Carbon-13 nuclear magnetic resonance studies of cobalamins

The carbon-13 nuclear magnetic resonance spectra of aquocobalamin, adenosylcobalamin, methylcobalamin, and (carboxymethyl)cobalamin have been interpreted. The assignments were made by a comparison of the spectra with that of cyanocobalamin, by a study of the pH dependence of the chemical shifts, by an analysis of the effect of the axial ligands on the carbon atoms of the corrin ring, and by a study of the specific line broadening effect of the paramagnetic ions Mn2+ and Gd3+. The chemical shift changes that accompany the "base-on"----"base-off" conversion of the organocobalamins demonstrate that the conformation of the "western" half of the corrin ring and the conformations of the a, b, c, d, f, and g side chains are relatively constant. In contrast, the conformations of the "eastern" half of the corrin ring and the e propionamide side chain are highly variable.     

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.     

Carbon-13 nuclear magnetic resonance studies of cerebroside derivatives and their properties in lecithin bilayers (1)

The ¹³C NMR chemical shifts and spin-lattice relaxation times of D-galactosylsphingosine derivatives in CDCl₃-CD₃OD and in egg-yolk lecithin vesicles in D₂O, and of N-acetylpsychosine micelles, are reported. Results with sonicated, unilamellar vesicles containing cerebroside and EYL^a show that (1) cerebrosides decrease the fluidity of the lecithin bilayer membrane and have the greatest effect on the glycerol backbone and choline methyl carbons. (2) N-acetylpsychosine experiences a greater freedom of motion in the galactose region than does cerebroside and does not reduce the fluidity of the lecithin as much as cerebroside. (3) Ac-Psy/EYL vesicles formed are permeable to Yb³⁺ but cerebroside/lecithin vesicles are not. (4) The choline groups on the inner bilayer surface are less mobile than those on the outer surface according to preliminary T₁ measurements of the Yb³⁺-separated resonances. (5) Yb³⁺-induced chemical shifts of choline methyl and choline $\text{C}\text{H}{}_2$OP peaks in mixed cerebroside-lecithin vesicle systems indicate a small preference for cerebroside in the outside monolayer. The data show that these molecules have significant effects on bilayer conformational mobilities, particularly near the surface, and thus demonstrate one mechanism for modulation of cell surface properties by glycosphingolipids.     

Carbon-13 and proton nuclear magnetic resonance of unsonicated model and mitochondrial membranes

Proton nuclear magnetic resonance (PMR) spectra at 270 MHz of aqueous dispersions of nonsonicated egg lecithin, dipalmitoyl lecithin, egg lecithin-cholesterol (1 : 1) and dipalmitoyl lecithin-cholesterol (1 : 1), together with PMR spectra of mitochondrial membranes and their extracted lipids, have been obtained.Carbon-13 nuclear magnetic resonance (CMR) spectra at 25.2 MHz of egg lecithin, egg lecithin-cholesterol (1 : 1) and sphingomyelin, together with CMR spectra of chloroplast and mitochondrial membranes, and erythrocyte ghosts, have also been obtained. The results obtained using CMR appear very promising for further study of intact membrane structure.It is suggested, on the basis of CMR evidence, that the proteins in mitochondrial membranes may be considerably less mobile than the lipids.     

Carbon-13 nuclear magnetic resonance studies of spironolactone and several related steroids

The ¹³c chemical shifts for all the carbon atoms in spironolactone have been assigned. Assignments for nine additional steroids which include the C-7β isomer of spironolactone, its C-7 thiol hydrolysis product, the 7α-thioacetate derivative of testosterone and its thiol hydrolysis product are also reported.     

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).     

Carbon-13 nuclear magnetic resonance studies on tobacco mosaic virus and its protein.

Fourier transform nuclear magnetic resonance studies on 12% 13C-enriched tobacco mosaic virus (TMV) and its rod-like protein oligomers in solution with molecular weights up to 42 X 10(6) are reported. In the virus approximately 17% of the carbons of the protein subunit have line widths of less than or equal to 300 Hz and T1 less than or equal to 1 s and are concluded to be mobile with more than one degree of freedom of internal rotation about a carbon--carbon bond. In the rodlike polymer of TMV protein at pH 5.3, 30% of the carbons are mobile, which implies rotational motions about carbon--carbon bonds and/or motions of the protein subunits within the polymer. The presence of internal mobility is supported by the observation that 20% of the carbons in the double disklike oligomer show decreasing line width upon increasing temperature; the remaining resonances have line widths which are temperature independent during the double disklike polymerization process. Since the molecular weight of TMV protein polymers increases with increasing temperature, this demonstrates that all nuclei within the double dislike oligomer are mobile. NMR and X-ray data on the double disklike polymer reveal differences with respect to internal mobility.     

Carbon-13 and proton magnetic resonance of mouse muscle.

It is shown that roughly 4 mmol carbon atoms/g mouse muscle can give rise to a "high resolution" 13C NMR spectrum. From the 13C spectrum, it is estimated that the protons from mobile organic molecules or molecular segments amount to 6-8%of total nonrigid protons (organic plus water) in muscle. Their spin-spin relaxation times (T2) are of the order of 0.4-2 ms. At 37 degrees C, the proton spin-echo decay of mouse muscle changes rapidly with time after death, while that of mouse brain does not.     

Metabolism of methanol in plant cells. Carbon-13 nuclear magnetic resonance studies

Using (13)C-NMR, we demonstrate that [(13)C]methanol readily entered sycamore (Acer pseudoplatanus L.) cells to be slowly metabolized to [3-(13)C]serine, [(13)CH(3)]methionine, and [(13)CH(3)]phosphatidylcholine. We conclude that the assimilation of [(13)C]methanol occurs through the formation of (13)CH(3)H(4)Pte-glutamate (Glu)(n) and S-adenosyl-methionine, because feeding plant cells with [3-(13)CH(3)]serine, the direct precursor of (13)CH(2)H(4)Pte-Glu(n), can perfectly mimic [(13)CH(3)]methanol for folate-mediated single-carbon metabolism. On the other hand, the metabolism of [(13)C]methanol in plant cells revealed assimilation of label into a new cellular product that was identified as [(13)CH(3)]methyl-beta-D-glucopyranoside. The de novo synthesis of methyl-beta-D-glucopyranoside induced by methanol did not require the formation of (13)CH(3)H(4)Pte-Glu(n) and was very likely catalyzed by a "transglycosylation" process.     

Carbon-13 nuclear magnetic resonance studies of adenosylcobalamin and alkylcorrinoids, selectively enriched with carbon-13.

The carbon-13 nuclear magnetic resonance spectra of a series of alkylcorrinoids, selectively enriched with 13C in the alkyl ligand, were recorded at 25.2 MHz and 25 degrees. The nature of the axial ligands markedly affects the chemical shift of the labeled alkyl moiety (trans effect) as well as the 13C resonances of selected carbon atoms of the corrin ring (cis effect). Although a number of factors appear to influence the trans effect on the chemical shift of the alkyl ligand (important among them being electric field effects), the cis effect appears to be dominated by changes in charge density (at the methine bridge carbon atoms, C-5, C-10, C-15) and by steric effects (at the methyl groups at C-1, C-5, and C-15) accompanying axial ligation. Spin-latice relaxation times of several organocorrinoids, selectively labeled with 13C in the ligands attached to cobalt, were also measured. The T1 values of the methylene carbons of [5'-13C]adenosylcobalamin and [2-13C]carboxymethylcobalamin are very similar to that of the methine bridge carbon atom C-10 of the corrin ring, indicating that rotation about the carbon-cobalt bond of these two corrinoids is severely restricted. On the other hand, internal rotation about the carbon-cobalt bond of methylcobalamin is rapid.     

Carbon-13 nuclear magnetic resonance studies of myocardial glycogen metabolism in live guinea pigs

Myocardial glycogen metabolism was studied in live guinea pigs by 13C NMR at 20.19 MHz. Open-chest surgery was used to expose the heart, which was then positioned within a solenoidal radio frequency coil for NMR measurements. The time course of myocardial glycogen synthesis during 1-h infusions of 0.5 g of D-[1-13C]glucose (and insulin) into the jugular vein was investigated. The possible turnover of the 13C-labeled glycogen was also studied in vivo by following the labeled glucose infusion with a similar infusion of unlabeled glucose. The degree of 13C enrichment of the C-1 glycogen carbons during these infusions was measured in heart extracts by 1H NMR at 360 MHz. High-quality proton-decoupled 13C NMR spectra of the labeled C-1 carbons of myocardial glycogen in vivo were obtained in 1 min of data accumulation. This time resolution allowed measurement of the time course of glycogenolysis of the 13C-labeled glycogen during anoxia by 13C NMR in vivo. With the solenoidal coil used for 13C NMR, the spin-lattice relaxation time of the labeled C-1 carbons of myocardial glycogen could be measured in vivo. For a comparison, spin-lattice relaxation times of heart glycogen were measured in vitro at 90.55 MHz. Natural abundance 13C NMR studies of the quantitative hydrolysis of extracted heart glycogen in vitro at 90.55 MHz showed that virtually all the carbons in heart glycogen contribute to the 13C NMR signals. The same result was obtained in 13C NMR studies of glycogen hydrolysis in excised guinea pig heart.     

Carbon-13 nuclear magnetic resonance spectroscopy of flavonoid and isoflavonoid compounds

The ¹³C NMR spectra of 15 flavonoid and 9 isoflavonoid substances of various ring C oxidiation states were analyzed and their carbon shifts assigned. In the case of 3 terpenic flavones and two glycoflavones linewidths were related qualitatively to molecular segmental motion.     

Carbon-13 nuclear magnetic resonance studies of polyamino acids: the helix-coil transition of poly-L-lysine

The helix-coil transition of poly-l-lysine hydrochloride ((Lys)_n) in aqueous solution has been studied by ¹³C Fourier-transform nuclear magnetic resonance spectroscopy. As reference compounds dodeca-l-lysine hydrobromide ((Lys)^?₁₂, tri-l-lysine hydrochloride ((Lys)₃), and l-lysine hydrochloride (Lys), have been also studied by the same method. It is found that ¹³C spin-lattice relaxation times t₁ of the carbonyl and the side-chain carbons decrease sharply at pD 10.2 which is the midpoint of the transition from the random-coil to the α-helix. Similarly the T₁ values of the carbonyl groups of (Lys)^?₁₂ decrease at this point in a more moderate way, while no change is observed for those of the side-chain carbons. This is interpreted in terms of the reduced α-helicity involved for (Lys)^?₁₂.The variation of ¹³C chemical shifts with pD for (Lys)_n and (Lys)^?₁₂ show the same trend:downfield shifts at higher pD. Furthermore, nonterminal and C-terminal residues of (Lys)₃ show similar behavior. Thus it is concluded that the ¹³C chemical shift changes are caused mainly by the pD changes and not by the conformational transition. Conversion from α-helix to β-structure by elevation of temperature at pD 11.2 results in narrowing and downfield shifts of the ¹³C resonances of (Lys)_n.     

Proton and carbon-13 nuclear magnetic resonance studies on methyl (methyl d-galactosid)uronates and their per-O-acetyl derivatives

The ¹H- and ¹³C-n.m.r. spectra of the anomeric methyl (methyl d-galactosid)uronates, as well as the ¹H-n.m.r. spectra of their acetyl derivatives, were analyzed. The spectra of the unacetylated d-galactopyranosiduronates showed good correlation with those of the corresponding anomeric d-galactopyranuronic acids and their methyl esters, and with those of the anomeric methyl d-galactopyranosides. From the values of the chemical shifts and coupling constants, it was concluded that the anomeric methyl (methyl d-galactopyranosid)uronates and their corresponding peracetates are in the ⁴C₁(d) conformation. The chemical shifts in the ¹³C-n.m.r. spectra show good correlation with those of the methyl d-galactosides. The signals of the furanose derivatives appear at fields lower than those of the corresponding pyranose compounds.     

Carbon-13 nuclear magnetic resonance of heme carbonyls. Cytochrome c and carboxymethyl derivatives of cytochrome c.

Carbonyl complexes of horse cytochrome c and various carboxymethylated derivatives have been examined using 13C NMR (carbon-13 nuclear magnetic resonance) spectroscopy. The multiplicity and chemical shift of the 13CO resonance were found to be functions of pH and the extent of alkylation. Correlations have been made among prominent features of the chemical shift titration curves and changes in the environment of the heme. A simple model compatible with the bulk of previous observations has been suggested to account for the several carbonyl resonance peaks and the complex behavior of the chemical shift with changes in pH.     

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.     

Carbon-13 nuclear magnetic resonance studies of acetate metabolism in intact cells of Rhodopseudomonas sphaeroides

¹³C-nuclear magnetic resonance was used to study the metabolism of [2-¹³C]acetate in suspensions of Rhodopseudomonas sphaeroides. In the dark, in logarithmic-phase cells the ¹³C label appeared first in butyrate C-2 and C-4 and subsequently in glutamate C-4 and succinate C-2 and C-3. In the light, synthesis of poly(β-hydroxybutyrate) (PHB) takes place. Butyrate synthesis seems to be independent of PHB synthesis or degradation activity. Starved, logarithmic-phase cells also show massive synthesis of PHB in the dark. Stationary-phase cells incorporate ¹³C predominantly into glutamate and succinate. No significant butyrate biosynthesis can be detected in the dark or during illumination. The incorporation of label in PHB is very slow in these cells and most probably originates from exchange of ¹²C for ¹³C into PHB. This might indicate slow turnover without net synthesis of the polymer occurring under these conditions. The results are discussed in relation to the redox state and the availability of metabolic energy for biosynthetic reactions in the dark and during illumination of cell suspensions of Rps. sphaeroides.     

High-resolution proton nuclear magnetic resonance studies of human gastrin

High-resolution 1H nuclear magnetic resonance (NMR) spectroscopy at 300 MHz has been used to study the behavior of human gastrin in aqueous solution. A large number of resonances have been assigned by analysis of one- and two-dimensional NMR spectra and the effects of pH and by comparison with the spectrum of des-less than Glu1-gastrin. In gastrin, the ratio of cis to trans conformations around the Gly-2 to Pro-3 peptide bond is 3:7. This is reflected in splitting of the resonances of several neighboring residues and of a residue distant in the sequence, Tyr-12. The pKa of Tyr-12 is 10.7. Sulfation of this residue perturbs the resonances of Tyr-12 and Gly-13 but has very little effect on the rest of the spectrum. A study of the temperature dependence shows that several perturbed resonances move toward their expected positions as the temperature is raised but with a linear dependence on temperature, consistent with a redistribution of populations among accessible local conformations rather than a cooperative conformational change. Addition of Na+ or Ca2+ causes only minor changes in the spectrum. The paramagnetic metal ion Co2+ produces a number of spectral changes, reflecting strong binding to at least one site involving the Glu residues and weaker binding to Asp-16.