Natural Abundance Carbon Research Articles

Spectroscopy and imaging with a 4 tesla whole-body MR system.

Magnetic resonance (MR) spectroscopy and imaging experiments on humans were performed with a whole-body MR system at a static field of 4 tesla. Spectroscopic studies focussed on 1H, 13C, and 31P. Imaging of humans turned out to be possible, although below the optimum at this field. This holds especially for body imaging, since RF penetration effects and dielectric resonances influence the RF field homogeneity. Excellent volume selective proton spectra of the human cerebrum and cerebellum were obtained using the stimulated echo method. Natural abundance carbon spectra of the human calf were acquired both undecoupled and with narrowband decoupling, resolving the various triglyceride resonances. Broadband decoupling, however, would have violated SAR guidelines. Liver glycogen was detected on natural abundance 13C spectra.

Carbon isotopic fractionation in heterotrophic microbial metabolism

Differences in the natural-abundance carbon stable isotopic compositions between products from aerobic cultures of Escherichia coli K-12 were measured. Respired CO2 was 3.4% depleted in 13C relative to the glucose used as the carbon source, whereas the acetate was 12.3% enriched in 13C. The acetate 13C enrichment was solely in the carboxyl group. Even though the total cellular carbon was only 0.6% depleted in 13C, intracellular components exhibited a significant isotopic heterogeneity. The protein and lipid fractions were -1.1 and -2.7%, respectively. Aspartic and glutamic acids were -1.6 and +2.7%, respectively, yet citrate was isotopically identical to the glucose. Probable sites of carbon isotopic fractionation include the enzyme, phosphotransacetylase, and the Krebs cycle.

ADP-ribosylation of elongation factor 2 by diphtheria toxin. NMR spectra and proposed structures of ribosyl-diphthamide and its hydrolysis products.

NMR spectral analysis of the novel amino acid, diphthamide, in elongation factor 2 which is ADP-ribosylated by diphtheria toxin suggests that it is 2-[3-carboxyamido-3-(trimethylammonio)propyl]histidine. Ribosyl-diphthamide was prepared by enzymatic hydrolysis of ADP-ribosyl-elongation factor 2 and three compounds were produced by its chemical hydrolysis (Van Ness, B. G., Howard, J. B., and Bodley, J. W. (1980) J. Biol. Chem. 255, 10717-10720). Proton NMR spectroscopy in 2H2O of diphthine demonstrated the elements of histidine minus the carbon 2 proton plus 14 additional nonexchangeable protons. These protons were attributed to an extensive modification at carbon 2 of the imidazole ring. Proton NMR spectroscopy in 2H2O of ribosyl-diphthamide showed only those protons seen in diphthine plus those expected of a ribofuranosylmoiety. Chemical shift dependence on pH was demonstrated for the histidine-derived protons as well as several protons of the modifying side chain. Natural abundance carbon 13 NMR spectroscopy of ribosyldiphthamide also showed the elements of histidine and ribose plus 7 additional carbon atoms attributed to the modification of the imidazole ring. Based on the NMR spectral properties of the anomeric proton of ribosyldiphthamide we propose that the ribose is linked to one of the nitrogens of the histidine imidazole ring.

Studies of the carbohydrate residues of glycoproteins by natural abundance carbon 13 nuclear magnetic resonance spectroscopy. Glucoamylase from Aspergillus niger.

Studies of the carbohydrate residues of glycoproteins by natural abundance carbon 13 nuclear magnetic resonance spectroscopy. Glucoamylase from Aspergillus niger.

Titration behavior and tautomeric states of individual histidine residues of myoglobins. Application of natural abundance carbon 13 nuclear magnetic resonance spectroscopy.

Titration behavior and tautomeric states of individual histidine residues of myoglobins. Application of natural abundance carbon 13 nuclear magnetic resonance spectroscopy.

Studies of individual carbon sites of hen egg white lysozyme by natural abundance carbon 13 nuclear magnetic resonance spectroscopy. Assignment of the nonprotonated aromatic carbon resonances to specific residues in the sequence.

The resonances of nonprotonated aromatic carbons in natural abundance 13C NMR spectra of hen egg white lysozyme are assigned to specific residues of the amino acid sequence. Chemical shift considerations, the effect of pH, and partially relaxed Fourier transform NMR spectra are used to assign each resonance to one of the seven types of nonprotonated aromatic carbons of amino acid residues. Spectra of chemically modified lysozyme samples yield various assignments to specific residues in the sequence. Line-broadening effects caused by binding of the relaxation probes Gd3+ and 4-N-acetamido-2,2,6,6-tetramethylipiperidine-1-oxyl yield specific assignments which are fully consistent with those based on chemical modifications. The effects of paramagnetic shift reagents and amino sugar inhibitors do not yield any obvious specific assignments. The effect of pH on the chemical shift of Cgamma of His-15 yields a pKalpha in agreement with published values, and indicates that the imidazole form of His-15 exists mainly (or entirely) as the Nepsilon3-H tautomer. The effect of pH on the chemical shifts (measured up to pH 8.8, at 38 degrees) of Czeta and Cgamma of the 3 tyrosine residues yields crude pKalpha values of 9.5 and 10 for Tyr-23 and one of the other tyrosines, respectively. The 3rd tyrosine residue does not exhibit titration behavior.

Participation of tryptophan 62 in the self-association of hen egg white lysozyme. Application of natural abundance carbon 13 nuclear magnetic resonance spectroscopy.

Self-association of hen egg white lysozyme in solution of 38 degrees) is examined by means of natural abundance 13C nuclear magnetic resonance spectroscopy. The effect of pH on the resonances of the nonprotonated aromatic carbons of 9 mM lysozyme, and the effect of protein concentration (at pH 7) on these resonances, both indicate that self-association significantly affects the chemical shift of Cgamma of Trp-62, but not the chemical shifts of the other nonprotonated aromatic carbons. This result is consistent with the reported participation of Trp-62 in the intermolecular contact (Banerjee, S.K., Pogolotti, A., and Rupley, J.A. (1975) J. Biol. Chem. 250, 8260-8266). Our results indicate that the resonance of Cgamma or Trp-62 is a convenient monitor of lysozyme self-association. The chemical shift of this resonance reflects the extent of aggregation, while the line width yields information about the lifetime of the intermolecular contact. This lifetime is 1 to 2 ms at 38 degrees (9 mM protein, 0.1 M NaCl, pH 7). Our results also indicate that self-association of lysozyme is not accompanied by any general conformational change, and that binding of a lanthanide ion (at the metal ion binding site near the carboxylate groups of ASP-52 AND Glu-35) strongly suppresses self-association.

Studies of individual carbon sites of hemoglobins in solution by natural abundance carbon 13 nuclear magnetic resonance spectroscopy

Proton-decoupled natural abundance 13C NMR spectra of carbon monoxide hemoglobins were recorded at 15.18 MHz by the Fourier transform method, under conditions of spectrometer sensitivity sufficient for detection of individual carbon resonances. The aromatic region of each spectrum contains broad bands of methine carbon resonances, and some relatively narrow peaks arising from nonprotonated carbons. Resonances of heme carbons were detected in spectra of carbon monoxide hemoglobins, but not in spectra of ferrihemoglobin (as a result of paramagnetic effects). Spectra of carbon monoxide hemoglobins from various species yielded only a few well resolved individual carbon resonances, most notably those of Cgamma of tryptophan residues. A comparison of the spectra of human adult, human fetal, chicken AII, and bovine fetal hemoglobins yielded specific assignments for all resonances of Cgamma of tryptophan residues. In the cases of human fetal, chicken AII, and bovine fetal hemoglobins, each tryptophan yielded a completely resolved individual carbon resonance. The chemical shift difference between the resonances of Cgamma of Trp-130beta and Cgamma of Trp-37beta is about 6 ppm. The chemical shift difference between Trp A12[14]alpha and Trp A12[15]beta is 1 ppm or less. A comparison of the chemical shifts of analogous tryptophan residues of the four carbon monoxide hemoglobins suggests very similar conformations in solution.

Studies of individual carbon sites of proteins in solution by natural abundance carbon 13 nuclear magnetic resonance spectroscopy. Strategies for assignments

Natural abundance 13C Fourier transform NMR spectra (at 15.18 MHz, in 20-mm sample tubes) of aqueous native proteins yield numerous narrow single carbon resonances of nonprotonated aromatic carbons. Techniques for the assignment of these resonances are presented. Each technique is applied to one or more of the following proteins: ferricytochrome c from horse heart and Candida krusei, ferrocytochrome c and cyanoferricytochrome c from horse heart, lysozyme from hen egg white, cyanoferrimyoglobins from horse and sperm whale skeletal muscle, and carbon monoxide myoglobin from horse. In all of the protein spectra we have examined, methine aromatic carbons give rise to broad bands. Studies of the narrow resonances of nonprotonated aromatic carbons of proteins are facilitated by removal of these broad bands by means of the convolution-difference method, preferably from spectra recorded under conditions of noise-modulated off-resonance proton decoupling. We present a summary of the chemical shift ranges for the various types of nonprotonated aromatic carbons of amino acid residues and hemes of diamagnetic proteins, based on our results for hen egg white lysozyme, horse heart ferrocytochrome c, horse carbon monoxide myoglobin, and carbon monoxide hemoglobins from various species...

Studies of individual carbon sites of proteins in solution by natural abundance carbon 13 nuclear magnetic resonance spectroscopy. Relaxation behavior

The aromatic regions in proton-decoupled natural abundance 13C Fourier transform nuclear magnetic resonance spectra (at 14.2 kG) of small native proteins contain broad methine carbon bands and narrow nonprotonated carbon resonances. Some factors that affect the use of natural abundance 13C Fourier transform NMR spectroscopy for monitoring individual nonprotonated aromatic carbon sites of native proteins in solution are discussed. The effect of protein size is evaluated by comparing the 13C NMR spectra of horse heart ferrocytochrome c, hen egg white lysozyme, horse carbon monoxide myoglobin, and human adult carbon monoxide hemoglobin. Numerous single carbon resonances are observed in the aromatic regions of 13C NMR spectra of cytochrome c, lysozyme, and myoglobin. The much larger hemoglobin yields few resolved individual carbon resonances. Theoretical and some experimental values are presented for the natural linewidths (W), spin-lattice relaxation times (T1), and nuclear Overhauser enhancements (NOE) of nonprotonated aromatic carbons and Czeta of arginine residues. In general, the 13C-1H dipolar mechanism dominates the relaxation of these carbons. 13C-14N dipolar relaxation contributes significantly to 1/T1 of C epsilon2 of tryptophan residues and Czeta of arginine residues of proteins in D2O. The NOE of each nonprotonated aromatic carbon is within experimental error of the calculated value of about 1.2. As a result, integrated intensities can be used for making a carbon count. Theoretical results are presented for the effect of internal rotation on W, T1, and the NOE. A comparison with the experimental T1 and NOE values indicates that if there is internal rotation of aromatic amino acid side chains, it is not fast relative to the over-all rotational motion of the protein.

Natural Abundance Carbon 13 Nuclear Magnetic Resonance of Cyanoferrimyoglobins and of Some Carboxymethyl Derivatives

Abstract Proton-decoupled Fourier transform nuclear magnetic resonance of natural abundance 13C was used to obtain spectra of cyanoferrimyoglobins of harbor seal (Phoca vitulina) and sperm whale (Physeter catodon). These spectra were compared with those of hen egg white lysozyme and of bovine pancreatic ribonuclease A to show differences reflecting gross composition as well as differences tentatively attributable to more subtle conformational constraints. The cyanoferrimyoglobin spectra were much alike but not identical. Carboxymethylation of the native seal protein produced barely detectable changes at the chemical shift positions of the major adducts but no systematic alterations elsewhere in the spectrum. However, extensive modification of histidine and methionine residues by carboxymethylation of seal apomyoglobin in the presence of 8 m urea yielded a denatured product, judged by circular dichroism measurements, for which the 13C spectrum was sharp and characteristic of a denatured protein. This spectrum was used to tabulate tentative assignments. Partially relaxed spectra were used to obtain spin-lattice relaxation times (T1) for certain carbon types. These values are compared for the various myoglobin preparations mentioned above. The similarities between the native forms are striking. The denatured preparation gives many evidences of increased segmental mobility, especially for those nuclei that appear relatively restrained in the native state.

Carbon 13 Nuclear Magnetic Resonance of Peptides in the Amino-terminal Sequence of Sperm Whale Myoglobin

Abstract The natural abundance carbon 13 nuclear magnetic resonance spectra of the peptides l-valyl-l-leucyl-l-seryl-l-glutamylglycine, comprising the first 5 residues of the amino terminus of sperm whale myoglobin, l-leucyl-l-seryl-l-glutamic acid, and l-seryl-l-glutamylglycine have been recorded by continuous wave and pulsed Fourier transform techniques. Signal assignments were made by comparison of the peptides with each other and with the free amino acids, by the pH dependence of the chemical shifts, and by modification of the degree of proton decoupling in certain cases. The chemical shift dependence of specific carbon centers on their position in the oligopeptide sequences and the influence of the state of ionization of various groups on neighboring carbon sites were explored.