The measurement of muscle protein synthesis in broilers with a flooding dose technique: use of 15N-labelled phenylalanine, GC-MS and GC-C-IRMS

An experiment was carried out to measure fractional muscle protein synthesis rates (k(s)) in broilers with injection of a flooding dose of phenylalanine (1 ml/100 g body weight of 150 mM phenylalanine; 38 atom percent excess (APE) [15N]phenylalanine). K(s) was calculated from the [15N] enrichment in phenylalanine of tissue-free and protein-bound phenylalanine using both gas chromatography mass spectrometry (GC-MS) and gas chromatography combustion isotope ratio mass spectrometry (GC-C-IRMS) for measurements after a 10 min isotope incorporation period. The tertiary-butyldimethylsilyl (t-BDMS) derivatives of phenylalanine were used for gas chromatographic separation in both systems. GC-MS and GC-C-IRMS were calibrated for a range of 7 to 37 [15N]APE and 0 to 0.62 [15N]APE, respectively, and for sample sizes of 0.45 to 4.5 nmol phenylalanine and 7 to 40 nmol phenylalanine, respectively. Reproducibility of standards as a measure of precision varied from 0.06 to 0.29 [15N]APE and from 0.0004 to 0.0018 [15N]APE in GC-MS and GC-C-IRMS, respectively. K(s) was measured in the m. pectoralis major of broilers fed rye based diets (56%) which were provided either unsupplemented (-) or supplemented (+) with an enzyme preparation containing xylanase. K(s) in breast muscles was significantly increased from 21.8%/d to 23.9%/d due to enzyme supplementation. It can be concluded from the study that the measurement of protein synthesis in broilers with the flooding dose technique can be carried out by using [15N]phenylalanine, GC-MS and GC-C-IRMS.     

Incorporation of 15NO2 Nitrogen into Individual Amino Acids by Sunflowers Using Gc-C-Irms

Abstract

The nitrogen isotopic composition of individual amino acids in sunflower leaves after exposures to ¹⁵NO₂ in the range of ambient NO₂ concentrations (5–37 ppb) was analysed by Gas Chromatography-Combustion-Isotope Ratio Mass Spectrometry (GC-C-IRMS). Amino acids as well as the amides glutamine and asparagine were converted with MTBSTFA (N-methyl-N-(tert.-butyldimethylsilyl)-tri-fluoroacetamid) in pyridine to their corresponding TBDMS derivatives (N, O-tert.-butyldimethylsilyl) in a simple one-step silylation reaction. The derivatized amino acids were separated by gas chromatography, combusted on-line, and the products were sent continuously to an isotope ratio mass spectrometer. Accurate measurements were obtained, when more than 7 nmol N₂ were introduced into the ion source of the mass spectrometer per gas chromatographically separated and combusted compound. No interferences of the silicate and fluor containing derivatization agents on the performance of the system were observed.

In the range of ambient NO₂ concentrations sunflower leaves predominately incorporate the nitrogen derived from atmospheric NO₂ into soluble amino acids. The highest δ¹⁵N values were measured for alanine. The ¹⁵N enrichments of the detectable amino acids increased with increasing ¹⁵NO₂ concentration.     

The use of stable isotopes in ecosystem research. First results of a field study with 15N

Terrestrial ecosystems, e.g. forest ecosystems, are characterized by a complex and sensitive network of biotic and abiotic factors and their interactions. By using stable isotopes (e.g. labelled nitrogen compounds), very small addition rates of highly enriched compounds can be applied, which do not change or disturb the investigated system, but provide information about single processes, their interactions and especially about their dynamics.

First results of a field study in the Fichtelgebirge, Northeast-Bavaria, Germany, are presented. The distribution of labelled nitrogen (as ¹⁵N-NH₄ ⁺ and ¹⁵N[sbnd]NO₃ ^?) within a spruce ecosystem (Picea abies (L.) Karst. in competition with understory vegetation of Vaccinium myrtillus, Calluna vulgaris and Deschampsia flexuosa) showed maximum ¹⁵N concentrations in tissues of the understory vegetation. During the first six weeks after the ¹⁵N application, the nitrogen uptake of all investigated species was higher after the ¹⁵N[sbnd]NO₃ ^? treatment than after the ¹⁵N[sbnd]NH₄ ⁺ treatment.     

15N/14N Analysis of Amino Acids with GC-C-IRMS - Methodical Investigations and Ecotoxicological Applications

Abstract

In order to perform the ¹⁵N/¹⁴N analysis of amino acids using a gas chromatograph and isotope ratio mass spectrometer linked up via a combustion interface (GC-C-IRMS), the amino acids must be derivatized. Tert-butyldimethylsilylation is examined using various techniques (direct conversion to nitrogen gas, ConFlo-IRMS [1], GC-C-IRMS [2]) and subsequently applied to isotopic characterization of amino acids from wheat protein hydrolyzate obtained from plants exposed to ozone. The method provides a reliable tool for studying ecotoxicological effects on plants at a molecular level in addition to the investigation of the natural variations of different N fractions.     

Synthese und Qualitätskontrolle 15N-markierter Stickoxide mit hoher 15N-Häufigkeit für umweltrelevante Tracer-Untersuchungen

Für die Untersuchung ausgewählter Probleme des Verhaltens und der Wirkung der Stickoxide NO _x (NO + NO₂) in Ökosystemen, z.B. die Aufnahme und Freisetzung von NOx durch das System “Boden-Pflanze”, bietet sich der Einsatz ¹⁵N-markierter Stickoxide an. Die dazu benötigten ¹⁵N-markierten Gasgemische hoher Reinheit werden aus eigens dafür synthetisiertem [¹⁵N]Stickstoffmonoxid oder [¹⁵N]Stickstoffdioxid mit hoher ¹⁵N-Häufigkeit hergestellt. Beide Synthesen gehen jeweils von der kostengünstig kommerziell erhältlichen [¹⁵N]Salpetersäure aus.

Im Falle des [¹⁵N]Stickstoffdioxids erfolgt die Herstellung über die Präparation von Bleinitrat und dessen thermische Zersetzung. Die Ausbeute liegt bei 70–75% bezogen auf eingesetzte [¹⁵N]Salpetersäure.

Die Herstellung von [¹⁵N]Stickstoffmonoxid erfolgt durch Reduktion von [¹⁵N]Salpetersäure mit Eisen-II-sulfat in stark saurer Lösung. Die Ausbeute beträgt 60–70%, bezogen auf eingesetzte Salpetersäure.

The application of ¹⁵N is very useful for the investigation of the behavior and the effect of the nitrogen oxides NO _x (nitric oxide + nitrogen dioxide) in ecosystems, e.g. the uptake and release of NO _x by the soil-plant system. The ¹⁵N labelled gas mixture needed for that purpose has to be prepared from synthesized highly enriched [¹⁵N]nitric oxide and [¹⁵N]nitrogen dioxide. These two syntheses both use the commercially available and reasonable [¹⁵N]nitric acid.

In the case of [¹⁵N]nitrogen dioxide the synthesis is carried out via [¹⁵N]lead nitrate and its decomposition with increasing temperature. The yield is 70–75% related to the [¹⁵N]nitric acid input. The preparation of [¹⁵N]nitric oxide is done by reduction of [¹⁵N]nitric acid by means of FeSO₄ in strong acid solution. The yield amounts to 60–70%.     

GC-MS-gestützte Synthese und Qualitätskontrolle von 15N-markiertem Stickstoffmonoxid mit hoher 15N-Häufigkeit für umweltrelevante Tracer-Untersuchungen

Abstract

[¹⁵N]nitric oxide for investigations on the effect of nitric oxide in biological systems can be synthesized economically from the commercially available [¹⁵N]nitric acid. Difficulties arise, however, for the preparation of highly enriched ¹⁵N-labelled nitric oxide on a lower mmol scale. These difficulties are caused by oxygen and result in both, yield depression and insufficient long-time stability of the NO-containing gas mixture due to its oxidation to NO₂. Therefore, a sensitive in situ oxygen detection during synthesis was carried out by on line coupling of the synthesis apparatus with a GC-MS. Additionally, this equipment offers the advantage that both, conversion and ¹⁵N enrichment may be measured almost continuously and simultaneously. In this way, the yield can be increased up to 85% without increasing by-products. The gas mixture prepared from this [¹⁵N]nitric oxide showed an adequate stability for several months at a concentration of about 100 ppm(v) NO.     

Estimation of protein synthesis rates using the flooding method and [15N] lysine

After injection of a single dose of double labelled lysine (1 MBq L-[u-¹⁴C]lysine and 150 μmol L-[α¹⁵N]lysine (95 atom% ¹⁵N)) to growing rats the fractional protein synthesis rates (FSR) of some organs were estimated by the “large dose-” or “flooding method”. Data obtained with both substances were compared and the following conclusions can be drawn:

—In principle lysine is suited as a flooding substance.

—In flooding experiments [¹⁴C]lysine and [¹⁵N]lysine gave identical FSR-values for the investigated organs.

—By application of [¹⁵N]lysine instead of the ¹⁴C labelled amino acid as flooding substance this method is suited for larger animals (pigs, sheep, ruminants) too, because of the absence of any radioactive burden.

Nach einmaliger Verabfolgung von doppelt markiertem Lysin (1 MBq L-[u-¹⁴C]Lysin und 150 μmol L-[α¹⁵N]Lysin (95 Atom% ¹⁵N)) an wachsende Ratten wurden nach der “Large dose-” oder “Flooding-Methode” die fraktionellen Proteinsyntheseraten (FSR) einiger Organe bestimmt und die für beide Substanzen erhaltenen Werte verglichen. Es ergaben sich folgende Schlußfolgergungen:

—Lysin ist grundsätzlich als Flooding-Substanz geeignet.

—[¹⁴C]Lysin und [¹⁵N]Lysin ergeben im Floodingversuch am gleichen Tier für die FSR der Organe identische Werte.

—Durch den Fortfall der radioaktiven Belastung bei Einsatz von [¹⁵N]Lysin anstelle von [¹⁴C]Lysin als Flooding-Substanz ist die Methode auch bei Croßtieren anwendbar.     

The First in Vivo Observation of C–N Coupling in Mammalian Brain

[5-¹³C,¹⁵N]Glutamine, with ¹J(¹³C–¹⁵N) of 16 Hz, was observed in vivo in the brain of spontaneously breathing rats by ¹³C MRS at 4.7 T. The brain [5-¹³C]glutamine peak consisted of the doublet from [5-¹³C,¹⁵N]glutamine and the center [5-¹³C,¹⁴N]glutamine peak, resulting in an apparent triplet with a separation of 8 Hz. The time course of formation of brain [5-¹³C,¹⁵N]glutamine was monitored in vivo with a time resolution of 20–35 min. This [5-¹³C,¹⁵N]glutamine was formed by glial uptake of released neurotransmitter [5-¹³C]glutamate and its reaction with ¹⁵NH₃ catalyzed by the glia-specific glutamine synthetase. The neurotransmitter glutamate C5 was selectively¹³C-enriched by intravenous [2,5-¹³C]glucose infusion to ¹³C-label whole-brain glutamate C5, followed by [¹²C]glucose infusion to chase ¹³C from the small and rapidly turning-over glial glutamate pool, leaving ¹³C mainly in the neurotransmitter [5-¹³C]glutamate pool, which is sequestered in vesicles until release. Hence, the observed [5-¹³C,¹⁵N]glutamine arises from a coupling between ¹³C of neuronal origin and ¹⁵N of glial origin. Measurement of the rate of brain [5-¹³C,¹⁵N]glutamine formation provides a novel noninvasive method of studying the kinetics of neurotransmitter uptake into glia in vivo, a process that is crucial for protecting the brain from glutamate excitotoxicity.     

Magnitudes and Orientations of the N Chemical Shift Tensor of [1-N]-2′-Deoxyguanosine Determined on a Polycrystalline Sample by Two-Dimensional Solid-State NMR Spectroscopy

The magnitudes and orientations of the ¹⁵N chemical shift tensor of [1-¹⁵N]-2′-deoxyguanosine were determined from a polycrystalline sample using the two-dimensional PISEMA experiment. The magnitudes of the principal values of the ¹⁵N chemical shift tensor of the N1 nitrogen of [1-¹⁵N]-2′-deoxyguanosine were found to be ς₁₁ = 54 ppm, ς₂₂ = 148 ppm, and ς₃₃ = 201 ppm with respect to (¹⁵NH₄)₂SO₄ in aqueous solution. Comparisons of experimental and simulated two-dimensional powder pattern spectra show that ς_33N is approximately collinear with the N–H bond. The tensor orientation of ς_33N for N1 of [1-¹⁵N]-2′-deoxyguanosine is similar to the values obtained for the side chain residues of ¹⁵N_ε1-tryptophan and ¹⁵N_π-histidine even though the magnitudes differ significantly.     

The Fate of [15N]Ammonium and [15N]Nitrate in the Soil of a 140-Year-Old Spruce Stand (Picea Abies) in the Fichtelgebirge (NE-Bavaria)

Abstract

A ¹⁵N tracer-experiment was carried out in a 140-year-old spruce stand (Picea abies (L.) Karst.) in the Fichtelgebirge (NE-Bavaria, Germany). Highly enriched (98 at%) [¹⁵N]ammonium and [¹⁵N]nitrate were applied as tracers by simulation of a deposition of 41.3 mol N ha^?1 with 11 water m^?2. To examine seasonal variations of uptake by spruce and understorey vegetation, different plots were labelled in spring, summer and autumn 1994.

One aim of the present study was to perfect a method of preparation of soil extracts for isotope ratio mass spectrometry (IRMS) measurements. Ammonium and nitrate from soil extracts were prepared for IRMS measurements by steam distillation and subsequent freeze drying. Additionally, tracer distribution and transformations in the soil nitrogen pools were examined. Ammonium, nitrate and total nitrogen were examined in the organic layer and the upper 10 cm of the mineral soil during 3 months after the first tracer application in spring 1994.

In July 1994, three months after tracer application, 40% of the [¹⁵N]ammonium label and 29% of the [¹⁵N]nitrate label, respectively, were recovered in the total N pool of the investigated soil horizons. In the organic layer the L/Of horizon retained most of the recovered tracers. Nitrification, immobilisation and mineralisation occurred even under the conditions of high soil acidity at the study site.     

Determination of Internucleotide JHN Couplings by the Modified 2D JNN-Correlated [N, H] TROSY

Recent developments in the direct observation of J couplings across hydrogen bonds in proteins and nucleic acids provide additional information for structure and function studies of these molecules by NMR spectroscopy. A J_NN-correlated [¹⁵N, ¹H] TROSY experiment proposed by Pervushin et al. (Proc. Natl. Acad. Sci. USA 95, 14147–14151, 1998) can be applied to measure ^hJ_HN in smaller nucleic acids in an E.COSY manner. However, it cannot be effectively applied to large nucleic acids, such as tRNA^Trp, since one of the peaks corresponding to a fast relaxing component will be too weak to be observed in the spectra of large molecules. In this Communication, we proposed a modified J_NN-correlated [¹⁵N, ¹H] TROSY experiment which enables direct measurement of ^hJ_HN in large nucleic acids.     

In Vivo Detection of N-Coupled Protons in Rat Brain by ISIS Localization and Multiple-Quantum Editing

Three-dimensional image-selected in vivo spectroscopy (ISIS) was combined with phase-cycled ¹H–¹⁵N heteronuclear multiple-quantum coherence (HMQC) transfer NMR for localized selective observation of protons J-coupled to ¹⁵N in phantoms and in vivo. The ISIS–HMQC sequence, supplemented by jump–return water suppression, permitted localized selective observation of 2–5 μmol of [¹⁵N_indole]tryptophan, a precursor of the neurotransmitter serotonin, through the ¹⁵N-coupled proton in 20–40 min of acquisition in vitro at 4.7 T. In vivo, the amide proton of [5-¹⁵N]glutamine was selectively observed in the brain of spontaneously breathing ¹⁵NH₄⁺-infused rats, using a volume probe with homogeneous ¹H and ¹⁵N fields. Signal recovery after three-dimensional localization was 72–82% in phantoms and 59 ± 4% in vivo. The result demonstrates that localized selective observation of ¹⁵N-coupled protons, with complete cancellation of all other protons except water, can be achieved in spontaneously breathing animals by the ISIS–HMQC sequence. This sequence performs both volume selection and heteronuclear editing through an addition/subtraction scheme and predicts the highest intrinsic sensitivity for detection of ¹⁵N-coupled protons in the selected volume. The advantages and limitations of this method for in vivo application are compared to those of other localized editing techniques currently in use for non-exchanging protons.     

15N-Traceruntersuchungen zum Mechanismus der N2O-Bildung in Böden

Nitrous oxide is a potential environmental hazard responsible for the green house effect and the destruction of the ozone layer in the lower stratosphere. Biological denitrification under anaerobic conditions in soils results in the formation of both N₂O and N₂, whereby highly nitrogen-fertilized agricultural soils contribute to a considerable extent of the N₂O emission. Latest results in the literature indicate that nitrous oxide can also be formed as a byproduct of the microbial nitrification. This is of importance for soils in central Germany because of the non-existence of typical denitrification conditions in a semiaride climate.

This study was conducted to measure the path of N₂O formation in Haplic Phaeozen: using [¹⁵N] ammonium and [¹⁵N] nitrat and a GC-MS aided incubation system. The kinetic isotope method was used to evaluate the experimental data. The results are:

- Under anaerobic conditions (～ 90% of the water holding capacity = WHC) N₂O originates mainly from the nitrate pool by denitrification.

- As expected, the N₂O formation is low under aerobic conditions (～ 80% WHC) but the gas originates directly from the ammonium and not from the nitrate pool, probably as a byproduct of the nitrification process.     

Towards an Inhalative 13C Breath Test Method

Abstract

Customary ¹³CO₂ breath tests—and also ¹⁵N urine tests—always start with an oral administration of a test substrate. The test person swallows a stable isotope labelled diagnostic agent. This technique has been used to study several pathophysiological changes in gastrointestinal organs. However, to study pathophysiological changes of the bronchial and lung epithelium, the inhalative administration of a stable isotope labelled agent appeared more suitable to us. [1-¹³C]Hexadecanol and [1-¹³C]glucose were chosen. Inhaled [1-¹³C]hexadecanol did not yield ¹³CO₂ in the exhaled air, but [1-¹³C]glucose did. To study the practicability of the [1-¹³C]glucose method and the reproducibility of the results, 18 inhalation tests were performed with healthy subjects. In 6 self-tests, the optimum inhalative dose of [¹³C]glucose was determined to be 205 mg. Using the APS aerosol provocation system with the nebulizer ‘Medic Aid’ (Erich Jaeger Würzburg), a 25% aqueous solution was inhaled. Then, breath samples were collected at 15 min. intervals and analysed for ¹³CO₂. 75–120min after the end of inhalation a well-reproducible maximum δ¹³C value of 6‰ over baseline (DOB) was detected for 12 healthy probands.

Speculating that the pulmonary resorption of the [¹³C]glucose is the rate-limiting step of elimination, decompensations in the epithelium ought to be reflected in changed [1-¹³C]glucose resorption rates and changed ¹³CO₂ output.

Therefore, we speculate that the inhalation of suitable ¹³C-labelled substrates will pave the way for a new group of ¹³CO₂ breath tests aiding investigations of specific pathophysiological changes in the pulmonary tract, such as inflammations of certain sections and decompensations of cell functions.     

In vivo NMR spectroscopy: in situ 15N pulse labelling NMR spectroscopy with photoautotrophic microorganisms

For studying the nitrogen metabolism in plants ¹⁵N NMR spectroscopy can be used. For in vivo ¹⁵N NMR (natural abundance of ¹⁵N: 0.37%) enrichment of the sample with the isotope ¹⁵N is compulsory. The detection of time courses of ¹⁵N assimilation from cells, which are enriched in culture is restricted in scope. Here, a method, the ¹⁵N pulse labelling NMR spectroscopy, is demonstrated, which permits labelling of different nitrogen compounds in photoautotrophic microorganisms during the NMR spectroscopic measurement. Using an effective illumination system it is possible to maintain photosynthesis in plant samples of high biomass densities in the magnet necessary for ammonia assimilation. The technique thus enables to directly observe ammonia assimilation pathways by application of a ¹⁵NO₃ ^? or ¹⁵NH₄ ^? pulses.

Für das Studium des Stickstoffstoffwechsels der Pflanzen kann die ¹⁵N-NMR-Spektroskopie herangezogen werden. Hierzu ist bei der in-vivo-¹⁵N-NMR (natürliche Häufigkeit von ¹⁵N: 0.37%) eine Anreicherung der Probe mit dem Isotop ¹⁵N unerläßlich. Eine Verfolgung der ¹⁵N-Assimilationskinetik mit Zellen, die in der Kultur angereichert wurden, ist jedoch nur bedingt möglich. In dieser Arbeit wird die ¹⁵N-Pulsmarkierungs-NMR-Spektroskopie als eine Methode vorgestellt, die es erlaubt, eine Markierung von Stickstoffverbindungen in photoautotrophen einzelligen Mikroorganismen während der NMR-Messung im Magneten vorzunehmen. Es wird ein spezielles Beleuchtungssystem verwendet, das eine für die Stickstoffassimilation ausreichende Photosyntheseleistung der Zellen unter NMR-Bedingungen bei hoher Biomassedichte ermöglicht. Diese Technik erlaubt durch die Applikation eines ¹⁵NO₃ ^?-oder ¹⁵NH₄ ⁺-Pulses eine direkte Verfolgung von Ammonium-Assimilationswegen.     

Relationships between Nitrogen Translocation and Accumulation in Growing Plant Parts of Grain Legumes

Abstract

Grain legumes absorbed mineral ¹⁵N at every stage of development and transported it directedly (just as symbiotically fixed ¹⁵N₂) into the plant parts growing at the respective stage. The ¹⁵N accumulation in the grains after long-lasting ¹⁵N supply can be ascribed, for the major part, to a secondary ¹⁵N translocation after a temporary incorporation into older plant parts (leaves, stem). Inhibition experiments with antibiotics revealed no direct relation between the accumulation of amino acid ¹⁵N in growing pods and seeds and the protein synthesis in this target organs. It may include, however, processes of (active ?) uptake and transport with a possible contribution of carrier systems specific for distinct amino acids.     

Formation of gold nanoparticles in gold ruby glass: The influence of tin

A ¹¹⁹Sn Mössbauer study was carried out of tin(IV) complexes with 2-benzoylpyridine thiosemicarbazone (H2Bz4DH) and its N(4)-methyl (H2Bz4M) and N(4)-phenyl (H2Bz4Ph) derivatives: [Sn(2Bz4DH)Cl₃] (1), [Sn(2Bz4DH)PhCl₂] (2), [Sn(2Bz4M)Cl₃] (3), [H₂2Bz4M]₂[Ph₂SnCl₄] (4), [Sn(2Bz4Ph)PhCl₂] (5), [Sn(2Bz4Ph)Ph₂Cl] (6), in which H2Bz4R stands for the neutral ligand and 2Bz4R stands for the anionic thiosemicarbazone. In addition, ¹¹⁹Sn Mössbauer studies of the tin(IV) complexes [Sn(H4Bz4DH)₂Cl₄H₂O] (7), [Sn(H4BzPS)₂Cl₄H₂O] (8) with 4-benzoylpyridine thiosemicarbazone (H4Bz4DH) and the correspondent semicarbazone (H4BzPS) were performed. The isomer shifts decrease upon coordination due to the variation in the percentage of s character as tin changes from approximately sp³ hybridization in the tin salts to sp³d² in the octahedral or sp³d³ in the heptahedral complexes. The Mössbauer parameters of compound (4) showed the existence of two tin(IV) sites, which have been attributed to the presence of the cis and trans isomers.     

Non-Kramers ENDOR and ESEEM of the S = 2 Ferrous Ion of [Fe(II)EDTA]

We report here the first non-Kramers (NK) ESEEM and ENDOR study of a mononuclear NK center, presenting extensive parallel-mode ESEEM and ENDOR measurements on the S_t = 2 ferrous center of [Fe(II)ethylenediamine-N,N,N′,N′-tetraacetato]²⁻; [Fe(II)EDTA)]²⁻. The results disclose an anomalous equivalence of the experimental patterns produced by the two techniques. A simple theoretical treatment of the frequency-domain patterns expected for NK-ESEEM and NK-ENDOR rationalizes this correspondence and further suggests that the very observation of NK-ENDOR is the result of an unprecedentedly large hyperfine enhancement effect. The mixed nitrogen–carboxylato oxygen coordination of [Fe(II)EDTA]²⁻ models that of the protein-bound diiron centers, although with a higher coordination number. Analysis of the NK-ESEEM measurements yields the quadrupole parameters for the ¹⁴N ligands of [Fe(II)EDTA]²⁻, K = 1.16(1) MHz, 0 ≤ η ≤ 0.05, and the analysis indicates that the electronic zero-field splitting tetragonal axis lies along the N–N direction.     

Separation of Anisotropy and Exchange Broadening Using N CSA–N–H Dipole–Dipole Relaxation Cross-Correlation Experiments

Based on the measurement of cross-correlation rates between ¹⁵N CSA and ¹⁵N–¹H dipole–dipole relaxation we propose a procedure for separating exchange contributions to transverse relaxation rates (R₂ = 1/T₂) from effects caused by anisotropic rotational diffusion of the protein molecule. This approach determines the influence of anisotropy and chemical exchange processes independently and therefore circumvents difficulties associated with the currently standard use of T₁/T₂ ratios to determine the rotational diffusion tensor. We find from computer simulations that, in the presence of even small amounts of internal flexibility, fitting T₁/T₂ ratios tends to underestimate the anisotropy of overall tumbling. An additional problem exists when the N–H bond vector directions are not distributed homogeneously over the surface of a unit sphere, such as in helix bundles or β-sheets. Such a case was found in segment 4 of the gelation factor (ABP 120), an F-actin cross-linking protein, in which the diffusion tensor cannot be calculated from T₁/T₂ ratios. The ¹⁵N CSA tensor of the residues for this β-sheet protein was found to vary even within secondary structure elements. The use of a common value for the whole protein molecule therefore might be an oversimplification. Using our approach it is immediately apparent that no exchange broadening exists for segment 4 although strongly reduced T₂ relaxation times for several residues could be mistaken as indications for exchange processes.     

The Influence of the Auxiliary Water and of Nitric Acid Flow-Rate about 15N Separation in Nitrox System

The experimental results obtained for ¹⁵N separation by Spindel-Taylor [1] method, in a laboratory exchange column [2] are presented. The influence of the auxiliary water flow and of the nitric acid flow-rate (0.6–2.6 ml/cm² min) on the ¹⁵N separation has been stuided. All the experimental points were obtained in unsteady state, thus giving information about the rate of the steady state achievement.