-dimensional heteronuclear single quantum coherence) spectroscopy, resulted in the assignment of low concentration carboxyl-containing metabolites from a library of isotope labeled compounds. The quantitative nature of this strategy is also demonstrated.

Dissecting electrostatic interactions in Bacillus circulans xylanase through NMR-monitored pH titrations

NMR-monitored pH titration curves of proteins provide a rich source of structural and electrostatic information. Although relatively straightforward to measure, interpreting pH-dependent chemical shift changes to obtain site-specific acid dissociation constants (pK (A) values) is challenging. In order to analyze the biphasic titrations exhibited by the side chain (13)C(γ) nuclei of the nucleophilic Glu78 and general acid/base Glu172 in Bacillus circulans xylanase, we have revisited the formalism for the ionization equilibria of two coupled acidic residues. In general, fitting NMR-monitored pH titration curves for such a system will only yield the two macroscopic pK (A) values that reflect the combined effects of both deprotonation reactions. However, through the use of mutations complemented with ionic strength-dependent measurements, we are able to extract the four microscopic pK (Ai) values governing the branched acid/base equilibria of Glu78 and Glu172 in BcX. These data, confirmed through theoretical calculations, help explain the pH-dependent mechanism of this model GH11 xylanase by demonstrating that the kinetically determined pK (A) values and hence catalytic roles of these two residues result from their electrostatic coupling.

Remeasuring HEWL pKa values by NMR spectroscopy: Methods, analysis, accuracy, and implications for theoretical pKa calculations

Site-specific pK(a) values measured by NMR spectroscopy provide essential information on protein electrostatics, the pH-dependence of protein structure, dynamics and function, and constitute an important benchmark for protein pK(a) calculation algorithms. Titration curves can be measured by tracking the NMR chemical shifts of several reporter nuclei versus sample pH. However, careful analysis of these curves is needed to extract residue-specific pK(a) values since pH-dependent chemical shift changes can arise from many sources, including through-bond inductive effects, through-space electric field effects, and conformational changes. We have re-measured titration curves for all carboxylates and His 15 in Hen Egg White Lysozyme (HEWL) by recording the pH-dependent chemical shifts of all backbone amide nitrogens and protons, Asp/Glu side chain protons and carboxyl carbons, and imidazole protonated carbons and protons in this protein. We extracted pK(a) values from the resulting titration curves using standard fitting methods, and compared these values to each other, and with those measured previously by ¹H NMR (Bartik et al., Biophys J 1994;66:1180–1184). This analysis gives insights into the true accuracy associated with experimentally measured pK(a) values. We find that apparent pK(a) values frequently differ by 0.5–1.0 units depending upon the nuclei monitored, and that larger differences occasionally can be observed. The variation in measured pK(a) values, which reflects the difficulty in fitting and assigning pH-dependent chemical shifts to specific ionization equilibria, has significant implications for the experimental procedures used for measuring protein pK(a) values, for the benchmarking of protein pK(a) calculation algorithms, and for the understanding of protein electrostatics in general.

Bimodal MR–PET Agent for Quantitative pH Imaging

Bimodal MR–PET Agent for Quantitative pH Imaging

Design Principles and Theory of Paramagnetic Fluorine‐Labelled Lanthanide Complexes as Probes for 19F Magnetic Resonance: A Proof‐of‐Concept Study

The synthesis and spectroscopic properties of a series of CF(3)-labelled lanthanide(III) complexes (Ln=Gd, Tb, Dy, Ho, Er, Tm) with amide-substituted ligands based on 1,4,7,10-tetraazacyclododecane are described. The theoretical contributions of the (19)F magnetic relaxation processes in these systems are critically assessed and selected volumetric plots are presented. These plots allow an accurate estimation of the increase in the rates of longitudinal and transverse relaxation as a function of the distance between the Ln(III) ion and the fluorine nucleus, the applied magnetic field, and the re-rotational correlation time of the complex, for a given Ln(III) ion. Selected complexes exhibit pH-dependent chemical shift behaviour, and a pK(a) of 7.0 was determined in one example based on the holmium complex of an ortho-cyano DO3A-monoamide ligand, which allowed the pH to be assessed by measuring the difference in chemical shift (varying by over 14 ppm) between two (19)F resonances. Relaxation analyses of variable-temperature and variable-field (19)F, (17)O and (1)H NMR spectroscopy experiments are reported, aided by identification of salient low-energy conformers by using density functional theory. The study of fluorine relaxation rates, over a field range of 4.7 to 16.5 T allowed precise computation of the distance between the Ln(III) ion and the CF(3) reporter group by using global fitting methods. The sensitivity benefits of using such paramagnetic fluorinated probes in (19)F NMR spectroscopic studies are quantified in preliminary spectroscopic and imaging experiments with respect to a diamagnetic yttrium(III) analogue.

NMR Spectroscopic Characterization of the Sialyltransferase CstII from Campylobacter jejuni: Histidine 188 Is the General Base

Cell surface glycans are often terminated by sialic acid, which is incorporated onto sugar acceptors by sialyltransferases. The crystal structure of the GT family 42 Campylobacter jejuni alpha-2,3/2,8-sialyltransferase (CstII) provides key insights into the sialyl-transfer mechanism, including tentative identification of His188 as the catalytic base. In support of this hypothesis, the CstII-H188A mutant is able to catalyze sialyl transfer from CMP-Neu5Ac to added anions such as azide and formate but not to its natural sugar acceptor lactose. Complementing this work, NMR spectroscopy was used to investigate the structure and dynamics of CstII and to measure the intrinsic pK(a) value of His188 for comparison with the pK(a) determined from the pH-dependent k(cat)/K(M) of the enzyme. By systematically introducing point mutations at the subunit interfaces, two active monomeric variants, CstII-F121D and CstII-Y125Q, were obtained and characterized. In contrast to the wild-type tetramer, the monomeric CstII variants yielded good quality (1)H/(15)N-HSQC and (1)H/(13)C-methyl-TROSY NMR spectra. However, the absence of signals from approximately one-half of the amides in the (1)H/(15)N-HSQC spectra of both monomeric forms suggests that the enzyme undergoes substantial conformational exchange on a millisecond to microsecond time scale. The histidine pK(a) values of CstII-F121D in its apo form were measured by monitoring the pH-dependent chemical shifts of [(13)C(epsilon1)]histidine, biosynthetically incorporated into the otherwise uniformly deuterated protein. Consistent with its proposed catalytic role, the site-specific pK(a) value approximately 6.6 of His188 matches the apparent pK(a) value approximately 6.5 governing the pH dependence of k(cat)/K(M) for CstII toward CMP-Neu5Ac in the presence of saturating acceptor substrate.

Ionization Properties of Phosphatidylinositol Polyphosphates in Mixed Model Membranes

Phosphatidylinositol polyphosphate lipids (phosphoinositides) form only a minor pool of membrane phospholipids but are involved in many intracellular signaling processes, including membrane trafficking, cytoskeletal remodeling, and receptor signal transduction. Phosphoinositide properties are largely determined by the characteristics of their headgroup, which at physiological pH is highly charged but also capable of forming hydrogen bonds. Many proteins have developed special binding domains that facilitate specific binding to particular phosphoinositides, while other proteins interact with phosphoinositides via nonspecific electrostatic interactions. Despite its importance, only limited information is available about the ionization properties of phosphoinositides. We have investigated the pH-dependent ionization behavior of all three naturally occurring phosphatidylinositol bisphosphates as well as of phosphatidylinositol 3,4,5-trisphosphate in mixed phosphoinositide/phosphatidylcholine vesicles using magic angle spinning (31)P NMR spectroscopy. For phosphatidylinositol 3,5-bisphosphate, where the two phosphomonoester groups are separated by a hydroxyl group at the 4-position, the pH-dependent chemical shift variation can be fitted with a Henderson-Hasselbalch-type formalism, yielding pK(a)(2) values of 6.96 +/- 0.04 and 6.58 +/- 0.04 for the 3- and 5-phosphates, respectively. In contrast, phosphatidylinositol 3,4-bisphosphate [PI(3,4)P(2)] as well as phosphatidylinositol 4,5-bisphosphate [PI(4,5)P(2)] show a biphasic pH-dependent ionization behavior that cannot be explained by a Henderson-Hasselbalch-type formalism. This biphasic behavior can be attributed to the sharing of the last remaining proton between the vicinal phosphomonoester groups. At pH 7.0, the overall charge (including the phosphodiester group charge) is found to be -3.96 +/- 0.10 for PI(3,4)P(2) and -3.99 +/- 0.10 for PI(4,5)P(2). While for PI(3,5)P(2) and PI(4,5)P(2) the charges of the individual phosphate groups in the molecule differ, they are equal for PI(3,4)P(2). Differences in the charges of the phosphomonoester groups can be rationalized on the basis of the ability of the respective phosphomonoester group to form intramolecular hydrogen bonds with adjacent hydroxyl groups. Phosphatidylinositol 3,4,5-trisphosphate shows an extraordinary complex ionization behavior. While at pH 4 the (31)P NMR peak of the 4-phosphate is found downfield from the other two phosphomonoester group peaks, an increase in pH leads to a crossover of the 4-phosphate, which positions this peak eventually upfield from the other two peaks. As a result, the 4-phosphate group shows a significantly lower charge at pH values between 7 and 9.5 than the other two phosphomonoester groups. The charge of the respective phosphomonoester group in PI(3,4,5)P(3) is lower than the corresponding charge of the phosphatidylinositol bisphosphate phosphomonoester groups, leading to an overall charge of PI(3,4,5)P(3) of -5.05 +/- 0.15 at pH 7.0. The charge of all investigated phosphoinositides at pH 7.0 is equal or higher than the corresponding charge of soluble inositol polyphosphate headgroup analogues, which is the opposite of what is expected on the basis of simple electrostatic considerations. This higher than expected headgroup charge can be rationalized with mutual intermolecular hydrogen bond formation. Measurements using different concentrations of PI(4,5)P(2) in the lipid vesicles (1, 5, and 20 mol %) did not reveal any significant concentration-dependent shift of the two phosphomonoester peaks, suggesting that PI(4,5)P(2) is clustered even at 1 mol %.

Identification and Characterization of the Slowly Exchanging pH-dependent Conformational Rearrangement in KcsA

Gating of ion channels is strictly regulated by physiological conditions as well as intra/extracellular ligands. To understand the underlying structures mediating ion channel gating, we investigated the pH-dependent gating of the K(+) channel KcsA under near-physiological conditions, using solution-state NMR. In a series of (1)H(15)N-TROSY HSQC (transverse relaxation optimized spectroscopy-heteronuclear single quantum coherence) spectra measured at various pH values, significant chemical shift changes were detected between pH 3.9 and 5.2, reflecting a conformational rearrangement associated with the gating. The pH-dependent chemical shift changes were mainly observed for the resonances from the residues near the intracellular helix bundle, which has been considered to form the primary gate in the K(+) channel, as well as the intracellular extension of the inner helix. The substitution of His-25 by Ala abolished this pH-dependent conformational rearrangement, indicating that the residue serves as a "pH-sensor" for the channel. Although the electrophysiological open probability of KcsA is less than 10%, the conformations of the intracellular helix bundle between the acidic and neutral conditions seem to be remarkably different. This supports the recently proposed "dual gating" properties of the K(+) channel, in which the activation-coupled inactivation at the selectivity filter determines the channel open probability of the channel. Indeed, a pH-dependent chemical shift change was also observed for the signal from the Trp-67 indole, which is involved in a hydrogen bond network related to the activation-coupled inactivation. The slow kinetic parameter obtained for the intracellular bundle seems to fit better into the time scale for burst duration than very fast fluctuations within a burst period, indicating the existence of another gating element with faster kinetic properties.

Titration of E. coli transhydrogenase domain III with bound NADP + or NADPH studied by NMR reveals no pH-dependent conformational change in the physiological pH range

A pH-titration 2D NMR study of Escherichia coli transhydrogenase domain III with bound NADP + or NADPH has been carried out, in which the pH was varied between 5.4 and 12. In this analysis, individual amide protons served as reporter groups. The apparent p K a values of the amide protons, determined from the pH-dependent chemical shift changes, were attributed to actual p K a values for several titrating residues in the protein. The essential Asp392 is shown to be protonated at neutral pH in both the NADP + and NADPH forms of domain III, but with a marked difference in p K a not only attributable to the charge difference between the substrates. Titrating residues found in loop D/α5 point to a conformational difference of these structural elements that is redox-dependent, but not pH dependent. The observed apparent p K a values of these residues are discussed in relation to the crystal structure of Rhodospirillum rubrum domain III, the solution structure of E. coli domain III and the mechanism of intact proton-translocating transhydrogenase.

A repertoire of pyridinium-phenyl-methyl cross-talk through a cascade of intramolecular electrostatic interactions.

Direct intramolecular cation-pi interaction between phenyl and pyridinium moieties in 1a(+) has been experimentally evidenced through pH-dependent (1)H NMR titration. The basicity of the pyridinyl group (pK(a) 2.9) in 1a can be measured both from the pH-dependent chemical shifts of the pyridinyl protons as well as from the protons of the neighboring phenyl and methyl groups as a result of electrostatic interaction between the phenyl and the pyridinium ion in 1a(+) at the ground state. The net result of this nearest neighbor electrostatic interaction is that the pyridinium moiety in 1a becomes more basic (pK(a) 2.92) compared to that in the standard 2a (pK(a) 2.56) as a consequence of edge-to-face cation (pyridinium)-pi (phenyl) interaction, giving a free energy of stabilization (DeltaDeltaG(o)pKa) of -2.1 kJ mol(-1). The fact that the pH-dependent downfield shifts of the phenyl and methyl protons give the pK(a) of the pyridine moiety of 1a also suggests that the nearest neighbor cation (pyridinium)-pi (phenyl) interaction also steers the CH (methyl)-pi (phenyl) interaction in tandem. This means that the whole pyridine-phenyl-methyl system in 1a(+) is electronically coupled at the ground state, cross-modulating the physicochemical property of the next neighbor by using the electrostatics as the engine, and the origin of this electrostatics is a far away point in the molecule-the pyridinyl-nitrogen. The relative chemical shift changes and the pK(a) differences show that the cation (pyridinium)-pi (phenyl) interaction is indeed more stable (DeltaDeltaG(o)pKa = -2.1 kJ mol(-1)) than that of the CH (methyl)-pi (phenyl) interaction (DeltaDeltaG(o)pKa = -0.8 kJ mol(-1)). Since the pK(a) of the pyridine moiety in 1a is also obtained through the pH-dependent shifts of both phenyl and methyl protons, it suggests that the net electrostatic mediated charge transfer from the phenyl to the pyridinium and its effect on the CH (methyl)-pi (phenyl) interaction corresponds to DeltaG(o)pKa of the pyridinium ion (approximately 17.5 kJ mol(-1)), which means that the aromatic characters of the phenyl and the pyridinium rings in 1a(+) have been cross-modulated owing to the edge-to-face interaction proportional to this DeltaG(o)pKa change.

NMR STUDIES AND SEMI-EMPIRICAL ENERGY CALCULATIONS FOR CYCLIC ADP-RIBOSE

A possible pH-dependent conformational switch was investigated for cyclic ADP-ribose. NMR signals for the exchangeable protons were observed in H2O at low temperature, but there was no direct evidence for the protonation of N-3 at neutral pH that has previously been postulated. MNDO calculations indicated that pH dependent 31P chemical shift changes are attributable to protonation of the phosphate adjacent to the N-1 of adenine, and not due to trans-annular hydrogen bonding with a protonated N-3.

31P NMR for the study of P metabolism and translocation in arbuscular mycorrhizal fungi

31P nuclear magnetic resonance (NMR) spectroscopy was used to study phosphate (P) metabolism in mycorrhizal and nonmycorrhizal roots of cucumber (Cucumis sativus L) and in external mycelium of the arbuscular mycorrhizal (AM) fungus Glomus intraradices Schenck & Smith. The in vivo NMR method allows biological systems to be studied non-invasively and non-destructively. 31P NMR experiments provide information about cytoplasmic and vacuolar pH, based on the pH-dependent chemical shifts of the signals arising from the inorganic P (Pi) located in the two compartments. Similarly, the resonances arising from α, β and γ phosphates of nucleoside triphosphates (NTP) and nucleoside diphosphates (NDP) supply knowledge about the metabolic activity and the energetic status of the tissue. In addition, the kinetic behaviour of P uptake and storage can be determined with this method. The 31P NMR spectra of excised AM fungi and mycorrhizal roots contained signals from polyphosphate (PolyP), which were absent in the spectra of nonmycorrhizal roots. This demonstrated that the Pi taken up by the fungus was transformed into PolyP with a short chain length. The spectra of excised AM fungi revealed only a small signal from the cytoplasmic Pi, suggesting a low cytoplasmic volume in this AM fungus.

Structure of dicarboxyl malto-oligomers isolated from hypochlorite-oxidised potato starch studied by 1H and 13C NMR spectroscopy

The main oxidised component in hypochlorite-oxidised potato starch was isolated by anion-exchange chromatography after enzymatic hydrolysis. The primary structure of the isolated oligosaccharides was determined by 1H and 13C NMR spectroscopy, using homonuclear and heteronuclear two-dimensional techniques. The isolated pentamer and hexamer contained one glucose unit oxidised to a dicarboxyl residue. As the hypochlorite oxidation has occurred at positions C-2 and C-3 of a glucose unit, the introduced carboxyl groups caused ring cleavage between the carbons C-2 and C-3. The ring-cleaved dicarboxyl residue had glycosidic linkages on both sides, implying that this oxidation pathway does not result in depolymerisation. The vicinal coupling constant between H-4 and H-5 in the ring-cleaved dicarboxyl residue was 3.2 Hz, showing that the gauche orientations are preferred. As a result, a different bending of the starch chain is observed and is probably, therefore, one of the reasons why hypochlorite oxidation reduces the tendency to retrogradation. The p K a values (3.0) were determined from the pH-dependent chemical shifts of H-1, H-4 and H-5 of the dicarboxylic residue.