Kinetic Studies on the Initial Crystallization Process of Lysozyme in the Presence of D2O and H2O

In the initial stages of the crystallization of egg-white lysozyme, monomeric lysozyme aggregates rapidly and forms a nucleus in the presence of high salt concentrations. The formation process of the aggregates was examined to make clear the difference between the situations in heavy water and in water at the same sodium ion concentration. The aggregation in both cases was observed at unsaturated and/or saturated lysozyme concentrations. The turbidity at 350 nm of lysozyme increased remarkably within 60 min under each experimental condition and showed no appreciable changes over 60 min. The increase of turbidity in H₂O was much slower than in D₂O at the same salt concentration (3%). Lysozyme showed a critical concentration for nucleus formation whose value in H₂O was lower than in D₂O at 3% salt concentration. There are two different aggregation models, depending on the concentration of lysozyme. However, similar results were not obtained at 3% sodium ions in H₂O. The initial aggregation rate was also dependent on the concentrations of both lysozyme and NaCI. Therefore, the effect of lysozyme concentration on the aggregation process in H₂O may be smaller than in D₂O.     

Effect of Na+ and K+ Ions on the Initial Crystallization Process of Lysozyme in the Presence of D2O and H2O

In the initial stage of the crystallization of egg-white lysozyme, monomeric lysozyme aggregated rapidly to form a nucleus in the presence of high salt concentrations. In the present studies, we examined the initial aggregation process of lysozyme (initial crystallization process of lysozyme) in D₂O/H₂O with sodium ions or potassium ions, and investigated the relationship between the surface hydrophobicity and the aggregation rate of lysozyme. The effect of sodium ions or potassium ions on the initial aggregation process of lysozyme in D₂O was clearly different from H₂O. The initial aggregation rate of lysozyme in H₂O was slower than in D₂O. In the case of H₂O, the initial aggregation rate was about the same in both ions. But in the case of D₂O, the initial aggregation rate was affected by the ion species and the value was lower in potassium ions than in sodium ions. These results suggest that the interaction between lysozyme molecules is stronger in D₂O than in H₂O. Furthermore, sodium ions have a stronger effect on the interaction than potassium ions in the case of D₂O. There was a good correlation among the initial aggregation rate, surface hydrophobicity, and -potential of lysozyme. The hydrophobic interaction may be an important active force in the initial aggregation process of lysozyme.     

Effect of Na+ and K+ Ions on the Initial Crystallization Process of Lysozyme in the Presence of D2O and H2O

In the initial stage of the crystallization of egg-white lysozyme, monomeric lysozyme aggregated rapidly to form a nucleus in the presence of high salt concentrations. In the present studies, we examined the initial aggregation process of lysozyme (initial crystallization process of lysozyme) in D₂O/H₂O with sodium ions or potassium ions, and investigated the relationship between the surface hydrophobicity and the aggregation rate of lysozyme. The effect of sodium ions or potassium ions on the initial aggregation process of lysozyme in D₂O was clearly different from H₂O. The initial aggregation rate of lysozyme in H₂O was slower than in D₂O. In the case of H₂O, the initial aggregation rate was about the same in both ions. But in the case of D₂O, the initial aggregation rate was affected by the ion species and the value was lower in potassium ions than in sodium ions. These results suggest that the interaction between lysozyme molecules is stronger in D₂O than in H₂O. Furthermore, sodium ions have a stronger effect on the interaction than potassium ions in the case of D₂O. There was a good correlation among the initial aggregation rate, surface hydrophobicity, and ζ-potential of lysozyme. The hydrophobic interaction may be an important active force in the initial aggregation process of lysozyme.     

Self-association of oxyhaemoglobin. A nuclear magnetic relaxation study in H2O/D2O solutions

The proton and deuterium longitudinal relaxation rates were Studied at room temperature up to the highest protein concentrations in oxyhaemoglobin solutions of different H₂O/D₂O composition. The deuterium relaxation rates followed the experimentally well known single linear dependence on protein concentration, the slopes being little influenced by solvent (D₂O/H₂O) composition. The proton ralaxation rates show two different liner dependences on haemoglobin concentration. The entire concentration range is described by two straight lines with the threshold concentration about 11 mM (in haem), The ratio of the slopes is 1.6 (high-to-low Hb-conc.). Only in the higher concentration range two T₁'s were observed if the solvent contained more than half of D₂O. The slow relaxation phase of protons has T₁'s similar to those measured in solutions with less than half of D₂O. The relaxation of the other phase was ten times faster. The ratio of the proton populations in these two phases was equal to 2 (slow-to-fast) and independent of protein concentration. The fast relaxing protons are attributed to water molecules encaged within two or more haemoglobin molecules which associate for times long enough on the PMR time-scale.     

The effects of deuterium oxide (2H2O) on the polymerization of tubulin in vitro

The initial rate and final extent of polymerization of both bovine brain tubulin and sea urchin egg tubulin were enhanced in the presence of ²H₂O. The yields were increased in association with the elevation of the ²H₂O concentration. ²H₂O also reduced the critical concentration for polymerization of brain tubulin. Thermodynamic analysis was attempted using the temperature dependence of the critical concentration for polymerization in the presence of ²H₂O. We obtained linear van 't Hoff plots and calculated thermodynamic parameters which were positive and were increased with the elevation of the ²H₂O concentration. The enhancement of the polymerization of tubulin by ²H₂O could, therefore, be the result of the strenghening of intra-and/or inter-molecular hydrophobic interactions of the tubulin molecules. We believe that the increase in lenghth and number of microtubules of the mitotic spindles in the dividing cells of the eukaryotes with ²H₂O may be caused by the direct involvement of ²H₂O in the polymerization of tubulin.     

Physiological Studies with Heavy Water. II. Germination and Growth Inhibition of Barley by D2O

Seed germination and barley seedling growth in various D₂O concentrations have been studied. It was observed that the emergence of root and shoot was delayed, there being greater delay in shoot than in root emergence. A complete block was observed in germination in pure D₂O and the germination rate was slowed down significantly in lower concentrations. An initial germination delay by different D₂O concentrations seemed to cause a subsequent retardation in the growth measured as shoot and root length. A comparison of root and shoot length with their respective dry weights suggested that the growth by cell division/elongation might have been affected more than the transport of food materials from the endosperm to the embryo. Analysis of the total sugars of the endosperm and the embryo at 8 hour intervals showed that while the hydrolysis of starch to sugars was progressively decreased by increasing D₂O concentrations, the transport rate from endosperm to embryo showed a sharp inhibition in 50% D₂O and above. This indicated that the inhibition in the transport of materials, besides less hydrolysis of reserve food materials, may also be a causal factor of germination and growth inhibition in D₂O.     

Photocatalytical decomposition of hydrazine in K4[Mo(CN)8] solution: X-ray crystal structure of (PPh4)2[Mo(CN)4O(NH3)] · 2H2O

Ligand-field photolysis of K₄[Mo(CN)₈] · 2H₂O in 98% N₂H₄ · H₂O yields the catalytical decomposition of N₂H₄ into NH₃ and N₂. From irradiated solutions of octacyanomolybdate(IV) both in NH₃(aq) and 98% N₂H₄ · H₂O(l) as solvents, the salt of the formula (PPh₄)₂[Mo(CN)₄O(NH₃)] · 2H₂O was isolated. The salt is not formed by direct ligand replacement in tetracyanooxomolybdate(IV) ions derived from K₃Na[Mo(CN)₄O₂] · 6H₂O as the solute in similar conditions. The X-ray crystal structure and spectral properties of (PPh₄)₂[Mo(CN)₄O(NH₃)] · 2H₂O are described. The improved method of the synthesis of K₄[Mo(CN)₈] · 2H₂O is also presented.     

Charcoal-catalyzed transfer of tritium into 3H2O from regiospecifically-labeled 2-hydroxyestradiol in the presence of thiols

Charcoal was found to catalyze the release of ³H₂O from [1-³H]2-hydroxyestradiol-17β ([1-³H]2-OHE₂) or [4-³H]2-hydroxy-estradiol-17β ([4-³H]2-OHE₂) and this effect was shown to occur in the presence of glutathione or other thiols and to depend on the concentration of free steroid. The radiometric assay for measuring the formation of ³H₂O was not affected significantly by subsequent treatment of the incubation mixture with charcoal if the ratio of steroid to tissue (rat brain or liver microsomes) was low and only initial rates of ³H release were measured. 2-Hydroxyestradiol did not show the charcoal effect in the presence of tyrosinase, either when it was generated from its parent estrogen or added to the enzyme. The formation of ³H₂O from [4-³H]2-OHE₂ in the presence of glutathione was inhibited by ascorbic acid but the addition of dextran or albumin did not protect the catechol estrogen from the charcoal-catalyzed loss of tritium. The reaction with glutathione and charcoal occurred even at 4°C but other adsorbants such as alumina, silica or hydroxylapatite were without effect.     

Oscillatory Transpiration and CO2 Exchange of Citrus Leaves at the CO2 Compensation Concentration

CO₂ and H₂O vapor exchange were measured by enclosing citrus (Citrus sinensis cv. Sour Orange) leaves in a temperature controlled transparent leaf chamber. Introduction of dry air into the closed circuit gas flow caused cyclic oscillation in CO₂ and H₂O vapor exchange. It is suggested that oscillation in the CO₂ exchange at the CO₂ compensation concentration is due to oscillation in non-stomatal resistance to CO₂. Three types of oscillation were observed: 3–6 min (peak to peak) in young leaves, 30 min in mature leaves, and 160 min in old leaves.     

Quasielastic light scattering,electrophoresis, and fluorescence studies of lysozyme-poly(2-acrylamido-methylpropylsulfate) complexes

Complexation between lysozyme and sodium poly(2-acrylamido-2-methylpropanesulfonate) (PAMPS) was studied by quasielastic light scattering, electrophoretic light scattering, fluorescence, and turbidimetry in electrolyte solution. These techniques show that complexation occurs at pH 9.6 in an ionic strength buffer of 0.25M NaCl + 25 mM Na₂B₄O₇. At constant lysozyme concentration (C_pro). The structure of the complex depends on the polymer concentration. At low polymer concentration (relative to C_pro), an intrapolymer complex is formed. This intrapolymer complex aggregates to an interpolymer species upon increase in polymer concentration. Complex formation was also studied by fluorescence using pyrene-labeled PAMPS (Py-PAMPS). Energy transfer from singlet-excited tryptophan residues in lysozyme to the pyrene label occurs when the complex is formed. Fluorescence and turbidity data indicate that lysozyme interacts with Py-PAMPS preferentially at pyrene sites, which leads to static quenching of tryptophan fluorescence via energy transfer to the pyrene label. © 1995 John Wiley & Sons, Inc.     

土壤溶解性有机物对CO_2和N_2O排放的影响

农田土壤是温室气体的重要排放源,溶解性有机物作为土壤微生物容易利用的基质,其含量变化与温室气体的产生和排放密切相关。基于室内培养试验,对溶解性有机物影响土壤CO2、N2O的排放过程进行了分析。设置空白(CK)、单施秸秆(S)、单施氮肥(N)、秸秆和氮肥(S+N)4个不同的处理,对添加不同物质条件下土壤溶解性有机碳(DOC)、溶解性有机氮(DON)和CO2、N2O的排放动态进行了研究,对DOC和DON影响CO2、N2O的排放过程进行了探讨。结果表明:不同处理的温室气体排放通量和土壤DOC、DON含量差异显著;各处理的CO2排放通量和DOC动态随培养时间的延长呈现逐渐减小的趋势,S和S+N处理的N2O排放和DON动态呈现先增大后减小的趋势;S+N处理的CO2排放量最高,DON含量也显著高于其他处理,单施秸秆(S)处理的N2O排放量和DOC含量显著高于其它处理,单施氮肥(N)对土壤CO2的排放量和DOC含量的影响较小;土壤CO2和N2O的排放通量与土壤DOC和DON含量呈显著的相关性,相关系数(R2)达0.6以上,说明溶解性有机物的含量和动态对CO2、N2O的排放过程产生显著影响。     

Synthesis and structure of a lead(II)-citrate: {Na(H2O)3}[Pb5(C6H5O7)3(C6H6O7)(H2O)3]·9.5H2O

The reaction of lead(II) nitrate with trisodium citrate Na₃(C₆H₅O₇) in a 1:22.5 ratio at pH 4.8 provides crystals of {Na(H₂O)₃}[Pb₅(H₂O)₃(C₆H₅O₇)₃(C₆H₆O₇)]·9.5H₂O (1). The structure of 1 is two-dimensional and exhibits five distinct Pb(II) sites and four different modes of citrate bonding. The five lead sites all display hemidirected coordination geometries, that is, irregular distribution of neighboring oxygen atoms resulting in obvious gaps in the coordination spheres. Consequently, the lead coordination geometries exhibit proximal bonding to a number of oxygen donors, as well as distal interactions with nearest neighbors. The coordination numbers vary from 8 to 10, with ‘5+3’, ‘5+4’, ‘6+4’ and ‘7+3’ coordination modes where the first number refers to the proximal ligands and the second to the distal set. The four crystallographically distinct citrate groups include three with deprotonated carboxylate groups (C₆H₅O₇)³⁻ and one with a single protonated carboxyl group (C₆H₆O₇)². The citrate ligands bridge 3, 5, 7 and 7 lead sites. Three of the citrate groups exhibit tridentate chelation coordination to a lead site through two carboxylate oxygen donors and the hydroxyl groups. One citrate group projects an uncoordinated -OH group and a pendant protonated carboxyl group into the interlamellar domain. This latter carboxyl group coordinates to a sodium cation, which exhibits five coordinate geometry defined by three aqua ligands and the carbonyl oxygen of the -CO₂H groups in the basal plane and a citrate -OH donor in the apical position.     

Robust anionic building blocks [M(malonate)2(H2O)2] for crystal engineering of inorganic-organic hybrid materials

One-pot reactions of transition metal (Cu^II, Ni^II, Co^II, or Cd^II) salt with malonic acid (H₂mal) in the presence of mesocyclic diamine generate three supramolecular complexes and a coordination polymer. [Cu(mal)₂(H₂O)₂](H₂O)₂(H₂DACH) (1) and [M(mal)₂(H₂O)₂](H₂DACO) (M = Ni for 2, and Co for 3) are ion-pair products and managed by charge-assistant noncovalent interactions (DACO = 1,5-diazacyclooctane, and DACH = 1,4-diazacycloheptane). In these structures, the similar mononuclear [M(mal)₂(H₂O)₂]²⁻ building blocks are connected by hydrogen bonds to form 2D networks (with the aid of one lattice water in the case of 1), which are further extended by the cationic diamine components to yield 3D pillar-layered solids. While [Cd(mal)(H₂O)₂]_n (4) is a neutral polymeric complex, in which the similar [Cd(mal)₂(H₂O)₂]²⁻ subunits are propagated by additional Cd-O coordinative forces to result in the final 2D layer.     

A seven-coordinate Fe compound: [Fe{O(CH2CO2)2}(H2O)2(NO3)]. Preparation, structure and magnetic properties

A seven-coordinate Fe^III complex, [Fe(oda)(H₂O)₂(NO₃)], was obtained after dissolving Fe(NO₃)₃ · 9H₂O in an aqueous solution of oxydiacetic acid (H₂oda) at room temperature. In the solid state, the Fe^III center adopts a pentagonal bipyramid geometry with an {FeO₇} core formed by a tridentate oda²⁻ and a bidentate in the equatorial plane, and two axial water molecules. Magnetic measurements and EPR spectra revealed the presence of S = 5/2 Fe^III centers with rhombic zero field splitting parameters (D = 0.81 cm⁻¹, E/D = 0.33 ). Weak antiferromagnetic interactions with J ≈ −0.06 cm⁻¹ operating between neighboring Fe ions connected through Fe-O-C-O?H-O-Fe paths are estimated using the molecular field approximation.     

Zinc complexes of 6-methyl-2-pyridinecarboxylic acid. Crystal structure of [Zn(MeC5H3NCOO)2(H2O)] · H2O

Reaction of Zn(AcO)₂ · 2H₂O with 6-methyl-2-pyridinecarboxylic acid (L) yielded a new compound [Zn(MeC₅H₃NCOO)₂(H₂O)] · H₂O. This complex was characterised by elemental analyses, conductivity measurements, infrared, ¹H and ¹³C{¹H} NMR spectroscopies and single-crystal X-ray diffraction. The crystal structure consists of discrete molecules involving a pentacoordinated Zn atom with a geometry intermediate between a trigonal bipyramid and a square pyramid and with the two N_py atoms occupying the apical sites. Treatment of the complex [Zn(MeC₅H₃NCOO)₂(H₂O)] · H₂O with 2,2^′-bipyridine (bpy) produced [Zn(MeC₅H₃NCOO)₂(bpy)]. The metallic atom in this complex displays a distorted octahedral geometry and is coordinated to two ligands (L) via the pyridine nitrogen and the carbonyl oxygen atoms and to one 2,2^′-bypyridine (bpy).     

Preparation and structural characterisation of the compound [MeC5H4NCOOH][ZnCl3(H2O)] · [MeC5H4NCOO]H2O

The reaction of the ZnCl₂ with 6-methyl-2-pyridinecarboxylic acid, with a 1:2 metal-to-ligand molar ratio, affords optimum yields for the synthesis of [MeC₅H₄NCOOH][ZnCl₃(H₂O)] · [MeC₅H₄NCOO]H₂O. The new complex has been characterised by elemental analyses, IR, ¹H and ¹³C{¹H} NMR, and single crystal X-ray diffraction methods. The X-ray structure analysis revealed that this structure consists of [ZnCl₃(H₂O)]⁻ anions, [MeC₅H₄NCOOH]⁺ cations, [MeC₅H₄NCOO] zwitterions, and solvent molecules (H₂O) by means of hydrogen bonds.     

A Raman and calorimetric investigation of 3′, 5′ GpG

The Raman spectra of guanylyl (3′-5′) guanosine (GpG) in solution in H₂O and D₂O at pH 3–7 have been recorded at various temperatures between 0 and 80°C. The results are consistent with the existence in the lower temperature range of stable aggregates formed by the stacking of GpG tetramers. The aggregates melt cooperatively near 60°C, which results in important changes in the spectra. Among these, a large increase in intensity of some of the bands assigned to the guanine residues shows that unstacking of the bases occurs at the melting. Also apparent in the spectra are changes in the intensity and frequency of band attributable to molecular groups involved in intermolecular hydrogen bonding between adjacent molecules in the complex. The melting temperature of GpG decreases by approximately 15°C upon lowering the concentration from 5 × 10^?2 to 5 × 10^?4M, as shown by Raman, calorimetric, CD, and uv measurements. The experimentally determined ΔH and ΔS for the melting transition are 9 Kcal/mol and 28 e.u./mol, respectively. The aggregation of GpG in 1.5 × 10^?3M solutions was found to be very slow. The half-time of the process, which roughly follows first-order kinetics, is approximately 3 min at 10°C and 21 min at 35°C. The negative energy of activation associated with this reaction (?143 Kcal) indicated that the process involves intermediates whose concentrations decrease the temperatures raised, thus slowing down the overall process. The rate of disaggregation of GpG upon dilution to very low concentration is also extremely slow, indicating that the GpG aggregates, once formed, are very stable.     

Selective synthesis of triangular cluster oxido-sulfidocomplexes of Mo and W: High yield preparations of [Mo3O2S2(H2O)9], [W3O2S2(H2O)9], [W2MoO2S2(H2O)9] and their derivatization

Reaction of [Mo₂O₂(μ-S)₂(H₂O)₆]²⁺ with Mo(CO)₆ or metallic Mo under hydrothermal conditions (140 °C, 4 M HCl) gives oxido-sulfido cluster aqua complex [Mo₃(μ₃-S)(μ-O)₂(μ-S)(H₂O)₉]⁴⁺ (1). Similarly, [W₃(μ₃-S)(μ-O)₂(μ-S)(H₂O)₉]⁴⁺ (2) is obtained from [W₂O₂(μ-S)₂(H₂O)₆]²⁺ and W(CO)₆. While reaction of [Mo₂O₂(μ-S)₂(H₂O)₆]²⁺ with W(CO)₆ mainly proceeds as simple reduction to give 1, [W₂O₂(μ-S)₂(H₂O)₆]²⁺ with Mo(CO)₆ produces new mixed-metal cluster [W₂Mo(μ₃-S)(μ-O)₂(μ-S)(H₂O)₉]⁴⁺ (3) as main product. From solutions of 1 in HCl supramolecular adduct with cucurbit[6]uril (CB[6]) {[Mo₃O₂S₂(H₂O)₆Cl₃]₂CB[6]}Cl₂⋅18H₂O (4) was isolated and structurally characterized. The aqua complexes were converted into acetylacetonates [M₃O₂S₂(acac)₃(py)₃]PF₆ (M₃ = Mo₃, W₃, W₂Mo; 5a-c), which were characterized by X-ray single crystal analysis, electrospray ionization mass spectrometry and ¹H NMR spectroscopy. Crystal structure of (H₅O₂)(Me₄N)₄[W₃(μ₃-S)(μ₂-S)(μ₂-O)₂(NCS)₉] (6), obtained from 2, is also reported.     

Preparation and characterization of the layered borophosphates M(H2O)2[B2P2O8(OH)2]·H2O (M = Fe, Co, Ni)

The isotypic layered transition metal borophosphates M^II(H₂O)₂[B₂P₂O₈(OH)₂]·H₂O (M^II = Fe, Co, Ni) were prepared under hydrothermal conditions. Their crystal structures were determined by single-crystal X-ray diffraction data and revealed an isotypic relationship to Mg(H₂O)₂[B₂P₂O₈(OH)₂]·H₂O, a structure containing wavy 6³ nets formed by tetrahedral phosphate and hydrogenborate groups interconnected in an alternating fashion by sharing common apices. The crystalline compounds were also characterized by chemical analyses, scanning electron microscopy, energy dispersive X-ray analyses, thermal analyses, IR-spectroscopy and magnetic susceptibility measurements.