Self-association of puromycin as studied by proton magnetic resonance spectroscopy

The self-association of puromycin has been studied using proton magnetic resonance spectroscopy. The concentration, temperature and pH dependence studies of the proton chemical shifts of the adenine protons indicate that puromycin in aqueous solution at pD 7.4 self associates predominantly through adenine-adenine interaction. At this pD, the amino group of the aminoacyl segment of puromycin has been demonstrated to exist in a equilibrium blend of protonated and non-protonated forms. At pD 2.6, PM is found to exist predominantly in the monomeric from in which the methyl groups of the 6N-dimethyladenine are found to be non-equivalent due to hindered rotation about the C6-N6 bond.     

Analysis of brain metabolism by proton magnetic resonance spectroscopy (1H-MRS) in attention-deficit/hyperactivity disorder suggests a generalized differential ontogenic pattern from controls

Attention-deficit/hyperactivity disorder (ADHD) is the most common behavioral disorder of childhood. Preliminary studies with proton magnetic resonance spectroscopy (¹H-MRS) of the brain have reported differences in brain metabolite concentration-to-Cr ratios between individuals with ADHD and unaffected controls in several frontal brain regions including anterior cingulate cortex. Using multivoxel ¹H-MRS, we compared 14 individuals affected with ADHD to 20 individuals without ADHD from the same genetic isolate. After controlling by sex, age, and multiple testing, we found significant differences at the right posterior cingulate of the Glx/Cr ratio density distribution function between ADHD cases and controls (P?<?0.05). Furthermore, we found several interactions of metabolite concentration-to-Cr ratio, age, and ADHD status: Ins/Cr and Glx/Cr ratios at the left posterior cingulate, and NAA/Cr at the splenius, right posterior cingulate, and at the left posterior cingulate. We also found a differential metabolite ratio interaction between ADHD cases and controls for Ins/Cr and NAA/Cr at the right striatum. These results show that: (1) NAA/Cr, Glx/Cr, and Ins/Cr ratios, as reported in other studies, exhibit significant differences between ADHD cases and controls; (2) differences of these metabolite ratios between ADHD cases and controls evolve in specific and recognizable patterns throughout age, a finding that replicates previous results obtained by structural MRI, where is demonstrated that brain ontogeny follows a different program in ADHD cases and controls; (3) Ins/Cr and NAA/Cr ratios, at the right striatum, interact in a differential way between ADHD cases and controls. As a whole, these results replicate previous 1H-MRS findings and add new intriguing differential metabolic and ontogeny patterns between ADHD cases and controls that warrant further pursue.     

Content of intramyocellular lipids derived by electron microscopy, biochemical assays, and (1)H-MR spectroscopy.

Three different methods to determine intramyocellular lipid (IMCL) contents in human skeletal muscle have been compared. (1)H-magnetic resonance spectroscopy (MRS) was evaluated against electron microscopic morphometry and biochemical assays of biopsy samples from m. tibialis anterior of 10 healthy subjects. The results of (1)H-MRS and morphometry were strongly correlated, proving the validity of the (1)H-MRS results for the noninvasive determination of IMCL. Biochemical assays yielded results that did not significantly correlate with the results of the other methods. When IMCL levels obtained from the three methods are expressed in common units, it was found that (1)H-MRS yielded IMCL average levels that were 1.8 times lower than those found by morphometry. Potential reasons for the discrepancy are discussed. It is expected that (1)H-MRS will be suitable to replace invasive techniques for IMCL determination, whenever noninvasiveness is crucial, e.g., for repeated investigations in studies of substrate recruitment and recovery in exercise.     

Intracellular metabolic compartmentation assessed by 13C magnetic resonance spectroscopy

Our understanding of the brain has developed from the theory that it is one continuous cell to the knowledge that there are many brain cells originally termed neurons and, furthermore to the discovery of glial cells and their multiple functions. Thus, an increasing complexity was unraveled and we have not reached a complete understanding of the phenomenon which comprises the compartmentation of metabolic pathways and metabolites. This is an important principle needed to fully understand the metabolic processes of the brain. At the cellular level this concept is well established whereas intracellular compartmentation has yet to be explored. Using magnetic resonance spectroscopy (MRS) for analysis of isotopomer composition combined with quantification of amino acid contents it is possible to construct models that describe intracellular compartmentation. Results of studies of cultures of astrocytes and neurons incubated in media containing [U- 13C]glutamate in the presence or absence of thiopental may be used to propose an intracellular three compartment model of mitochondrial function. Due to the experimental paradigm only certain aspects of metabolism can be described. The present model consists of compartments assigned to CO(2) production, glutamate synthesis from ketoglutarate and finally synthesis of a four-carbon metabolite which is shuttled between compartments. It is likely that metabolism may be far more complex than this and we are only beginning to glimpse some aspects of compartmentation at the cellular level.     

Elucidation of glycolipid structure by proton nuclear magnetic resonance spectroscopy

The primary structure of the oligosaccharide moiety of a glycosphingolipid can be elucidated by employing high-field proton nuclear magnetic resonance (NMR) spectroscopy. Information with respect to the composition and configuration of its sugar residues, and the sequence and linkage sites of the oligosaccharide chain can be obtained by employing a variety of one- and two-dimensional techniques. The latter include both scalar and dipolar correlated two-dimensional NMR spectroscopy. These techniques are also useful in establishing the solution conformation (secondary structure) of the oligosaccharide moiety. Examples in utilizing these techniques in elucidating the primary and secondary structures of glycolipids are presented.     

Nucleophilic selectivity and reaction kinetics of chloroethylene oxide assessed by the 4-(p-nitrobenzyl)pyridine assay and proton nuclear magnetic resonance spectroscopy

The nucleophilic selectivity (Swain-Scott's constant s) of chloroethylene oxide (CEO), an ultimate carcinogenic metabolite of vinyl chloride, was determined to be 0.71 using the 4-(p-nitrobenzyl)pyridine (NBP) assay (Spears method). The molar extinction coefficient of the adduct formed between NBP and CEO was measured; and the second-order rate constants for the reactions of CEO with NBP and with thiosulfate were estimated at three temperatures. The disappearance of CEO and the formation of chloroacetaldehyde (CAA) and glycolaldehyde (GCA) were followed in D2O or a mixture of D2O/hexadeuterated acetone (acetone-d6), using Fourier transform proton nuclear magnetic resonance spectroscopy (1H-FTNMR). Evidence was obtained that CEO reacts with chloride ions to yield CAA at a rate constant of about 17 M-1 h-1 in D2O/acetone-d6 (1 : 1, v/v) at 280 K. Under the same conditions, the first-order rate constant kr for the thermal rearrangement of CEO into CAA was estimated to be approximately 0.41 h-1. These data suggest that the isomerization of CEO may be a minor reaction in physiological saline. These chemical properties of CEO are discussed in relation to the mechanism of vinyl chloride-induced carcinogenesis.     

Biotransformations monitored in situ by proton nuclear magnetic resonance spectroscopy

One-dimensional Fourier-transform proton nuclear magnetic resonance (1H-NMR) spectroscopy can be used to study biotransformations in situ, in vivo and in aqua (1H2O). Although an insensitive method, it rapidly provides solution-structural information of mixtures of diverse compounds that are used and formed during enzymic reactions and culture fermentations; the samples do not require any physical or chemical processing for analysis. The absolute stereochemistry of some reactions can also be determined, and assessments of metabolic fluxes made. This technique, with appropriate modifications, is of obvious value for on-line assessments of industrial fermentation processes.     

Amyloid oligomer formation probed by water proton magnetic resonance spectroscopy

Formation of amyloid oligomers, the most toxic species of amyloids in degenerative diseases, is critically coupled to the interplay with surrounding water. The hydrophobic force driving the oligomerization causes water removal from interfaces, changing the surface-hydration properties. Here, we show that such effects alter the magnetic relaxation response of local water in ways that may enable oligomer detection. By using water proton magnetic resonance spectroscopy, we measured significantly longer transverse magnetic relaxation (T₂) times in mixtures of serum and amyloidogenic Aβ_1-42 peptides versus similar concentration solutions of serum and nonamyloidogenic scrambled Aβ_42-1 peptides. Immunochemistry with oligomer-specific antibodies, electron microscopy and computer simulations demonstrated that the hyperintense magnetic signal correlates with Aβ_1-42 oligomerization. Finding early biophysical markers of the oligomerization process is crucial for guiding the development of new noninvasive imaging techniques, enabling timely diagnosis of amyloid-related diseases and pharmacological intervention.     

Conformational analysis of thyrotropin releasing factor by proton magnetic resonance spectroscopy

The proton magnetic resonance spectrum of thyrotropin releasing factor (TRF) in solution in deuterium oxide and deuterated dimethylsulfoxide (DMSO–d₆) has been analyzed. Two forms differing in cis–trans isomerism about the His-Pro peptide bond are observed. From the temperature dependence of chemical shift of the amide protons, it is concluded that TRF in DMSO–d₆ does not contain intramolecular hydrogen bonds. Measurement of NH? C_αH coupling constant provides an estimate of the histidine dihedral angle ?. Structural information about the histidine side-chain is deduced from C_αH? C_βH coupling constants and from the nonequivalence of the two prolyl δ-protons. In DMSO–d₆, there is evidence for a tautomeric equilibrium corresponding to an exchange of imidazole proton between the two nitrogen atoms N-δ and N-ε. In water, the N-εH tautomer is found to be the predominant tautomeric form of the imidazole ring. These results in combination with energy calculation, vibrational analysis, and carbon nmr studies allow the determination of the conformationof TRF.     

Partial characterization of dermatan sulfate by proton nuclear magnetic resonance spectroscopy

The degree of galactosamine N-acetylation, iduronic acid composition, and total uronic acid/hexosamine ratios of the three dermatan sulfates of human skin, DS18, DS28, and DS35 (M. O. Longas et al. (1987) Carbohydr. Res. 159, 127-136), were determined by Fourier transform, proton nuclear magnetic resonance (FT 1H NMR) spectroscopy. Analysis of DS of varying ages was conducted at 400 MHz and 60 degrees C. Chemical shifts for H-1, H-2, H-4, and H-5 of L-IdUA were independent of those for the respective protons of D-GalNAc and D-GlcUA. The resonance intensities of H-1 and acetamido methyl protons of D-GalNac did not display the expected 1:3 ratios. Therefore, their integration values were employed to estimate the percentage N-acetylation (N-CH3/3 H-1) which was corroborated chemically. The L-IdUA content, relative to total uronic acid, was calculated from signal intensities of H-1 of L-IdUA and D-GlcUA and ascertained by quantitative chemical methods. Total uronic acid/hexosamine ratios were determined from both 1H NMR spectroscopy and chemical analyses. The data show the following N-acetylation (N-CH3/3 H-1) of galactosamine in DS:DS18, 61-72% between 17 and 60 years, unaffected by senescence; DS28, 78-86% with no age-related trend; DS35, 101% at 19 years. Furthermore, in all ages investigated, the percentage (wt/wt) L-IdUA relative to total uronic acid was 42-44% for DS18 and 37-40% for DS28. At age 19 years, DS35 had a 29% (wt/wt) L-IdUA. The total uronic acid/hexosamine ratios for DS18 and DS28 varied from 1.40:1.0 to 1.70:1.0 irrespective of age.     

Identification and measurement of methylamines in elasmobranch tissues using proton nuclear magnetic resonance (1H-NMR) spectroscopy

Methylamines are frequently present in high concentrations in biological samples, but their separation and quantification are difficult. Data presented show that methylamines commonly occurring in biological material can be uniquely identified and quantified by proton nuclear magnetic resonance spectroscopy by recording spectra at both neutral and acid pH. Use of a high sensitivity probe permits this analysis even in the presence of high water concentrations, allowing accurate quantification with minimum preparative technique. The method was tested on tissues of the dogfish. Trimethylamine oxide was found in amounts ranging from 42 mmol kg⁻¹ fresh weight in liver, up to 115 mmol kg⁻¹ fresh weight in heart. Betaine was found to range from 10 mmol kg⁻¹ fresh weight in liver to 49 mmol kg⁻¹ fresh weight in brain. Creatine was not found in heart or liver, but was present in body wall muscle and in brain. Further analysis using high-performance liquid chromatography allowed determination of urea/methylamine ratios, which ranged from 1.9 in liver to 3.7 in body wall muscle. Accepted: 7 October 1997     

Reversible trifluoroacetic acid-induced conformational changes in glycophorin as detected by proton nuclear magnetic resonance spectroscopy

High-field (270 MHz) ¹H-NMR has been employed to study the solution conformation of glycophorin A, a sialoglycoprotein which spans the human erythrocyte membrane. Glycophorin A is one of the most fully characterized integral membrane proteins known, making it an excellent model for the study of membrane-bound proteins. This protein consists of three distinct domains: a glycosylated extracellular N-terminus, a hydrophobic intramembranous segment, and a polar cytoplasmic C-terminus. These domains contain aromatic residues which serve as convenient ¹H-NMR conformational probes. The aromatic region of the NMR spectrum of glycophorin A in ²H₂O shows single, well-resolved His and Tyr resonances. No resonances are observed, however, for the Phe residues which are located in or near the hydrophobic domain. These observations suggest that considerable heterogeneity with respect to segmental motions exists within the protein. This is consistent with circular dichroism data showing the intramembranous segment to be completely helical with the extremities of the protein being predominantly random coils. The helix of the hydrophrobic domain is remarkably resistant to conventional denaturing conditions including variations in pH, and temperature, and treatment with guanidine hydrochloride. However, in trifluoroacetic acid, which strongly solvates peptide backbones, there is extensive reversible unfolding of the helical structure as evidenced by the appearance of Phe resonances. Solvent titration experiments indicate that approximately a 1 : 1 volume ratio of trifluoroacetic acid to ²H₂O is required to initiate unfolding of the helix.     

High resolution proton nuclear magnetic resonance spectroscopy of lactoperoxidase

The first high resolution proton nuclear magnetic resonance spectra are reported for the native ferric and ferric cyano complexes of bovine lactoperoxidase. The spectrum of the native species exhibits broad heme signals in a far downfield region characteristic of the high-spin ferric state. The low-spin cyano complex yields a proton nuclear magnetic resonance spectrum with signals as far as 68.5 ppm downfield and as far as -28 ppm upfield of the tetramethylsilane reference. These peak positions are anomalous with respect to those seen only as far as 35 ppm downfield in other cyano hemoprotein complexes. An extreme asymmetry in the unpaired spin delocalization pattern of the iron porphyrin is suggested. The unusual proton nuclear magnetic resonance properties parallel distinctive optical spectral properties and the exceptional resistance to heme displacement from the enzyme. Lactoperoxidase utilized in these studies was isolated from raw milk and purified by an improved, rapid chromatographic procedure.