C-13 nuclear magnetic resonance spectroscopy of humic substances

Summary Evidence is presented from a preliminary study showing that C-13 N.M.R. spectroscopy is a useful new tool for the elucidation of the structure of soil organic matter.     

Carbon-13 nuclear magnetic resonance spectra of lignins

From the ¹³C-nmr spectra of a large number of dimeric and monomeric lignin model compounds the chemical shifts of the carbon atoms of the C₉-units in lignin with different substitution patterns were determined. The absorption peaks of the carbon-13 spectra of two lignins (beech and spruce) could be assigned by comparison (Table 3).     

Proton and carbon-13 nuclear magnetic resonance studies of rhodopsin-phospholipid interactions

Proton and carbon-13 nuclear magnetic resonance (1H and 13C NMR) spectra of rhodopsin-phospholipid membrane vesicles and sonicated disk membranes are presented and discussed. The presence of rhodopsin in egg phosphatidylcholine vesicles results in homogeneous broadening of the methylene and methyl resonances. This effect is enhanced with increasing rhodopsin content and decreased by increasing temperature. The proton NMR data indicate the phospholipid molecules exchange rapidly (less than 10(-3) s) between the bulk membrane lipid and the lipid in the immediate proximity of the rhodopsin. These interactions result in a reduction in either or both the frequency and amplitude of the tilting motion of the acyl chains. The 13C NMR spectra identify the acyl chains and the glycerol backbone as the major sites of protein lipid interaction. In the disk membranes the saturated sn-1 acyl chain is significantly more strongly immobilized than the polyunsaturated sn-2 acyl chain. This suggest a membrane model in which the lipid molecules preferentially solvate the protein with the sn-1 chain, which we term an edge-on orientation. The NMR data on rhodopsin-asolectin membrane vesicles demonstrate that the lipid composition is not altered during reconstitution of the membranes from purified rhodopsin and lipids in detergent.     

Steady-state carbon-13 nuclear magnetic resonance spectra of acyl-alpha-chymotrypsin

When [13C]carbonyl-enriched p-nitrophenyl 5-n-propyl-2-furoate is incubated with alpha-chymotrypsin, a new peak appears in the 13C NMR spectrum. On the basis of its position and the fact that it is "chased" with unlabeled substrate, we conclude that this new signal is due to the acyl-enzyme intermediate. In spectra taken during steady-state turnover, the acyl-enzyme ester carbonyl 13C chemical shift displays a pH dependence that fits to a titration curve with an apparent pK of 7.1 (0.1). The apparent pK of the kcat vs. pH curve for enzyme-catalyzed hydrolysis of the same substrate under conditions differing only in reactant concentration is 7.0 (0.1). We have found no spectral evidence for a tetrahedral intermediate.     

H and 13C nuclear magnetic resonance spectra of some peptides with fibrinogen-like reactivity.

The 1H and 13C spectra of four peptides, L-Phe-Val-Arg-pNA, D-Phe-Val-Arg-pNA, L-Phe-Pip-Arg-pNA and D-Phe-Pip-Arg-pNA (pNA = p-nitroaniline, Pip = pipecolic acid residue), have been examined, and deductions made about their conformation in solution. The D-Phe peptides, which are cleaved especially rapidly by thrombin in water, have structures (in deuterated DMSO) in which the aromatic ring of the D-Phe residue is folded back over the Val or Pip residue. This arrangement is not found in the L-Phe peptides. It is proposed that this feature (in which Phe could be situated near Val and near the Arg-Gly bond of the A alpha chain in the three-dimensional structure of fibrinogen) may be especially advantageous for binding to the enzyme.     

Proton nuclear magnetic resonance spectra of Pseudomonas aeruginosa ferricytochrome cd1

Proton nuclear magnetic resonance spectra of ferricytochrome cd1 from the denitrifying bacterium Pseudomonas aeruginosa have been obtained. The normal 0-10-ppm chemical shift range shows many overlapping and nonresolvable peaks, as would be expected for a dimeric protein of molecular weight approximately 120,000. In the downfield region between 10 and 50 ppm, and in the upfield region between 0 and -20 ppm, resolvable resonances corresponding to a small number of protons are observed. The temperature and pH behavior of these resonances have been examined. For some of the resolved resonances, the pH behavior of chemical shifts and intensities indicates that the oxidized form of the enzyme undergoes a structural transition with a pK of 5.8 +/- 0.3. On the basis of several lines of evidence, some assignments are proposed in which resolvable resonances are assigned as originating from either the heme c or the heme d1 prosthetic groups of the enzyme.     

Carbon-13 nuclear magnetic resonance spectra of carbohydrate oxirane derivatives

The ¹³C-chemical shifts and ¹J_C,H values of two series of carbohydrate oxirane derivatives, namely methyl 2,3-anhydro-ribo- and -lyxofuranosides and methyl 2,3-anhydro-4,6-O-benzylidene-manno- and -allopyranosides have been determined. The assignment of ¹³C resonances has been established mainly by the examination of the proton-coupled and the selective proton-decoupled spectra. The effect of the oxirane rings on the chemical shifts of β and γ carbon atoms (from the oxirane ring oxygen atom) has been observed. Large ¹J_C,H values associated with cis CH bonds adjacent to the oxirane rings relative to those of trans counterparts have been found.     

FT-C13 nuclear magnetic resonance spectra of natural humic substances

The C13 resonance spectra of two humic acids extracted from Vertisol and Andosol soil and of a fulvic acid form Podzol soil are reported. The spectra were taken in 5 % W/W solution of the substances in 0.1 N NaOD in D₂O using the Fourier-transform-technique.     

Proton nuclear magnetic resonance spectra of compounds I and II of horseradish peroxidase

Enzymatic reaction intermediates of horseradish peroxidase, compounds I and II, were studied by high-resolution nuclear magnetic resonance spectroscopy at 220 MHz. The heme peripheral proton peaks were successfully obtained in the downfield region of 50 to 80 ppm from 4,4-dimethyl-4-silapentane-5-sulfonate for compound I and of 10 to 20 ppm for compound II at pH 9.2. This indicates that no isoporphyrin appears in the catalytic cycle of the enzyme. Temperature dependences of the spectra also were determined for these compounds between 7 and 32 degrees C. With increasing temperature, all the peaks in the downfield region for compound I shifted upfield, obeying the Curie law. These results suggest that the Fe atoms in compounds I and II are in ferryl high- and low-spin states, respectively. The spectrum was also observed in solutions of horse metmyoglobin to which hydrogen peroxide (H2O2) was added. The electron formulations of the hemes in their spectra. Evidence was found against a pi-cation radical on the heme ring as a source of the oxidizing equivalent in compound I.     

The carbon-13 nuclear magnetic resonance spectra of four eudesmane sesquiterpenols

The ¹³C NMR spectra of geosmin, selina-4(14),7(11)-diene-99-ol and two dihydroeudesmol isomers have been obtained and the individual resonances assigned. Several different empirical correlations developed by others have been combined in simple calculations to predict chemical shift values for sesquiterpenols of the eudesmane group.     

Proton nuclear magnetic resonance spectra of excised rat brain. Assignment of resonances

We have recorded 1H NMR spectra of excised rat brain at 361 MHz using two different water suppression pulse sequences. The assignment of the resonances has been carried out in perchloric acid extracts and subcellular fractions. Our results show that cytosolic proteins, membrane phospholipids and 16 different metabolites contribute to the observed spectra. The new resonances assigned allow the direct observation of myo-inositol and urea. Moreover, changes in the spectral pattern upon anesthesia, ischemic exposure of the brain and age of the rat have been recorded and correlated with the compounds producing the spectra.     

13C nuclear magnetic resonance spectra of 1,2-dioctadec-cis-enoyl-sn-glycero-3-phosphorylcholines.

The 13C NMR spectra of all sixteen 1,2-dioctade-cis-enoyl-sn-glycero-3-phosphorylcholines have been obtained. Resonance lines of the olefinic, methylene, methyl and carboxyl carbon nuclei are sufficiently characteristic to permit unequivocal designation of double bond position for each isomer. Two resonances of the sn-glycero-3-phosphorylcholine structure have been reassigned.     

Carbon-13 nuclear magnetic resonance spectra of D-homoannulated 17-hydroxypregnan-20-ones

The 13C-NMR spectra of several groups of isomeric D-homoannulated 17 alpha-hydroxypregnan-20-ones have been recorded. The chemical shifts of the various carbon atoms have been correlated with the structures of the different isomers.     

Proton nuclear magnetic resonance study of cobrotoxin.

Cobrotoxin (Mr 6949), which binds tightly to the acetylcholine receptors, contains no phenylalanines and only two histidines, two tyrosines, and one tryptophan that result in well-resolved aromatic proton resonances in D2O at 360 MHz. His-32, Tyr-25, and the Trp are essential for toxicity and may interact with the acetylcholine receptor. We assign two titratable resonances (pKa = 5.1) at delta = 9.0 and 7.5 ppm at pH 2.5 and at 7.7 and 7.1 ppm at pH 9.5 to the C-2 and C-4 ring protons, respectively, of His-4. Two other titratable resonances (pKa = 5.7) at delta = 8.8 and 6.9 ppm at pH 2.5 and at 7.8 and 6.7 ppm at pH 9.5 are assigned to the C-2 and C-4 ring protons of His-32, respectively. The differences in delta values of the two histidines reflect chemically different microenvironments while their low pKa values could arise from nearby positive charges. A methyl resonance gradually shifts upfield to delta approximately 0.4 ppm as His-4 is deprotonated and is tentatively assigned to the methyl group of Thr-14 or Thr-15 which, from published X-ray studies of neurotoxins, are located in the vicinity of His-4. Further, we have identified the aromatic resonances of the invariant tryptophan and individual tyrosines and the methyl resonance of one of the two isoleucines in the molecule. Several broad nontitrating resonances of labile protons which disappear at pH greater than 9 may arise from amide groups of the beta sheet in cobrotoxin.     

Proton nuclear magnetic resonance of neutral and acidic glycosphingolipids

A number of intact neutral glycosphingolipids (globo, asialoganglio, neolacto, and gala series), gangliosides, and sulfatide were analyzed by proton nuclear magnetic resonance (NMR) using dimethyl-d6 sulfoxide as a solvent at different conditions of measurement. The chemical shifts of amide proton of ceramide, N-acetylhexosamine and sialic acid moieties were positioned with regularity, thus providing their molar composition. The chemical shifts of amide proton in ceramide moiety differed with respect to constituent fatty acids; delta 7.45 to 7.52 ppm at 25 degrees C for the nonhydroxy acids and 7.32 to 7.42 ppm for the hydroxy acids. The chemical shifts of methyl proton in N-acetyl group were distinguished between N-acetylhexosamine and N-acetylneuraminic acid, and those in N-acetylgalactosamine were discriminated between neutral glycolipids and gangliosides. In the presence or absence of D2O in dimethyl sulfoxide at 110 degrees C, the anomeric protons resonated with regularity characteristic of respective monosaccharide linkages, and the anomeric protons of N-acetylgalactosamine in neutral glycolipids and gangliosides were clearly distinguished. The present study thus demonstrates the general applicability of NMR procedure to glycosphingolipids, providing the determination of chemical composition of both the lipophilic and carbohydrate moieties and the structural elucidation.     

Proton nuclear magnetic resonance of human muscle extracts

High resolution proton nuclear magnetic resonance (NMR) spectra of normal and diseased human muscle extracts were recorded at 470 MHz. Resonances from lactic acid, creatine, glucose, ribose, purine and pyrimidine bases were identified. The longitudinal relaxation times of these resonances were determined to allow quantitation of muscle metabolites. With aid of a standardized reference capillary, inserted into the NMR tube containing the muscle extracts, the lactic acid and total creatine content of the extracts was determined. After 5 h of incubation at 37 degrees C, normal muscles contained on average 103 mumol lactic acid and 36 mumol creatine/173 mg of noncollagenous protein, equivalent to 1.0 g of fresh muscle. The lactic acid and creatine contents decreased slightly in scoliosis and idiopathic scoliosis and they decreased significantly in cerebral palsy. In an extract of a patient whose illness was diagnosed as 'scoliosis' no creatine was present, and in an extract of a patient with unknown diagnosis the creatine content was reduced to 2 mumol/173 mg of noncollagenous protein. The short time (1.7 sec to 6.5 min) and the small amount of tissue (300 mg) needed for an analysis add to the potential of proton NMR as a new technique for the characterization of muscular diseases.