Electrophoretic mobility of lambda phage HIND III and HAE III DNA fragments in agarose gels: a detailed study

The electrophoretic mobility (μ) of DNA fragments from λ phage and ΦX 174, split by restriction enzyme to molecular lengths from 3 × 10² to 2.36 × 10⁴ base pairs, has been investigated in 0.6–4% agarose gels at various field strengths, ionic strengths, and temperatures. As already observed, μ is seen to be very sensitive to the field, increasing with field strength. The sensitivity increases with the molecular length of the DNA and decreases at high gel concentration. Our data are in qualitative agreement with recent theoretical predictions that concern the influence of the electric field on electrophoretic mobility. Mobility data have been extrapolated to zero field. This enables a comparison of our experimental results with theoretical predictions on the dependence of μ on the molecular weight of the DNA fragments. Our data fit, quite closely, a reptation model, where the tube path is described as a semiflexible entity with a persistence length equal to the pore diameter. The influence of the agarose concentration and the ionic strength of the buffer on the two parameters of the model—intrinsic electrophoretic mobility (μ₀) and the number of base pairs per element of the tube (g)—are well described by the model. The temperature dependence of the electrophoretic mobility, together with the influence of the agarose concentration on μ₀, indicate that the hydrodynamic drag is the leading frictional force on the DNA molecules in the gel.     

Charge effects in the sedimentation of polyelectrolytes

The theory for sedimentation of macromolecular polyelectrolytes in solutions of low-ionic strength has been modified for the case of a supporting low molecular weight electrolyte possessing a partial specific volume close to unity. The final equations simplify greatly, allowing sedimentation data to be plotted in a linear fashion. These data then may be readily extrapolated to infinite dilution, yielding a value, S^o, simply related to size and shape. From the initial slope of such plots, the net charge on the macro-molecular polyelectrolyte may be determined. It is suggested that tetramethyl-ammonium chloride may prove to be a satisfactory supporting electrolyte for such studies.     

Complex formation regulates the glycosylation of the reversibly glycosylated polypeptide

Reversible glycosylated polypeptides (RGPs) are highly conserved plant-specific proteins, which can perform self-glycosylation. These proteins have been shown essential in plants yet its precise function remains unknown. In order to understand the function of this self-glycosylating polypeptide, it is important to establish what factors are involved in the regulation of the RGP activity. Here we show that incubation at high ionic strength produced a high self-glycosylation level and a high glycosylation reversibility of RGP from Solanum tuberosum L. In contrast, incubation at low ionic strength led to a low level of glycosylation and a low glycosylation reversibility of RGP. The incubation at low ionic strength favored the formation of high molecular weight RGP-containing forms, whereas incubation at high ionic strength produced active RGP with a molecular weight similar to the one expected for the monomer. Our data also showed that glycosylation of RGP, in its monomeric form, was highly reversible, whereas, a low reversibility of the protein glycosylation was observed when RGP was part of high molecular weight structures. In addition, glycosylation of RGP increased the occurrence of non-monomeric RGP-containing forms, suggesting that glycosylation may favor multimer formation. Finally, our results indicated that RGP from Arabidopsis thaliana and Pisum sativum are associated to golgi membranes, as part of protein complexes. A model for the regulation of the RGP activity and its binding to golgi membranes based on the glycosylation of the protein is proposed where the sugars linked to oligomeric form of RGP in the golgi may be transferred to acceptors involved in polysaccharide biosynthesis.     

Heteroxylans from maize bran in aqueous solution. Part II: Studies of polyelectrolyte behaviour

Potentiometric and viscosimetric behaviours of four samples of heteroxylans, extracted from corn bran at different temperatures with varying type and concentration of alkali were studied in dilute solutions. All four samples behave as typical polyelectrolytes with a low charge density parameter (λ = 0.15). They could be described by the Lifson and Katchalsky model pK₀≈3.15), indicating a rather homogeneous repartition of the charges along the macromolecules. Their intrinsic viscosities varied linearly with the reciprocal of the square root of the ionic strength. Their flexibility parameters, B (≈0.024), were similar to those of semi-flexible macromolecules, and their intrinsic viscosities extrapolated to infinite ionic strength were between 144 and 177ml/g. Their conformational dimensions depended on the type of counter-ion in solution (dimensions minimal with divalent cations and decreasing with size of monovalent alkali metal ions), of the temperature and the pH of the solution. The radius of gyration for unchanged heteroxylans was estimated from intrinsic viscosities extrapolated to infinite ionic strength (R²_g∞^1/2 = 21−25 nm).     

Isolation and polymerization of brain actin

The studies presented here confirm earlier reports that an actin-like protein is abundant in brain. However, when the traditional procedures for isolating muscle actin are applied to brain, many different proteins are extracted. Tubulin, a major protein in brain with properties similar to actin, is the major constituent. A method is described for isolating the “brain actin” to a purity of 90–95%. The isolation method begins with an extraction of bovine brain in low ionic strength buffer with ATP and sucrose. The extract is treated with NH₄SO₄, MgCl, and KCl and incubated at 37°C. A precipitate is formed which contains primarily tubulin and brain actin. Resolubilization of the brain actin is achieved with a low ionic strength buffer solution with sucrose and ATP. Further purification is accomplished by a cycle of polymerization—depolymerization. This “brain actin” shares with muscle actin the following properties: (1) Similar molecular weight and molecular charge as determined by SDS polyacrylamide gel and ordinary disc electrophoresis; (2) Polymerization to a filamentous form under the same conditions; (3) Contains 3-methylhistidine; (4) Vinblastine sulfate will induce filament formation.     

Cumulative Title Index

Abstract

Quantitative data on the speciation of chitosan (310 kDa) with low and high molecular weight carboxylates in aqueous solution are reported. The following carboxylic ligands were considered: monocarboxylate (butyrate); dicarboxylates (malonate, succinate, azelate); tricarboxylate (1,2,3-propa-netricarboxylate); tetracarboxylate (1,2,3,4-butanetetracarboxylate); polyacrylates (2.0 and 20 kDa); polymethacrylate (5.4 kDa). The investigation was performed by potentiometry at t =25°C, at low ionic strength (without addition of supporting electrolyte) and at I =0.15mol L^?1 (NaCl). For all the systems the formation of (chitosan)LH_i species was found (L = carboxylic ligand; i = 1 to 4 depending on the carboxylic ligand considered). The stability of proton–chitosan–carboxylate species depends on the number of carboxylic groups involved in the complexation, and it was possible to calculate a rough free energy value per bond ΔG_n = ?15±2kJ mol^?1. By using the stability data, the quantitative sequestering capacity of chitosan towards the carboxylates here considered [expressed as the-log(total chitosan concentration) necessary to bind 50% of carboxylate, i.e., pL₅₀] was calculated for different pH values, at low ionic strength and at I =0.15 mol L^?1. The pL₅₀ values, ranging from 3 to 7, show that chitosan is quite a strong sequestering agent towards carboxylates. Evidences were also obtained for the different behaviour between low and high molecular weight carboxylates.     

Physicochemical investigation of k-carrageenan in the random state

Light-scattering, viscosity, and sedimentation experiments on aqueous solutions of k-carrageenan show that this sulfated polygalactose is an expanded flexible random coil. This expansion is due to long-range interactions that are predominantly electrostatic. Extrapolation of viscosity data to infinite ionic strength provided values for the intrinsic viscosity which were subjected to the Stockmayer-Fixman analysis, giving a value for the Mark-Houwink coefficient under theta-conditions, K_θ, of 0.27. The characteristic ratio, C_∞, under these conditions is 7.8, and the conformation factor σ is 2. In a solution of 0.118 ionic strength, where a Mark-Houwink exponent a_η of 0.86 is found, the radii of gyration calculated from viscosity data are lower than those found from the angular dependence of scattered light. On the other hand, the radius of gyration found from the sedimentation rate agrees well with the light-scattering radius. The relations between molecular parameters are corrected for the poly-dispersity of the sample.     

Electric birefringence of native DNA in an alternating field

The relaxation of the birefringence of native DNA in solution was investigated in a pulsed sine-wave electric field. Relaxation times were calculated from the degree of damping of the birefringence signal and were studied as a function of the strength and frequency of the applied field, the molecular weight of the DNA, and the viscosity and ionic strength of the solvent. Relaxation times decrease with increasing field strength. For high-molecular weight DNA (>10⁶ daltons), the relaxation times decreased with frequency and increased less than linearly with viscosity. For low-molecular-weight DNA (<6 × 10⁵ daltons), the relaxation times were independent of frequency, increased linearly with viscosity, and varied with the 1.65 ± 0.1 power of the molecular weight. The average birefringence of high-molecular-weight DNA decreased with frequency in 0.001M Na₂ EDTA plus NaOH, pH 7.0, but is much less frequency-dependent if the EDTA concentration is reduced tenfold, while the average birefringence of sonicated DNA increases in both solvents with increasing frequency.     

Effect of Screening Out Implausible Energy Intake Reports on Relationships between Diet and BMI

Objective: We present an updated method for identifying physiologically implausible dietary reports by comparing reported energy intake (rEI) with predicted energy requirements (pER), and we examine the impact of excluding these reports. Research Methods and Procedures: Adult data from the Continuing Survey of Food Intakes by Individuals 1994 to 1996 were used. pER was calculated from the dietary reference intake equations. Within‐subject variations and errors in rEI [coefficient of variation (CV) ～ 23%] over 2 days (d), pER (CV ～ 11%), and measured total energy expenditure (mTEE; doubly labeled water, CV ～ 8.2%) were propagated, where ±1 SD = . Thus, a report was identified as implausible if rEI was not within 78% to 122% of pER. Multiple cut‐offs between ±1 and ±2 SD were tested. Results: %rEI/pER = 81% in the total sample (n = 6499) and progressively increased to 95% in the ±1 SD sample (n = 2685). The ±1 to 1.4 SD samples yielded rEI‐weight associations closest to the theoretical relationship (mTEE to weight). Weak or spurious diet—BMI associations were present in the total sample; ±1 to 1.4 SD samples showed the strongest set of associations and provided the maximum n while maintaining biological plausibility. Discussion: Our methodology can be applied to different data sets to evaluate the impact of implausible rEIs on health outcomes. Implausible rEIs reduce the overall validity of a sample, and not excluding them may lead to inappropriate conclusions about potential dietary causes of health outcomes such as obesity.     

Dynamic light scattering studies of internal motions in DNA. II. Clean viral DNAs

Internal Brownian motions of clean ?29 and λ-DNAs have been studied using photon-correlation techniques at both visible (λ₀ = 632.8 nm) and uv (λ₀ = 363.8 nm) wavelengths. The present dynamic light scattering data, which extend to K² = 19 × 10¹⁰ cm^?2, can in every case be satisfactorily simulated by a Rouse-Zimm model polymer with an appropriate choice of the three model parameters. The effects of pH, salt concentration, single-strand breaks, and molecular weight on those model parameters have also been investigated. Intact clean DNAs exhibit surprisingly little variation with pH from 7.85 to 10.25, with salt concentration from 0.01 NaCl to 5.4M NH₄Cl, or with molecular weight or GC content. The single-strand breaks have no effect at pH 9.46, but produce dramatic changes in the model parameters at pH 10.0 and 10.25, indicating the introduction of titratable joints at those pHs. The failure of either single-strand breaks or a large change in GC content to alter the model parameters in the neutral pH range is a strong indication that local denaturation is not required for those flexions and torsions that dominate the relaxation of fluctuations in the scattered light. The Langevin relaxation time for the slowest internal mode of a particular Rouse-Zimm model derived from the dynamic light scattering data is compared with pertinent literature data extrapolated to the same molecular weight. The present algorithm for determining model parameters from the light-scattering D_app vs K² curve actually yields a Langevin time in fairly good agreement with the literature value. For unknown reasons the light-scattering D₀ values generally exceed those obtained from the molecular weight and sedimentation coefficient by about 20%.     

Comparison of Different Approaches for Calculation of Polyelectrolyte Free Energy

Abstract

We consider the problem of the mean field (Poisson-Boltzmann) calculation of the electrostatic free energy for a strongly charged polyelectrolyte such as DNA in a salt solution. We compare two approaches to calculate the free energy: (i) direct one, starting from the statistical-mechanical expression for the electrostatic free energy and (ii) the polyion charge variation method. In the infinite dilution limit (in respect to polyion) and in excess salt (IDLES) the two approaches are fully equivalent. This is shown by straight forward algebra. We have performed specific calculations of the free energy difference for the case of B-Z transition in DNA as a function of ionic strength. As expected, the two approaches led to identical results. The ionic strength dependence of the B-to-Z free energy proves to be concaved up and as a result Z-DNA is stabilized at low ionic concentration as well as at high salt in full agreement with our previous results (M.D. Frank- Kamenetskii et al, J. Biomol. Struct. Dyn. 3, 35–42 (1985)). Our data quantitatively agree with the results of Soumpasis (D. M. Soumpasis, J. Biomol. Struct. Dyn. 6, 563–574 (1988)). However, his claim about the absence of the effect of stabilization of Z-DNA at low salt proves to be groundless, and the criticism of our earlier approach seems to be irrelevant.     

The self-assembly of collagen molecules

The aggregation of native acid-soluble collagen (N-ASC) and of pronase-treated acid soluble collagen (P-ASC) was examined in solution under conditions which varied from those of minimum collagen-collagen interaction to those leading to incipient fiber formation. Molecular weights and weight distributions were determined in the analytical ultracentrifuge using the Yphantis high speed sedimentation equilibrium and Aarchiblad approach-to-equilibrim techniques. The aggregation was pH and ionic strength dependent in each case. Under conditions of minimum aggregation (low pH, low ionic strength), N-ASC showed the presence of permant aggregates. At higher pH and ionic strength, a higher fraction of aggregate was formed but these were of the same charcter and molecular weight as the permanent aggregates. The aggregates were of a single molecular size, with a weight of 1.5 × 10⁶ daltons, compared with a monomer collagen weight of 3.1 × 10⁵ daltons. The P-ASC formed aggregates also but to a much lower extent and the maximum aggregate size corresponded to dimers in molecular weight. These data show the major importance of molecular end-regions in collagen aggregation to form native type fibers and, by virtue of the discrete size of the N-ASC aggregates, support the microfibrillar hypothesis for the assembly of collagen fibrills.     

Factors Influencing Conformation Changes in a 7S Protein of Soybean Globulins by Ultracentrifugal Investigations

Effects of pH, ionic strength, kind of salts and disulfide bond cleaving agent (2-mercaptoethanol) on conformation changes revealed on ultracentrifugal patterns of a 7S protein in soybean globulins were investigated. In the solution with lower pH than isoelectric point, this protein dissociated into two components in low ionic strength, but showed a 7S sedimentation pattern in higher ionic strength than 0.1. On the other hand, in the solution with higher pH than isoelectric point, this protoin showed aggregation to a 9S isomer in lower ionic strength than 0.1. Between ionic strength of 0.1 and 0.5, the mixture of 7S and 9S forms existed and in higher ionic strength than 0.5, the protein kept a 7S form stablely. These reactions were reversible and effect of 2-mercaptoethanol was scarcely observed but those of salts were observed.

The molecular weight of the 9S isomer was approximately 370,000 and the s_20,w value was 12.30S. Therefore, the 9S isomer was considered to be a dimer of the 7S protein.     

A comparison of the effects of ionic strength on three preparations of acetylcholinesterase in the presence and absence of gallamine

The kinetic behaviour of three forms of acetylcholinesterase as a function of ionic strength of the medium was investigated. The forms of enzyme were that bound to human erythrocyte membranes, acetylcholinesterase solubilized from these by Triton X-100, and a commercial preparation of the enzyme from bovine erythrocytes. The properties investigated were hydrolysis of the substrate acetylthiocholine, decarbamylation of dimethylcarbamyl-acetylcholinesterase and ageing of isopropylmethylphosphonyl-acetylcholinesterase. The effect of 10^?5 M gallamine triethiodide on these properties was also examined as a function of ionic strength.Detailed results for the variation of kinetic behaviour with ionic strength and the presence of gallamine are presented. No unified theory to predict the influence of these variables on all three forms of the enzyme could be formulated. Thus, the enzyme conformation stabilized by gallamine at low ionic strength was not necessarily similar to that of the gallamine-free enzyme at physiological ionic strength. Nor was it useful to consider the free enzyme at low ionic strength to be a model of the membrane-bound enzyme in vivo (Crone, 1973).It was concluded that kinetic results for solubilized and partially or wholly purified acetylcholinesterase cannot be extrapolated to the membrane-bound enzyme. Prediction of the effect of drugs on the system in vivo requires the use of the membrane-bound enzyme.     

Carbohydrates of the antarctic brown seaweed Ascoseira mirabilis

Mannitol, sucrose and four monosaccharides were obtained from an ethanolic extract of Ascoseira mirabilis. Sequential extraction with aqueous calcium chloride, dilute acid and dilute alkali gave mixtures of laminaran, ‘fucan’ and alginic acid. Laminarans fractionated from the extracts contained different proportions of uniformly (1 → 3) and (1 → 6) linked chains of β-D-glucose residues. The ‘fucan’ contained varying proportions of fucose, galactose and glucuronic acid, small amounts of xylose, mannose, glucose, half ester sulphate and protein. Extraction of the weed under mild alkaline conditions gave a yield of 13.4% of low molecular weight calcium alginate with a mannuronate to guluronate ratio of 30:70 and only a small proportion of sequences of alternating residues. Selective extraction and fractionation gave alginate fractions rich (> 80%) in mannuronate or guluronate.     

Electron-transfer reactions of photoreduced flavin analogues with c-type cytochromes: quantitation of steric and electrostatic factors

We have found correlations between rate constants and the difference in redox potential of the reactants for electron-transfer reactions between oxidized cytochromes and either photoproduced riboflavin or flavin mononucleotide (FMN) semiquinones (the latter rate constants extrapolated to infinite ionic strength). The riboflavin-cytochrome rate constants are about 70% of those for reduction by lumiflavin, probably because of steric interference by the ribityl side chain. Reduction of cytochromes by FMN semiquinone was ionic strength dependent in all cases, due to electrostatic interactions. Extrapolation of rate constants to infinite ionic strength shows that the phosphate exerts a significant steric effect as well (rate constants average about 27% of those for lumiflavin, although part of this decrease is due to a difference in the semiquinone pK value). Differences in the magnitude of the FMN steric effect correlate well with surface topology differences for those cytochromes whose three-dimensional structures are known. Mitochondrial cytochromes c and the cytochromes c2 all showed attractive (plus-minus) interaction with FMN in spite of the fact that some of these proteins have large net negative charges. Four small c-type cytochromes (including Pseudomonas cytochrome c-551) show a weak repulsive interaction with FMN semiquinone. We conclude that flavosemiquinones interact at a site on the cytochromes that is near the exposed heme edge. There is a large positive electrostatic field at this site in mitochondrial cytochrome c and the cytochromes c2, but this region is primarily hydrophobic in Pseudomonas cytochrome c-551 and in the other small bacterial cytochromes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of nonspecific ion-protein interactions on the redox chemistry of cytochrome c

 The effects of the ionic atmosphere on the enthalpic and entropic contributions to the reduction potential of native (state III) beef heart cytochrome c have been determined through variable-temperature direct electrochemistry experiments. At neutral or slightly alkaline pH values, from 5 to 50  °C, the reduction enthalpy and entropy become less negative with decreasing ionic strength. The reduction entropy extrapolated at null ionic strength is approximately zero, indicating that, in the absence of the screening effects of the salt ions on the network of the electrostatic interactions at the protein-solvent interface, the solvation properties and the conformational flexibility of the two redox states are comparable. The moderate decrease in E°′ observed with increasing ionic strength [ΔE°′_IS =(E°′) _{I =0.1 M}–(E°′) _{I =0 M}=–0.035 V at 25  °C], once the compensating enthalpic and entropic effects of the salt-induced changes in the hydrogen bonding within the hydration sphere of the molecule in the two redox states are factorized out, results in being ultimately determined by the stabilizing enthalpic effect of the negatively charged ionic atmosphere on the ferri form. At pH 9, the ionic strength dependence of the reduction termodynamics of cytochrome c follows distinctive patterns, possibly as a result of specific binding of the hydroxide ion to the protein. A decrease in ionic strength at constant pH, as well as a pH increase at constant ionic strength, induces a depression of the temperature of the transition from the low-T to high-T conformer of cytochrome c, which suggests that a temperature-induced decrease in the pK _a for a residue deprotonation is the key event of this conformational change. Received: 7 April 1999 / Accepted: 19 July 1999     

Deamidation of glutaminyl residues: dependence on pH, temperature, and ionic strength

We have shown the dependence of the deamidation half-times of the peptides, GlyLeuGlnAlaGly and GlyArgGlnAlaGly upon pH, temperature, and ionic strength. Increase in temperature or ionic strength, variation of pH to pH′s higher or lower than pH 6, and the use of phosphate buffer rather than Tris buffer at high pH all decrease the half-time of dcamidation. Temperature increase of 20°C or pH change of 2 pH units decreases the half-time about fivefold, while increase of one ionic strength unit decreases the half-time about twofold. In pH 7.4, I = 0.2, 37.0°C phosphate buffer, the deamidation half-times are 663 ± 74 and 389 ± 56 days respectively for the two peptides, GlyLeuGlnAlaGly and GlyArgGlnAlaGly.These experiments should serve as a warning to peptide and protein experimenters that even the more stable glutaminyl residues are unstable with respect to deamidation in certain solvent conditions. These experiments also provide, along with previously reported experiments on asparaginyl peptides (7), some quantitative data to help with the extrapolation of in vitro deamidation experiments to in vivo deamidation conditions.     

Polyelectroyte behavior of a bacterial polysaccharide from Xanthomonas campestris: Comparison with carboxymethylcellulose

The main physicochemical properties of the polysaccharide called Xanthan produced by Xanthomonas compestris are discussed: the activity coefficient of the counter-ion, the pK(α), and the ionic selectivity are investigated and compared to those of a carboxymetholcellulose. The weight-average molecular weight (M _w = 2 × 10⁶), the intrinsic viscosity and the constant of sedimentation are determined as a function of the ionic strength. It is proved that in dilute solution, there is no intermolecular association, whatever the ionic strength. The conformation is proposed to be a rigid rodlike molecule whose length is 6000 Å, independent of ionic strength > 10^?2N.