Analysis of NMR spectra of sugar chains of glycolipids by multiple relayed COSY and 2D homonuclear Hartman-Hahn spectroscopy.

We applied multiple relayed COSY and 2D homonuclear Hartman-Hahn spectroscopy to globoside, a glycolipid purified from human red blood cells. The subspectra corresponding to individual sugar components were extracted even from overlapping proton resonances by taking the cross sections of 2D spectra parallel to the F2 axis at anomeric proton resonances, so that unambiguous assignments of sugar proton resonances were accomplished.

Newly developed circulating blood volume-monitoring system and its clinical application for measuring changes in blood volume during hemofiltration.

Blood and ultrafiltrate electrical resistivities were continuously monitored during hemofiltration. By substituting these values into a previously developed equation that was modified by the authors, the hematocrit value was determined; and blood volume change was obtained from the change in this value. The following facts were discovered as the result of monitoring the blood volume change during treatment: When the body position was changed from the reclining to the sitting position, the blood volume decreased by 4.

Hexacyanochromate ion as a paramagnetic anion probe for active sites of enzymes.

Hexacyanochromate ion, (Cr(CN)6)3-, was applied to ribonuclease T1 (RNase T1), which specifically cleaves RNA chains at guanylic acid residues. From kinetic studies, this anion was shown to bind to the active site of RNase T1 as a competitive inhibitor. Therefore, the line broadening effect of NMR resonances due to binding of (Cr(CN)6)3- was analyzed for the mapping of the active site of RNase T1.

Conformations of fibroblast and E. coli-derived recombinant human interferon-beta s as studied by nuclear magnetic resonance and circular dichroism.

The conformations of fibroblast and E. coli-derived recombinant human interferon-beta s were studied by circular dichroism and nuclear magnetic resonance spectroscopy in the acidic pH region of 4.6 to 1.

Molecular conformation of alpha-bungarotoxin as studied by nuclear magnetic resonance and circular dichroism.

We have examined the circular dichroism and nuclear magnetic resonance spectra of a long neurotoxin, alpha-bungarotoxin, over a wide range of pH values and temperatures, and under high salt conditions. The observations are interpreted partly in terms of the known crystal structure of this polypeptide. We support earlier findings of a greater degree of beta-sheet structure in solution than has been reported by X-ray crystallography and, importantly, the invariant residue associated with neurotoxicity, Trp29, is shown to be in a similar environment to that found in alpha-cobratoxin and LS III from Laticauda semifasciata.

Binding modes of inhibitors to ribonuclease T1 as studied by nuclear magnetic resonance.

The binding modes of inhibitors to ribonuclease T1 (RNase T1) were studied by the analyses of 270-MHz proton NMR spectra. The chemical shift changes upon binding of phosphate, guanosine, 2'-GMP, 3'-GMP, 5'-GMP, and guanosine 3',5'-bis(phosphate) were observed as high field shifted methyl proton resonances of RNase T1. One methyl resonance was shifted upon binding of phosphate and guanosine nucleotides but not upon binding of guanosine.

Structural dynamics of erabutoxin b. A 13C nuclear magnetic resonance relaxation study of methyl groups.

A detailed examination has been made of the 13C NMR relaxation times of the assigned methyl groups of erabutoxin b. Anisotropic rotation was analysed using a restricted diffusion model. The results are compared with a previous study of the 1H NMR relation times.

Local conformational transition of toxin B from Naja naja as studied by nuclear magnetic resonance and circular dichroism.

The 270-MHz proton NMR spectra of toxin B, a long neurotoxin from Naja naja, in 2H2O solution were analyzed. From the analysis of the discontinuous pH dependence of His-21 C-2 proton chemical shift a 23 degrees C, toxin B is found to exhibit a slow-exchange conformational transition between the states A and B with the transition midpoint at pH 5.75.

Individual assignments of the amide proton resonances involved in the triple-stranded antiparallel pleated beta-sheet structure of a long neurotoxin, Laticauda semifasciata III from Laticauda semifasciata.

THe characteristic feature of the crystal structure of erabutoxin b, a short neurotoxin from Laticauda semifasciata, and alpha-cobratoxin, a long neurotoxin from Naja naja siamensis, is the presence of a triple-stranded antiparallel pleated beta-sheet structure formed by the central and the third peptide loops. In the present study, we have studied the assignment of slowly exchangeable amide protons of Laticauda semifasciata III from L. semifasciata, using nuclear Overhauser effects (NOE) and spin-decoupling methods.

Two-dimensional NMR spectroscopy of siomycin A. Proton--carbon-13 chemical shift correlation.

A trial application of a recent two-dimensional nuclear magnetic resonance experiment to the polypeptide antibiotic siomycin A is described. Proton--carbon-13 chemical shift correlation measures the proton and carbon-13 chemical shift for each directly bonded CH group in a molecule, in a single experiment. The resultant map of correlated chemical shifts enables the carbon-13 spectrum to be assigned directly from the known proton shifts, and allows individual proton signals to be identified without problems of overlap.

Dynamics of erabutoxin b as studied by nuclear magnetic resonance. Relaxation studies of methyl proton resonances.

Longitudinal and transverse relaxation times were measured for well-resolved and assigned methyl proton resonances of erabutoxin b at 270 MHz, 300 MHz and 500 MHz. Both longitudinal and transverse magnetization decay curves are non-exponential due to cross-relaxation and cross-correlation effects. The longitudinal and transverse relaxation rates were obtained from the initial slope of both magnetization decay curves.

A proton-magnetic-resonance study on the molecular conformation and structure-function relationship of a long neurotoxin, laticauda semifasciata III from Laticauda semifasciata.

The 300-MHz and 500-MHz NMR spectra of a long neurotoxin laticauda semifasciata III (LS III) from Laticauda semifasciata have ben analysed. Comparison with the NMR spectra of alpha-cobratoxin from Naja naja siamensis, a homologous long neurotoxin to laticauda semifasciata III, allowed the assignment of all the aromatic protein resonances to specific amino acid residues. All the methyl proton resonances have been assigned to specific types of amino acid residues.

Proton-nuclear-magnetic-resonance study on molecular conformations of long neurotoxins. alpha-Bungarotoxin from Bungarus multicinctus and Toxin B from Naja naja.

The 270-MHz proton NMR spectra were analyzed of the long neurotoxins alpha-bungarotoxin from Bungarus multicinctus and Toxin B from Naja naja. The aromatic proton resonances were completely assigned to individual nuclei for alpha-bungarotoxin and in part for toxin B. The pH dependences of proton chemical shifts were analyzed by the nonlinear least-square method, for obtaining pKa values and protonation shifts.

Structural differences between erabutoxins in aqueous solution and in crystalline states.

The snake venom toxins erabutoxin a and b, have been studied in detail in aqueous solution over a wide pH range by proton NMR spectroscopy. Great care has been taken particularly to assign and locate in the structure residue histidine-7 with reference to other amino-acid sidechains. It lies in a pocket away from the surface in agreement with its lack of chemical reactivity.

Advanced nuclear magnetic resonance lanthanide probe analyses of short-range conformational interrelations controlling ribonucleic acid structures.

An advanced method was developed for lanthanide-probe analyses of the conformations of flexible biomolecules such as nucleotides. The new method is to determine structure parameters (such as internal-rotation angles) and population parameters for local conformational equilibria of flexible sites, together with standard deviations of these parameters. As the prominent advantage of this method, the interrelations among local conformations of flexible sites may be quantitatively elucidated from the experimental data of lanthanide-induced shifts and relaxations and vicinal coupling constants.

Nuclear magnetic resonance study on the microenvironments of histidine residues of ribonuclease T1 and carboxymethylated ribonuclease T1.

The 270-MHz 1H NMR spectra and fluorescence of ribonuclease T1 and carboxymethylated ribonuclease T1 were measured in aqueous solution. Histidine C4 proton resonances were assigned to individual residues. From the pH dependences of the chemical shifts of histidine C2 and C4 protons, the pKa values of histidine residues were obtained by the non-linear least-squares method.

Molecular conformation and function of erabutoxins as studied by nuclear magnetic resonance.

The 270-MHz proton NMR spectra of erabutoxins a, b and c from Laticauda semifasciata in 2H2O solution were observed together with [15-N6-acetyllysine]erabutoxin b, [27-N6-acetyllysine]-erabutoxin b and [47-N6-acetyllysine]erabutoxin b. The lysine epsilon-methylene proton resonances of erabutoxin b are assigned to individual residues. The epsilon-methylene proton resonance of Lys-27 is significantly broad, indicating that the mobility of this residue is restricted.