Chromatin fiber dimensions and nucleosome orientation: a neutron scattering investigation.

Chromatin fibers were studied in solutions of mM monovalent salt by small angle neutron scattering. The variation of the cross section radius of gyration with H2O/D2O contrast shows that DNA is at much larger average radial distances from the fiber axis than histone. Consequently, the coils of DNA in a core particle must be approximately parallel to the fiber direction. The radii of gyration suggest that the maximum diameter of chromatin and nucleosomes is approximately 14 nm and that the DNA id distributed in two radial layers. The concentration dependence of the scattering maxima near 14 nm spacings furnishes independent support for a 14 nm external diameter and can be interpreted by a double DNA layer configuration.     

Small angle neutron scattering from lysozyme solutions in unsaturated and supersaturated states (SANS from lysozyme solutions)

Small angle neutron scattering (SANS) method was used to study lysozyme solutions, with particular interest in an understanding of the crystallization process at the initial stage. It is found that (1) in the unsaturated solution, the protein molecules aggregate with a continuous increase in size when NaCl concentration is increased, and (2) in the supersaturated solution, an irreversible change, superimposed on the former process, occurs when the supersaturation is realized. These facts indicate the usefulness of SANS in detecting changes of protein molecules in solution on the nanometer scale. The reliability of the SANS results are indicated by (1) comparing them with those of small angle X-ray scattering (SAXS), and (2) comparing the effect of D(2)O and H(2)O as solvent. Since the interparticle interaction is essential in the crystallization process and a simple Guinier plot analysis is not allowed, a more rigorous framework of analyzing data with interference function is developed, through which both average interparticle distance and particle size are estimated.     

Interaction of SOS2 with nucleoside diphosphate kinase 2 and catalases reveals a point of connection between salt stress and H2O2 signaling in Arabidopsis thaliana

SOS2, a class 3 sucrose-nonfermenting 1-related kinase, has emerged as an important mediator of salt stress response and stress signaling through its interactions with proteins involved in membrane transport and in regulation of stress responses. We have identified additional SOS2-interacting proteins that suggest a connection between SOS2 and reactive oxygen signaling. SOS2 was found to interact with the H2O2 signaling protein nucleoside diphosphate kinase 2 (NDPK2) and to inhibit its autophosphorylation activity. A sos2-2 ndpk2 double mutant was more salt sensitive than a sos2-2 single mutant, suggesting that NDPK2 and H2O2 are involved in salt resistance. However, the double mutant did not hyperaccumulate H2O2 in response to salt stress, suggesting that it is altered signaling rather than H2O2 toxicity alone that is responsible for the increased salt sensitivity of the sos2-2 ndpk2 double mutant. SOS2 was also found to interact with catalase 2 (CAT2) and CAT3, further connecting SOS2 to H2O2 metabolism and signaling. The interaction of SOS2 with both NDPK2 and CATs reveals a point of cross talk between salt stress response and other signaling factors including H2O2.     

HNMR of succinate binding to aspartate transcarbamylase. A comparison of results in D2O and H2O.

The interaction of succinate with asparatete transcarbamylase from Escherichia coli has been studied by magnetic resonance relaxation measurements of the dicarboxylic acid methylene protons in H2O solutions. The pH and temperature dependence of the relaxation in the presence of either native asparte transcarbamylase or its catalytic subunit in H2O solutions is qualitatively very similar to the corresponding situation utilizing D2O as the solvent. From previous result of measurements in D2O[C.B. Beard and P.G. Schmidt, Biochemistry 12(1973)2255] a mechanism was proposed involving 2 protonated groups affecting succinate binding and titratable over the pH range 7-10. Quantitatively, fitting the data from H2O solutions to the mechanism yeilds values of the fitting parameters generally in good agreement with the D2O experiments. The main exceptions are the pKa values calculated for the two titratable groups. For these species the values obtained in the presence of the catalytic subunit are 6.7 and 7.8 in H2O solutions versus 7.3 and 8.6 in D2O solutions. In the presence of native enzyme the corresponding values are 6.8 and 8.3 in H2O versus 7.6 and 9.2 in D2O. These observed differences are consistent with differences in ionization constants of weak acids in D2O relative to H2O. The results imply that succinate interaction with the enzyme active site is similar in the two solvents.     

Polymerization and gelation of fibrinogen in D2O

The solution properties of fibrinogen and the thrombin-induced activation and gelation of fibrinogen in 95% D2O at pH 7.4 were compared to those in H2O under similar conditions. The initial release rates of fibrinopeptides A and B in D2O were slightly slower than those in H2O. However, the values of the Michaelis-Menten parameters Km and V for the release of the two peptides in D2O and H2O in the presence of 0.5 M NaCl were about the same. From turbidity measurements at 450 nm it is obvious that fibrinogen is soluble in a slightly more narrow range of NaCl concentration and that the fibrin gels have a higher degree of lateral aggregation in D2O than in H2O. The variation of fibrinogen concentration, thrombin concentration, pH and ionic a strength have a similar dependence on the final gel structure and clotting time in D2O and H2O. SDS-gel electrophoresis on fibrin samples, which were cross-linked by factor XIII, yielded results where the cross-linking of the gamma-chain appeared to be the same in D2O and H2O. The alpha-chain cross-linking was somewhat faster in D2O than in H2O. When fibrinogen solutions in 95% D2O were incubated at 20 mM CaCl2, a slow gelation of fibrinogen was observed, which was found to be induced by trace amounts of factor XIII. The final gel turbidity appeared to be about the same for this gelation as for that induced by thrombin. The differences in solubility for fibrinogen, kinetics for the enzyme reaction and optical properties for the fibrin gels in D2O and H2O may be explained by differences in electrostatic interactions, hydrogen bonding and hydration of fibrinogen in these two media.     

Chromatin very small angle neutron scattering: further evidence for a 30 nm diameter super coil in dilute solutions.

Intact chromatin, chromatin minus histone H1, and nuclease digestion fragments have been studied by very small angle neutron scattering. The results are not consistent with a straight chain of nucleosomes and require the presence of a higher order coiling in monovalent salt solutions. The data are interpretable by a structure having a cross section radius of gryration of 8.5 +/- 1 nm, which suggests an outer diameter for a coil of nucleosomes of 27 +/- 3 nm.     

Ribonuclease hydration and its heat stability in solutions of H2O and D2O

Ribonuclease hydration in wide concentration and temperature range using differential scanning microcalorimetry and NMR technique was studied. The temperature- concentration equilibrium diagram for H2O--ribonuclease system was suggested. Protein hydration in D2O was shown to be higher than in ordinary water. NMR measurements in dilute protein solutions showed that cooperative temperature denaturation was followed by solvation changes. Models of globular protein hydration are discussed.     

两价盐对C_8－卵磷脂微团溶液液－液相分离的影响

采用激光散射等方法测定了在添加不同的两价无机盐情况下,Ｃ８－卵磷脂微团溶液的液－液相分离曲线,及其相变临界温度随盐类型和盐离子强度的变化。并从理论上分析了两价盐对Ｃ８－卵磷脂微团溶液吉布斯自由能的影响,推导出－关于盐对该微团溶液相交临界温度影响的半经验半理论公式,可满意地描述该微团溶液的液－液相分离受益调控的规律。     

A formula for correlating pKa values determined in D2O and H2O

A linear correlation between pH-meter readings in equivalent D2O and H2O solutions, determined experimentally, leads to a novel equation, which allows for a direct recalculation of pKa values measured in D2O into a H2O equivalent: pKH=0.929pKH*+0.42. The comparison of this equation with the previously used approach is discussed.     

两价盐对C_8－卵磷脂微团溶液液－液相分离的影响

采用激光散射等方法测定了在添加不同的两价无机盐情况下,Ｃ８－卵磷脂微团溶液的液－液相分离曲线,及上变临界温度随盐类型和盐离子强度的变化,并从理论上分析两价盐对Ｃ８－卵磷脂微团溶液吉布斯自由能的影响,推导出－关于盐对该微团溶液相变临界温度影响的半经验半理论公式,可满意地描述该微团溶液的液－液相分离受盐调控的规律。     

Small angle neutron scattering studies of chromatin subunits in solution

Neutron scattering studies have been performed on dilute solutions of the fundamental subunit of chromatin, the nucleosome. The subunits contain approximately 195 base paris (bp) of DNA and histones H2A, H2B, H3, and H4. Measurements of the small angle scattering curves in various H2O/D2O solvents allow the contrast dependence of the radius of gyration of the subunits to be examined and give the mean scattering density of the particle. Further application of contrast variation to the higher angle scatter curves allows the contributions from the shape and internal structure of the subunits to be analyzed separately. From these results, we are able to propose a spherically averaged structure with most of the histones closely packed into a core of radius 3.2 nm surrounded by a loosely packed DNA-rich shell of 2.0 nm thickness resulting in a particle of 5.2 nm average radius. Model calculations for ellipsoids show that the outer shape of the subunit must have an axial ratio between 0.5 and 1.4 but is probably best described by more spherical particle. These results are correlated with the diffraction from chromatin films to provide an explanation for some of the diffraction rings.     

Study of interaction between aspartic acid and silver by surface-enhanced Raman scattering on H(2)O and D(2)O sols.

Three different surface-enhanced Raman scattering (SERS) spectra are recorded for aspartic acid on H(2)O silver sols under different concentrations and pH values. The analysis of the results shows that it interacts with the metal surface in its dianionic form in two different ways, depending on the pH and concentration. Moreover, in some cases the fumarate anion is detected, which results from the chemical surface transformation of the aspartate. The N-deuterated aspartic acid adsorbed on the D(2)O silver sols gives rise to only one SERS spectrum as a consequence of the interaction of amino and carboxylate functional groups of the dianion with the metal, independent of the concentration and pD.     

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.     

Roles of Extensibility and Turgor in Gibberellin- and Dark-stimulated Growth

The elongation response elicited by incubating excised hypocotyl sections of lettuce (Lactuca sativa L.) in light in gibberellin (GA) can be enhanced by the addition of Cl(-), Br(-), and NO(3) (-) salts of K(+) and Na(+). Sections incubated in light in the absence of GA do not elongate in response to the addition of salts. In contrast, excised hypocotyls incubated in darkness elongate equally in both GA and water, and their elongation can also be enhanced by KCl treatment. Growth stimulation by the salts of K(+) and Na(+) occurs optimally at 10 mm and the magnitude of the response is proportional to the duration of salt treatment. Although the growth of sections incubated in light in the absence of GA is not enhanced by various salts of K(+) and Na(+), the concentration of these cations exceeds that in GA-treated sections. In dark-grown tissue, uptake of K(+) also occurs in both GA- and H(2)O-treated sections incubated in 10 mm KCl. Since increased osmotic potential resulting from cation uptake does not correlate with growth stimulation resulting from salt treatments, we conclude that increased cell turgor is not the principal driving force for growth in hypocotyl sections. Changes in the extensibility of GA-treated, light-grown tissue and dark-grown tissue incubated with and without GA correlate with the increased growth rate of these sections. Incubation of sections in KCl results only in changes in water potential of sections without having a significant effect on extensibility. When changes in water potential are accompanied by increased extensibility, however, a marked increase in growth rate is observed.     

Structure of phenylalanine-accepting transfer ribonucleic acid and of its environment in aqueous solvents with different salts

Yeast tRNA(Phe) was studied in different salt-containing solvents by UV absorbance and small-angle neutron scattering (SANS). This extends results obtained previously in NaCl and KCl solutions [Li, Z.-Q., Giegé, R., Jacrot, B., Oberthür, R., Thierry, J. C., & Zaccai, G. (1983) Biochemistry 22, 4380-4388]. As expected, at low concentrations of all salts studied, the tRNA molecule is unfolded. The importance of specific counterion interactions and the flexibility of the macromolecule are emphasized by the observation that it cannot take up its folded structure in N(CH3)4Cl solvents, even when that salt concentration is increased to 1 M, in the absence of Mg ions. In CsCl solvents, on the other hand, the folded conformation is obtained in salt concentrations above about 0.2 M, similar to NaCl or KCl. By a comparison of SANS results in CsCl H2O and CsCl 2H2O solvents with the data from NaCl and KCl solvents, thermodynamic and structural parameters were derived for the solvated macromolecule. All the data are accounted for, quantitatively, by a model for the particle in NaCl, KCl, or CsCl solution made up of tRNA76-, closely associated with 76 positive hydrated counterions, surrounded by an aqueous solvent layer that excludes salt (and, therefore, of density different from that of bulk solvent). The mass of water in that layer depends on salt concentration, and the values found are consistent with those predicted by the Donnan effect.     

Stability of globular proteins in H2O and D2O

In several experimental techniques D2O rather then H2O is often used as a solvent for proteins. Concerning the influence of the solvent on the stability of the proteins, contradicting results have been reported in literature. In this paper the influence of H2O-D2O solvent substitution on the stability of globular protein structure is determined in a systematic way. The differential scanning calorimetry technique is applied to allow for a thermodynamic analysis of two types of globular proteins: hen's egg lysozyme (LSZ) with relatively strong internal cohesion ("hard" globular protein) and bovine serum albumin (BSA), which is known for its conformational adaptability ("soft" globular protein). Both proteins tend to be more stable in D2O compared to H2O. We explain the increase of protein stability in D2O by the observation that D2O is a poorer solvent for nonpolar amino acids than H2O, implying that the hydrophobic effect is larger in D2O. In case of BSA the transitions between different isomeric forms, at low pH values the Nm and F forms, and at higher pH values Nm and B, were observed by the presence of a supplementary peak in the DSC thermogram. It appears that the pH-range for which the Nm form is the preferred one is wider in D2O than in H2O.     

Use and abuse of exogenous H2O2 in studies of signal transduction

The goal of this review is to present a rationale for the use of exogenous H(2)O(2), which has been demonstrated to have both toxicological and physiological signaling roles. Reasons for the use of exogenous application of nontoxic concentrations of H(2)O(2) in model systems and caveats for interpretation of the data obtained will both be presented. Briefly, an argument for the cautious use of the addition of exogenous H(2)O(2) is that, because of the permeability of cell membranes to this neutral small molecule, a concentration that is produced locally and that is necessary for the physiological action can be mimicked. On the other hand, it must be recognized that the addition of an agent or its enzymatic generation in the medium may produce reactions that may not normally occur because the total dose of H(2)O(2) and the concentration of H(2)O(2) in some cellular locations will exceed what is normally achieved even under a pathophysiological state. For this reason, this review will try to provide an unbiased balanced pros- and -cons analysis of this issue.     

Mammalian antioxidant defenses are not inducible by H2O2

As an approach to understanding how mammals regulate H(2)O(2) toxicity, intracellular concentration to prevent its we analyzed the genome-wide mRNA profile changes of human cells after treatment with a non-toxic H(2)O(2) concentration. We identified a large and essentially late H(2)O(2) response of induced and repressed genes that unexpectedly comprise few or no antioxidants but mostly apoptosis and cell cycle control activities. The requirement of the p53 regulator for regulating about a third of this H(2)O(2) stimulon and the lack of an associated enhancement of total cellular H(2)O(2) scavenging activity further suggest that H(2)O(2) elicits a stress antiproliferative/repair response that does not increase antioxidant defenses. We conclude that mammalian antioxidant defenses are constitutive, a finding that contrasts with the oxidant-inducibility of such defenses in microorganisms. This finding might be important in understanding the role of H(2)O(2) as a key signaling molecule in mammals.     

An optical method for the determination of the isotopic composition of 1H2O/2H2O mixtures: Eu3+ luminescence lifetimes

A spectroscopic method employing pulsed dye laser instrumentation is described for the determination of the 1H2O/2H2O composition of aqueous solutions by the measurement of reciprocal excited state lifetimes of EuEDTA-. The reciprocal lifetimes, gamma-1, of the 1H2O/2H2O mixtures increase linearly with the mole fraction of 1H2O. For EuEDTA- the relationship between gamma-1 and the mole fraction, chi H, of 1H2O in 1H2O/2H2O mixtures is expressed by the equation chi H = 0.37 gamma-1-0.152, with a sensitivity in chi H of +/- 0.02. The reciprocal lifetimes are independent of pH in the range 5.1 to 10.5, changes in ionic strength, and the type of buffer used in EuEDTA- containing solutions.