Steven S. Danyluk

13C nuclear magnetic resonance study of the complexation of calcium by taurine

13C Nuclear magnetic resonance chemical shifts, 1JC-C scalar coupling constants, spin-lattice relaxation times, and nuclear Overhauser effects were determined for taurine-[1,2 13C] and a taurine-[1 13C] and taurine-[2 13C] mixture in the presence and absence of calcium. Ionization constants for taurine amino and sulfonic acid groups and chemical shifts of N-methylene and S-methylene carbons of the taurine cation, zwitterion, and anion were obtained from simultaneous least squares analysis of 13C titration curves of both taurine carbons. Comparison of taurine titration shifts to values for related compounds reveals some unusual electronic properties of the taurine molecule. Stability constants of 1:1 calcium complexes with taurine zwitterions and anions, as well as their 13C chemical shifts, were obtained by least squares analysis of titration curves measured in the presence of calcium. The stability constants of calcium-taurine complexes were significantly lower than previous values and led to estimates that only approximately one percent of intracellular calcium of mammalian myocardial cells would exist in a taurine complex. The implications of these results with respect to the effect of taurine on calcium ion flux are discussed.

Proton wide-line nuclear magnetic resonance spectra of hydrated proteins

Abstract The wide-line proton magnetic resonance spectra of ribonuclease and bovine serum albumin have been measured in the hydrated and vacuum-dried states over the temperature range −140° to −180°. A single, broad signal attributed to the protein protons was observed for the dried proteins up to 120°. Little variation in line-widths or second moments was noted over the entire temperature range. Above 120° the spectra showed an additional weak, narrow signal due to residual adsorbed water molecules. The spectra for hydrated proteins are essentially the same as for the dried proteins below −40°. Above −40° the spectra consist of a narrow signal superimposed upon a broad signal; the narrow signal can be removed by prolonged pumping in vacuo and is due to weakly adsorbed H2O molecules. The broad signals for both hydrated proteins undergo line-narrowing transitions in the temperature range −60° to −10°. A further more marked transition of the broad component is noted at approx. 60° for ribonuclease and approx. 20° for bovine serum albumin. From the magnitudes of the line-widths and second moments of the broad signals it is concluded that the hydrated and dried proteins have considerable motional freedom in the solid state at all of the temperatures studied. The present measurements also indicate that the extent of thermally induced configurational changes in the proteins at elevated temperatures is strongly linked to the amount and structural properties of the adsorbed water.

A direct assignment of all base anomeric H1′ signals in the proton spectrum of a trinucleoside diphosphate, ApApA: Structure implications

Trinucleotides can be considered as primitive models for triplet codons of t-RNA and there is accordingly considerable interest in defining structures and conformations for these molecules. An important step in this direction was made recently [ I] with the first successful crystal structure determination for a trinucleoside dephosphate, adenylyl-(3’-5’)-adenylyl(3’-5’)-adenosine, ApAp?A’. Because it is difficult to predict what effect solution forces will have on structure, particularly for smaller molecules with conformational flexibility, it is essential to obtain comparable structural information in solution. However, the use of NMR spectroscopy for this purpose is severely restricted because of extensive signal overlap, especially in homo-oligonucleotides, i.e. XpXpX. Selective labeling of nucleotidyl units [2,3] is an obvious solution to overlap problems and in this communication we describe the use of this approach to achieve the first direct assignment of base and anomeric Hl’ protons for a homo-oligonucleotide, ApApA.

17O NMR spectroscopy of nucleoside derivatives; bonding characteristics of pyrimidine carbonyls

Abstract Various uridine derivatives have been selectively enriched (≈ 50%) at the 04 and 02 oxygens, and the 17 O chemical shifts and linewidths measured. The chemical shifts, which are primarily dependent on π-bond-order and hydrogen bonding, show effects that are selective for the 02 and 04 oxygens. In particular, significantly more H-bonding to D 2 O and higher π bond order are found for the 04 oxygen.

A nuclear magnetic resonance study of thermal effects upon base stacking in oligonucleotides. ApA--p.

Abstract A proton magnetic resonance study has been made of the effect of temperature upon the spectrum of the dinucleotide ApA>p in aqueous solution at neutral pH. The base-ring proton signals are observed to shift down-field with increasing temperature in the range 5–95°. This down-field shift is attributed to a progressive destacking of the base-rings in the dinucleotide with increasing temperature. A simple analysis of the chemical shift-temperature curves, in terms of a two-state equilibrium, i.e. stacked dinucleotide ai unstacked dinucleotide, yields an enthalpy change of −8.2 ± 1.0 kcal·mole −1 for the stacking process in reasonable agreement with values reported for similar dimers using other techniques.

Auger electron spectroscopy of stoichiometric chromium carbides and carbide precipitates at grain boundaries of type 304 stainless steel

Auger electron spectroscopy (AES) data on carbide phases at the grain boundaries of type 304 SS are reported. The carbide phase was identified from the unique shape of the carbide peak in the AES spectrum and composition of the carbide was obtained from the Auger Spectra from stoichiometric chromium carbides, iron and nickel carbide and the pure graphite, iron, chromium, and nickel. (FS)

Coordination and Binding of Taurine as Determined by Nuclear Magnetic Resonance Measurements on 13C-Labeled Taurine

A number of pharmacological actions of taurine (5,8,9,16,17,19, 21,30,31) on central nervous and peripheral tissues have suggested that taurine might modulate cation flux (15) by either direct (10, 13) or indirect interactions (4) with calcium. The ability of taurine to influence calcium flux by direct coordination with calcium depends on the fraction of free intracellular calcium which can be trapped by taurine. The fraction of calcium that exists as a taurine complex can be estimated readily from intracellular taurine, calcium and hydrogen ion levels, and the formation constants of taurine-calcium complexes. The latter formation constants can be determined in vitro and applied to in vivo conditions. Dolara and coworkers (11,12) using calcium-taurine formation constants estimated from natural abundance 13C chemical shift titration curves, have calculated that approximately 8% of the total calcium in mammalian myocardium exists in taurine complexes. The inherent difficulties associated with making very precise measurements on noisy, low sensitivity natural abundance 13C NMR spectra suggested that the determination of calcium-taurine formation constants be repeated using 13C enriched taurine. We have determined 13C chemical shift titration curves of the calcium-taurine system using 13C-taurine and have obtained new formation constants for calcium-taurine complexes by rigorous complex equilibrium analysis of the titration curves. These formation constants predict that only neglible amounts of calcium are bound directly to taurine at the taurine and calcium levels found in myocardial cells. Additional 13C NMR measurements of taurine in solution have revealed some unexpected electronic properties of the molecule, which may be important to its biological function.

Proton magnetic resonance studies of 9-(β-d-xylofuranosyl)adenine 3′,5′-cyclic monophosphate and 9-(β-d-arabinofuranosyl)adenine 2′,5′-cyclic monophosphate

Abstract A detailed 1 H 220-MHz n.m.r. study of 9-(β- d -xylofuranosyl)adenine 3′,5′-cyclic monophosphate (3′,5′-xylo-cAMP, 1 ) and 9-(∇- d -arabinofuranosyl)adenine 2′,5′-cyclic monophosphate (2′,5′-ara-cAMP, 2 ) in D 2 O solution is described. The sugar-ring conformations in 1 and 2 are shown to be 3 E and 2 E , respectively, and the phosphate rings are in a chair form. An unusual 4 J P,H coupling of 2.4 Hz is observed between H-4′ and phosphorus in 1 and a vicinal J P,H of 30.8 Hz between H-5′ and phosphorus in 2 . This latter coupling verifies a similar value found previously in the ara-cytidine analog of 2 . A comparison of the conformational properties of cyclic nucleotides having fused phosphate and sugar rings has been made, together with an assessment of the use of the Karplus constants in such ring-systems.

Variation of 1J(C1′, H1′) with glycosidic bond conformation of pyrimidine nucleosides

1J(C1′, H1′) magnitudes of a series of pyrimidine cyclonucleosides are found to vary quantitatively with glycosidic bond conformation. Measurements of J(C1′, H1′) in pyrimidine nucleosides and nucleotides can be used to determine the range of allowed syn and anti conformations.

Application of a directly exposed self‐scanning photodiode array as a linear position sensitive detector in a small‐angle x‐ray scattering instrument

A linear position sensitive small‐angle x‐ray detector has been developed which utilizes a directly exposed self‐scanning photodiode array cooled to liquid nitrogen temperature to reduce fixed pattern noise. Direct exposure of the array improves the overall sensitivity to 8 keV photons by an order of magnitude over photoconversion techniques. With this improvement in sensitivity, the photodiode array has been effectively used to detect x rays from weakly scattering biological molecules in solution. The slit‐like geometry of the photodector elements ideally matches that of a Kratky collimator employed with a low‐flux x‐ray source to enhance greatly the data collection rate. The detective quantum efficiency and stability of the detector have been determined while taking small‐angle scattering measurements of proteins and the results are quantitatively applied to characterize the performance of the instrument.