NMR study of 17O from H217O in human erythrocytes

Human erythrocytes were incubated in a Ringer's solution enriched with 10–18% H₂¹⁷O. The longitudinal relaxation time (T₁) of the ¹⁷O was determined separately in samples of red cell suspesions, packed cells, and supernatant. The longitudinal relaxation of ¹⁷O in erythrocyte suspensions was non-exponential, reflecting water exchange across the cell membranes as well as relaxation processes inside and outside the cell.The T₁ of intracellular ¹⁷O is 4–5 times shorter than in the supernatant, similar to the enhancement of proton relaxation by hemoglobin in erythrocytes and free solution at the frequency applied (8.13 MHz). This datum is consistent with the thesis that hemoglobin modifies the NMR relaxation behavior of water inside cells and in free solution in the same way.The rate constant

for water exchange was calculated to be 60 and 107 s⁻¹ at 25 and at 37° C, respectively. The apparent activation energy for

over the temperature range 23–37° C was 8.7±1.0 kcal/mole.     

Acoustical properties of aqueous solutions of oxygenated and deoxygenated hemoglobin

The acoustical relaxation spectrum of aqueous solutions of oxygenated and deoxygenated hemoglobin solutions was investigated in the frequency range 0.5–1000 MHz. The abrupt high frequency cut off of the broad absorption spectrum is consistent with a heavily weighted, shortest relaxation time of 2 × 10^?9 sec. Both the high-frequency cut-off and high frequency residual absorption appear to be slightly different for oxygenated and deoxygenated hemoglobin solution.     

1H-NMR spin-spin relaxation time assessment of the reflection coefficient of the Triticale seed cell wall–plasmalemma barrier to mannitol

The ¹H-NMR spin-spin relaxation time (T₂) in Triticale seeds swelling in external osmotica, polyethylene glycol 8000 or mannitol can identify both bound and free water. At the same water content, the free water spin-spin relaxation time increases for seeds imbibed with the mannitol solution, demonstrating inadequate water potential adjustment. The exchange rate of free/bound water molecules is apparently influenced by the driving force for water flow. The reciprocal lifetime of free water molecules, as a measure of water flow through the main cell barrier, was obtained. From a model of the seed as a resistance–capacitor network for water flow, a method was derived for calculating the reflection coefficient σ as a lifetime ratio of the free water molecules in seeds imbibed with two different osmotica (one penetrating across the main cell barrier and one not penetrating) at the same water potential. The ¹H-NMR method and the classical method based on volume rate changes yielded reflection coefficients for mannitol for the cell wall–plasmalemma barrier of 0.78 ± 0.08 and 0.68 ± 0.06, respectively.     

Phosphorus nuclear magnetic resonance studies of phosphoglucomutase and its metal ion complexes.

The ³¹P nuclear magnetic resonance of the covalently bound phosphate group at the active site of phosphoglucomutase has been examined by means of Fourier transform nuclear magnetic resonance spectroscopy. At a pD of 7.9, the chemical shift of the ³¹P nucleus is 3.8 ± 0.1 ppm downfield from 85% H₃PO₄; this shift is close to that of phosphoserine (dianionic form). Proton decoupling experiments suggest that the phosphorus of the enzymic phosphate group is coupled to protons with chemical shifts similar to those of phosphoserine. In D₂O, with proton decoupling, the ratio of the longitudinal and transverse diamagnetic relaxation times in solutions of 1.6 mm phosphoenzyme yields an approximate correlation time of 10^?7s for the ³¹P nucleus of the enzyme. This is within the range of values expected for tumbling of the entire protein molecule and suggests that the covalently attached phosphate group is immobilized or “frozen” at the active site of the enzyme by means of noncovalent interactions with adjacent groups. Consistent with this, the pK_a of the enzymic phosphate is significantly lower than that of phosphoserine. Binding of the diamagnetic activator, Mg²⁺, causes little or no change in the chemical shift of the resonance of the enzymic phosphorus from pD = 5.3 to 7.6, a downfield shift (?0.5 ± 0.1 ppm) at pD = 8.6, but an upfield shift (0.8 ±0.1 ppm) for that of phosphoserine, suggesting that bound Mg²⁺ is not coordinated to the enzymic phosphate. Independent evidence against direct coordination is provided by the paramagnetic effects of Ni²⁺ bound at the active site on the relaxation rates of the enzymic phosphorus. By assessing the paramagnetic effect of bound Ni²⁺ on both the longitudinal and transverse relaxation rates of the observed resonance, and by using correlation times determined for water proton relaxation induced by the Ni²⁺ complex, a range of Ni²⁺ to phosphorus distances of 4 to 6 Å is calculated. These distances suggest a second sphere interaction between the enzyme-bound metal and the enzymic phosphate group. Bound Ni²⁺ also markedly decreases the integrated intensity of the ³¹P resonance. Although the reason for this intensity decrease is incompletely explained, the present data establish the close proximity of the bound metal ion and the active site phosphoserine on phosphoglucomutase.     

Proton magnetic relaxation and anion effect in solutions of acid ferricytochrome c.

Measurements of the longitudinal relaxation rates of water protons in aqueous solutions of ferricytochrome c and their temperature dependence, were used for the elucidation of the heme iron ligands at acid pH. The relaxation rates increased with a decrease in pH and pK values of 2.5 and 4.48 were evaluated for the aqueous and 6 m urea solutions, respectively. The results at acid pH are compatible with a structure in which two water molecules exchange rapidly between the coordination sphere of high spin heme iron and the bulk. They suggest that concomitantly with the low-high spin transition the histidine-18 and methionine-80 iron bonds break simultaneously. Addition of various anions, including methanesulfonate at pH 1.95 caused a 85% decrease in the net longitudinal relaxation rate. However, neither the chemical shift nor the width of the methyl proton nmr line of methanesulfonate in solution of acid ferricytochrome c were affected indicating that the effect of anions is not due to a direct binding to the heme iron. The relaxation mechanism of the water molecules in the first coordination sphere of the ferric ion in acid cytochrome c is discussed. It appears that the longitudial relaxation rate is modulated by the electronic correlation time of the ferric ion which was calculated to be τ_s = 6 × 10^?11 sec at 60 MHz.     

Lambda phage cro repressor. DNA sequence-dependent interactions seen by tyrosine fluorescence

The thermodynamics of sickle cell hemoglobin gelation in the presence of oxygen has been investigated by measuring the fractional saturation of the solution and polymer phases, and the solubility. The fractional saturation of the solution phase with oxygen and the solubility were measured by near infrared spectrophotometry after sedimentation of the polymers, while the fractional saturation of the polymer phase was determined from linear dichroism measurements on gels formed by nucleation with an argon ion laser. Using the solution binding data of Gill et al. (1979) to calculate the oxygen pressure corresponding to the solution phase saturation, the initial portion of the polymer binding curve was determined. The self-consistency of the data analysis in terms of the two-phase model for the gel was tested by comparing measured and calculated gel (i.e. solution plus polymer) binding curves, and by comparing the observed solubilities with those calculated from the solution and polymer binding curves using Gibbs-Duhem relations.Oxygen binding to the polymer was found to be non-co-operative up to the maximum measured fractional saturation of 0.14. The binding constant was 0.0059 ± 0.0015 torr^?1 (p₅₀ = 170 ± 40 torr), which is about three times smaller than that of hemoglobin in the low-affinity T quaternary structure. Both the non-co-operative binding and the low affinity could be qualitatively explained in terms of an allosteric model and the current information on the polymer structure.     

DNA replication of SV40-infected cells. VII. Formation of SV40 catenated and circular dimers

The binding of methyl isonitrile (CH₃Nandz.tbnd;C) to hemoglobin β chains has been studied by measuring the ¹H nuclear magnetic resonance transverse relaxation times for methyl isonitrile as a function of protein concentration, temperature and ¹⁴N decoupling. Binding of methyl isonitrile both at the heme iron and at a non-specific site (or sites) has an effect upon the measured nuclear spin relaxation times. The results yield a value of 57 ± 12 seconds^?1 (20 °C) for the “off” rate constant K_?1 for specific binding and an Arrhenius activation energy for k_?1 of 14 ± 3 kcal mol^?1.     

Thermodynamic analysis of the physical state of water during freezing in plant tissue, based on the temperature dependence of proton spin-spin relaxation

Multi-proton spin-echo images were collected from cold-acclimated winter wheat crowns (Triticum aestivum L.) cv. Cappelle Desprez at 400 MHz between 4 and ?4 °C. Water proton relaxation by the spin-spin (T2) mechanism from individual voxels in image slices was found to be mono-exponential. The temperature dependence of these relaxation rates was found to obey Arrhenius or absolute rate theory expressions relating temperature, activation energies and relaxation rates, Images whose contrast is proportional to the Arrhenius activation energy (E_a), Gibb's free energy of activation (ΔG^?), and the entropy of activation (ΔS^?) for water relaxation on a voxel basis were constructed by post-image processing. These new images exhibit contrast based on activation energies rather than rules of proton relaxation. The temperature dependence of water proton T₂ relaxation rates permits prediction of changes in the physical state of water in this tissue over modest temperature ranges. A simple model is proposed to predict the freezing temperature kof various tissue in wheat crowns. The average E_a and ΔH^? for water proton T₂ relaxation over the above temperature range in winter wheat tissue were ?6.4 ± 14.8 and ?8.6 ± 14.8kj mol^?1, respectively. This barrier is considerably lower than the E_a for proton translation in ice at 0°C, which is reported to be between 46.0 and 56.5 kj mol^?1     

A comparative spectroscopic and calorimetric investigation of the interaction of amsacrine with heme proteins,hemoglobin and myoglobin

The binding of the anilido aminoacridine derivative amsacrine with the heme proteins, hemoglobin, and myoglobin, was characterized by various spectroscopic and calorimetric methods. The binding affinity to hemoglobin was (1.21?±?.05) × 10⁵ M^?1, while that to myoglobin was three times higher (3.59?±?.15) × 10⁵ M^?1. The temperature-dependent fluorescence study confirmed the formation of ground-state complexes with both the proteins. The stronger binding to myoglobin was confirmed from both spectroscopic and calorimetric studies. The binding was exothermic in both cases at the three temperatures studied, and was favored by both enthalpy and entropy changes. Circular dichroism results, three-dimensional (3D) and synchronous fluorescence studies confirmed that the binding of amsacrine significantly changed the secondary structure of hemoglobin, while the change in the secondary structure of myoglobin was much less. New insights, in terms of structural and energetic aspects of the interaction of amsacrine with the heme proteins, presented here may help in understanding the structure-activity relationship, therapeutic efficacy, and drug design aspects of acridines.     

Determination of the correction factors used in Fricke dosimetry for HDR 192Ir sources employing the Monte Carlo method

PurposeFricke dosimetry has shown great potential in the direct measurement of the absolute absorbed dose for ¹⁹²Ir sources used in HDR brachytherapy. This work describes the determination of the correction factors necessary to convert the absorbed dose in the Fricke solution to the absorbed dose to water. Methods: The experimental setup for Fricke irradiation using a ¹⁹²Ir source was simulated. The holder geometry used for the Fricke solution irradiation was modelled for MC simulation, using the PENELOPE. Results: The values of the factors determined for validation purposes demonstrated differences of less than 0.2% when compared to the published values. Four factors were calculated to correct: the differences in the density of the solution (1.0004 ± 0.0004); the perturbations caused by the holder (0.9989 ± 0.0004); the source anisotropy and the water attenuation effects (1.0327 ± 0.0012); and the distance from the center of the detection volume to the source (7.1932 ± 0.0065). Conclusion: Calculated corrections in this work show that the largest correction comes from the inverse squared reduction of the dose due to the point of measurement shift from the reference position of 1 cm. This situation also causes the correction due to volume averaging and attenuation in water to be significant. Future versions of the holder will aim to reduce these effects by having a position of measurement closer to the reference point thus requiring smaller corrections.     

Reaction of cythchrome c with one-electron redox reagents. I. Reduction of ferricytochrome c by the hydrated electron produced by pulse radiolysis

Pulse radiolysis-kinetic spectrometry has been used to investigate the reaction of hydrated electrons with ferricytochrome c in dilute aqueous solution at pH 6.5–7.0. Time resolutions from 2·10^?7 to 1 s were employed. Transient spectra from 320 to 580 nm were characterized with a wavelength resolution of ±0.5 nm. 1 In neutral salt-free solution, k(ferricytochrome c+e^?_aq)=(6.0±0.9)·10¹⁰ M^?1·s^?1 and k(ferricytochrome c+H)=(1.2±0.2)·10¹⁰ M^?1·s^?1. The reaction of ferricytochrome c with hydrated electrons is sensitive to ionic strength; in 0.1 M NaClO₄, k(ferricytochrome c+e^?_aq)=(2.4±0.4)·10¹⁰ M^?1·s^?1. In contrast, k(ferricytochrome c+H) is insensitive to ionic strength. Time resolution of three spectral stages has been accomplished. The primary spectrum is the first observable spectrum detectable after irradiation and is formed in a second-order process. Its rate of formation is indisting-uishable from the rate of disappearance of the electron spectrum. The secondary spectrum is generated in a true first order intramolecular process, k(p→s)=(1.2±0.1)·10⁵ s^?1. The tertiary spectrum is also generated in a true first-order process, k(s→t)=(1.3±0.2)·10² s^?1. The specific rates of both transformations are independent of the wavelength of measurement. The tertiary spectrum, observable 50 ms after initial reaction and remaining unchanged thereafter for at least 1 s, shows that relaxed ferrocytochrome c is the only detectable product. This product is not autoxidizable, as expected for native reduced enzyme. It is more probable that the intramolecular changes responsible for the p→s and s→t spectral transformations involve the influence of conformational relaxation of ferrocytochrome c upon electronic energy states then that they are intramolecular transmission of reducing equivalents from primary sites of electron attachment.     

NMR relaxation processes of 31P in macromolecules

³¹P-Nmr relaxation parameters (spin-lattice relaxation time, linewidth, and nuclear Overhauser effect) were obtained at three different frequencies for poly(U) and a well-defined (145 ± 3 base-pair) fragment of DNA in solution. Data sets for the two samples were analyzed by theories which included relaxation by the mechanisms of ³¹P chemical shift anisotropy as well as by ¹H-³¹P dipole–dipole interaction. Neither data set could be satisfactorily described by a single correlation time. A model of a rigid rotor most nearly fits the data for the DNA molecule. Parameters obtained from the least-square fit indicate (1) that the DNA undergoes anisotropic reorientation with a correlation time τ₀ = 6.5 × 10^?7 sec for the end-to-end motion, (2) the ratio of diffusion constants D_∥/D_⊥ is 91, and (3) that the linewidth is due to chemical shift dispersion to the extent of 0.5 ppm. Some deviations of the calculated from the observed values suggested that significant torsional and bending motions may also take place for this DNA. Another model which contains isotropic motion but with a broad distribution of correlation times was required to fit the data for poly(U). A log ? χ² distribution function of correlation times [Scheafer, J. (1973) Macromolecules 6 , 881–888] described well the motion of poly(U) with the average correlation time τ = 3.3 × 10^?9 sec and a distribution parameter p = 14.     

NMR study of rapid water diffusion across lipid bilayers in dipalmitoyl lecithin vesicles

A method for measurement of rapid diffusional exchange between external and internal water in lecithin vesicles is described. Paramagnetic ions were inserted inside DPL vesicles and the NMR relaxation times for water protons were measured as a function of temperature. It was found that water diffusion rate is described by a single activation energy of 15±1 kcal/mole in the temperature range 16 – 35°C and exhibits a maximum at 44°C. The permeability of DPL vesicles to water was calculated to 16–18 × 10^?4 cm/s at 44°C and 1.7 × 10^?4 cm/s at 20°C.     

Evaluation of the water environments in deoxygenated sickle cells by longitudinal and transverse water proton relaxation rates

The longitudinal and transverse water proton relaxation rates of oxygenated and deoxygenated erythrocytes from both normal adults and individuals with sickle cell disease were measured as a function of temperature at two different frequencies. The simplest model which fits all of the data consists of three different environments for water molecules. The majority of the water (98%) has a correlation time indistinguishable from bulk water (3 × 10^?11 sec). Secondly, there is a small amount of water (1.3–1.5%) present which has a correlation time of 2–4 × 10 ^?9 sec and is apparently independent of the erythrocyte sample studied. Presumably this water is the hydration sphere around the hemoglobin molecules and its correlation time is significantly slower than bulk water. The third environment contains approximately 0.2% of the water present and has a correlation time≥ 10^?7 sec. This third environment is considered tightly bound to the hemoglobin because the water proton correlation time is very similar to the expected rotational correlation time for the hemoglobin molecules. The value of the transverse relaxation rate, f_b(T_2b)^?1, for the tightly bound water fraction decreases in oxy ($\text{S}\text{S}$), deoxy ($\text{A}\text{A}$), and oxy ($\text{A}\text{A}$) erythrocyte samples as the temperature is increased as expected for a rotational correlation time process. In dramatic contrast,f_b (T_2b)^?1 increases almost linearly as the temperature is increased over the whole 4 ° to 37 °C temperature range in samples of deoxy ($\text{S}\text{S}$) erythrocytes. The observation suggests a continual increase in the formation of deoxyhemoglobulin S polymers rather than a sudden transition from a homogeneous solution of deoxyhemoglobin S molecules to a solid gel.     

Recrystallization of Ice Crystals in Trehalose Solution at Isothermal Condition

An isothermal ice recrystallization behavior in trehalose solution was investigated. The isothermal recrystallization rate constants of ice crystals in trehalose solution were obtained at ?5 °C, ?7 °C, and ?10 °C. Then the results were compared to those of a sucrose solution used as a control sample. Simultaneous estimation of water mobility in the freeze-concentrated matrix was conducted by ¹H spin–spin relaxation time T₂ to investigate mechanisms causing the different ice crystal recrystallization behaviors of sucrose and trehalose. At lower temperatures, lower recrystallization rates were obtained for both trehalose and sucrose solutions. The ice crystallization rate constants in trahalose solution tended to be smaller than those in sucrose solution at the same temperature. Although different ice contents (less than 3.6%) were observed between trehalose and sucrose solutions at the same temperature, the recrystallization behaviors of ice crystals were not markedly different. The ¹H spin–spin relaxation time T₂ of water components in a freeze-concentrated matrix for trehalose solution was shorter than in a sucrose solution at the same temperature. Results show that the water mobility of trehalose solutions in freeze-concentrated matrix was less than that of sucrose solutions, which was suggested as the reason for retarded ice crystal growth in a trehalose solution. Results of this study suggest that the replacement of sucrose with trehalose will not negatively affect deterioration caused by ice crystal recrystallization in frozen foods and cryobiological materials.     

Light scattering studies of supercoiled and nicked DNA

Static and dynamic light scattering measurements were made of solutions of pGem1a plasmids (3730 base pairs) in the relaxed circular (nicked) and supercoiled forms. The static structure factor and the spectrum of decay modes in the autocorrelation function were examined in order to determine the salient differences between the behaviors of nicked DNA and supercoiled DNA. The concentrations studied are within the dilute regime, which is to say that the structure and dynamics of an isolated DNA molecule were probed. Static light scattering measurements yielded estimates for the molecular weight M, second virial coefficient A₂, and radius of gyration R_G. For the nicked DNA, M = (2.8 ± 0.4) × 10⁶g/mol, A₂ = (0.9 ± 0.2) × 10⁻³ mol cm³/g², and R_G = 90 ± 3 nm were obtained. For the supercoiled DNA, M = (2.5 ± 0.4) × 10⁶ g/mol, A₂ = (1.2 ± 0.2) × 10⁻³ mol cm³/g², and R_G = 82 ± 2.5 nm were obtained. The static structure factors for the nicked and supercoiled DNA were found to superpose when they were scaled by the radius of gyration. The intrinsic stiffness of DNA was evident in the static light scattering data. Homodyne intensity autocorrelation functions were collected for both DNAs at several angles, or scattering vectors. At the smallest scattering vectors the probe size was comparable to the longest intramolecular distance, while at the largest scattering vectors the probe size was smaller than the persistence length of the DNA. Values of the self-diffusion coefficients D were obtained from the low-angle data. For the nicked DNA, D = (2.9 ± 0.3) × 10⁻⁸ cm²/s, and for the supercoiled DNA, D = (4.11 ± 0.21) × 10⁻⁸ cm²/s. The contribution to the correlation function from the internal dynamics of the DNA was seen to result in a strictly bimodal decay function. The rates of the faster mode Γ_int, reached plateau values at low angles. For the nicked DNA, Γ_int = 2500 ± 500 s⁻¹, and for the supercoiled DNA, Γ_int = 5000 ± 500 s⁻¹. These rates correspond to the slowest internal relaxation modes of the DNAs. The dependence of the relaxation rates on scattering vector was monitored with the aid of cumulants analysis and compared with theoretical predictions for the semiflexible ring molecule. The internal mode rates and the dependence of the cumulants moments reflected the difference between the nicked DNA and the supercoiled DNA dynamical behavior. The supercoiled DNA behavior seen here indicates that conformational dynamics might play a larger role in DNA behavior than is suggested by the notion of a branched interwound structure. © 1996 John Wiley & Sons, Inc.     

Effect of Seawater–Sewage Cross-Transplants on Bacterial Metabolism and Diversity

Bioassays experiments were conducted to determine the metabolic and community composition response of bacteria to transplants between relatively pristine coastal seawater and sewage-impacted seawater. There were four treatments: (1) pristine seawater bacteria?+?pristine seawater (Pb?+?Pw), (2) sewage-impacted bacteria?+?sewage-impacted water (Sb?+?Sw), (3) pristine seawater bacteria?+?sewage-impacted water (Pb?+?Sw), and (4) sewage-impacted bacteria?+?pristine seawater (Sb?+?Pw). Sewage-derived DOC was more labile and readily utilized by bacteria, which favored the growth of high nucleic acid (HNA) bacteria, resulting in high bacterial production (BP, 113?±?4.92 to 130?±?15.8 μg C l^?1?day^?1) and low respiration rate (BR, <67?±?11.3 μg C l^?1?day^?1), as well as high bacterial growth efficiency (BGE, 0.68?±?0.09 to 0.71?±?0.05). In contrast, at the relatively pristine site, bacteria utilized natural marine-derived dissolved organic matter (DOM) at the expense of lowering their growth efficiency (BGE, <0.32?±?0.02) with low BP (<62?±?6.3 μg C l^?1?day^?1) and high BR 133?±?14.2 μg C l^?1?day^?1). Sewage DOM input appeared to alter the partitioning of carbon between respiration and production of bacteria, resulting in a shift toward higher BGE, which would not enhance oxygen consumption. Taxonomic classification based on 454 pyrosequencing reads of the 16S rRNA gene amplicons revealed that changes in bacterial community structure occurred when seawater bacteria were transferred to the eutrophic sewage-impacted water. Sewage DOM fueled the growth of Gammma-proteobacteria and Epsilson-proteobacteria and reduced the bacterial richness, but the changes in the community were not apparent when sewage-impacted bacteria were transferred to pristine seawater.     

Transient electric birefringence studies of T2 bacteriophage and T2 ghost

The transient electric birefringence behavior of bacteriophage T2 and the T2 ghost or protein coal was studied. The field free relaxation measurements show both the intact virus and its ghost to have two rotary diffusion coefficients. These coefficients have values of 555 ± 54 and 111 ± 22 sec.^?1 for the intact virus and 688 ± 89 and 161 ± 29 sec.^?l for the ghost. The equivalent ellipsoids for the fast and slow relaxation coefficients were obtained by use of Perrin's equation and were related to the bacteriophage structure in terms of a possible extension of the tail fibers or an enlargement of the head structure. The saturation of the specific birefringence of the phage and the ghost when compared with the specific birefringence of the free nucleic acid gave an average optical orientation of 10 to 18% of the nucleic acid parallel to the main axis of the phage. The analysis of the birefringence versus applied field strength in the Kerr region gave the following values for the anisotropy of the polarixability. α_e,33 – α_e,11 and intrinsic dipole, μ, of both phage and ghost : for T2 phage α_e,33 – α_e,11 = 5.0 × 10^?14 cm.³ and μ = 64,400 Debyes; for T2 ghost α_e,33 – α_e,11 = 7.9 × 10^?14cm.³ and μ = 57,200 Debyes. The high intrinsic dipole for phage and ghost is interpreted as to be associated with the mechanisms of the virus for attachment, to the host cell wall.     

Automatic <Superscript>13</Superscript>C chemical shift reference correction for unassigned protein NMR spectra

Poor chemical shift referencing, especially for ¹³C in protein Nuclear Magnetic Resonance (NMR) experiments, fundamentally limits and even prevents effective study of biomacromolecules via NMR, including protein structure determination and analysis of protein dynamics. To solve this problem, we constructed a Bayesian probabilistic framework that circumvents the limitations of previous reference correction methods that required protein resonance assignment and/or three-dimensional protein structure. Our algorithm named Bayesian Model Optimized Reference Correction (BaMORC) can detect and correct ¹³C chemical shift referencing errors before the protein resonance assignment step of analysis and without three-dimensional structure. By combining the BaMORC methodology with a new intra-peaklist grouping algorithm, we created a combined method called Unassigned BaMORC that utilizes only unassigned experimental peak lists and the amino acid sequence. Unassigned BaMORC kept all experimental three-dimensional HN(CO)CACB-type peak lists tested within ±?0.4 ppm of the correct ¹³C reference value. On a much larger unassigned chemical shift test set, the base method kept ¹³C chemical shift referencing errors to within ±?0.45 ppm at a 90% confidence interval. With chemical shift assignments, Assigned BaMORC can detect and correct ¹³C chemical shift referencing errors to within ±?0.22 at a 90% confidence interval. Therefore, Unassigned BaMORC can correct ¹³C chemical shift referencing errors when it will have the most impact, right before protein resonance assignment and other downstream analyses are started. After assignment, chemical shift reference correction can be further refined with Assigned BaMORC. These new methods will allow non-NMR experts to detect and correct ¹³C referencing error at critical early data analysis steps, lowering the bar of NMR expertise required for effective protein NMR analysis.     

Water proton magnetic resonance studies of normal and sickle erythrocytes Temperature and volume dependence

The temperature and cell volume dependence of the NMR water proton linewidth, spin-lattice, and spin-spin relaxation times have been studied for normal and sickle erythrocytes as well as hemoglobin A and hemoglobin S solutions. Upon deoxygenation, the spin-spin relaxation time (T₂) decreases by a factor of 2 for sickle cells and hemoglobin S solutions but remains relatively constant for normal cells and hemoglobin A solutions. The spin-lattice relaxation time (T₁) shows no significant change upon dexygenation for normal or sickle packed red cells. Studies of the change in the NMR linewidth, T₁ and T₂ as the cell hydration is changed indicate that these parameters only slightly by a 10–20% cell dehydration. This result suggests that the reported 10% cell dehydration observed with sickling is not important in the altered NMR properties. Low temperature studies of the linewidth and T₁ for oxy and deoxy hemoglobin A and hemoglobin S solutions suggest that the “bound” water possesses similar properties for all four species. The low temperature linewidth ranges from about 250 Hz at ?15°C to 500 Hz at ?36°C and analysis of the NMR curves yield hydration values near 0.4 g water/g hemoglobin for all four species. The low temperature T₁ data go through a minimum at ?35°C for measurements at 44.4 MHz and ?50°C for measurements at 17.1 MHz and are similar for oxy and deoxy hemoglobin A and hemoglobin S. These similarities in the low temperature NMR data for oxy and deoxy hemoglobin A and hemoglobin S suggest a hydrophobically driven sickling mechanism. The room temperature and low temperature relaxation time data for normal and sickle cells are interpreted in terms of a three-state model for intracellular water. In the context of this model the relaxation time data imply that type III, or irratationally bound water, is altered during the sickling process.