Thermostabilization of ovotransferrin by anions for pasteurization of liquid egg white

The effects of anions on the thermostability of ovotransferrin (oTf) were investigated. The temperature, T(m), causing aggregation of oTf was measured in the presence or absence of anions, and the denaturation temperature, T(m)(DSC), was also determined by differential scanning calorimetry (DSC) in the presence of the citrate anion. We found that some anions (phosphate, sulfate and citrate) raised temperature T(m) of oTf by about 5-7 degrees C. However, neither sodium chloride nor sodium bicarbonate raised T(m) by that much. Temperature T(m) was increased by increasing the concentration of the citrate anion, and was in good agreement with denaturation temperature T(m)(DSC), suggesting that denaturation of the oTf molecules resulted in aggregation of oTf. We also demonstrated that the anions, especially sulfate, repressed the heat-aggregation of liquid egg white.The Van't Hoff plot from the T(m) and DeltaH(d) values revealed that two anion-binding sites were concerned with heat stabilization. These binding sites may have been concerned with sulfate binding (not bicarbonate binding) that is found in the crystal structure of apo-form of oTf, since the bicarbonate anion did not raise T(m).     

On the hydration of DNA. I. Preferential hydration and stability of DNA in concentrated trifluoroacetate solution

The hydration of DNA is an important factor in the stability of its secondary structure. Methods for measuring the hydration of DNA in solution and the results of various techniques are compared and discussed critically. The buoyant density of native and denatured T-7 bacteriophage DNA in potassium trifluoroacetate (KTFA) solution has been measured as a function of temperature between 5 and 50°C. The buoyant density of native DNA increased linearly with temperature, with a dependence of (2.3 ± 0.5) × 10^?4 g/cc-°C. DNA which has been heat denatured and quenched at 0°C in the salt solution shows a similar dependence of buoyant density on temperature at temperatures far below the T_m, and above the T_m. However, there is an inflection region in the buoyant density versus T curve over a wide range of temperatures below the T_m. Optical density versus temperature studies showed that this is due to the. inhibition by KTFA of recovery of secondary structure on quenching. If the partial specific volume is assumed to be the same for native and denatured DNA, the loss of water of hydration on denaturation is calculated to be about 20% in KTFA at a water activity of 0.7 at 25°C. By treating the denaturation of DNA as a phase transition, an equation has immmi derived relating the destabilizing effect of trifluoroacetate to the loss of hydration on denaturation. The hydration of native DNA is abnormally high in the presence of this anion, and the loss of hydration on denaturation is greater than in CsCl. In addition, trifluoroacetate appears to decrease the ΔHof denaturation.     

A theory of aggregation in the thermal denaturation region of multistrand biopolymers

A theory explicitly allowing the possibility of aggregation of multistrand biopolymers is proposed. It is found that the same secondary bonds responsible for stabilizing the native structure at low temperature will promote aggregation in the thermal denaturation region for sufficiently long chains. A requirement for both open and zippered regions dictates that the aggregation region does not extend far below T_m. However, its width, or extension on the high-temperature side of T_m, is a strongly increasing function of chain length and also of the cooperativity parameter. The present theoretical results obtained for DNA and collagen with almost no adjustable parameters are in good qualitative agreement with a number of previously poorly understood experimental observations. The significance of such a spontaneous aggregation phenomenon for genetic recombination is noted.     

Studies on poly(L-lysine50, L-tyrosine50)-DNA interaction

Interaction between poly(Lys⁵⁰, Tyr⁵⁰) and DNA has been studied by absorption, circular dichroism (CD), and fluorescence spectroscopy and thermal denaturation in 0.001M Tris, pH 6.8. The binding of this copolypeptide to DNA results in an absorbance enhancement and fluorescence quenching on tyrosine. There is also an increase in the tyrosine CD at 230 nm. The CD of DNA above 250 nm is slightly shifted to the longer wavelength which is qualitatively similar to, but quantitatively much smaller than, that induced by polylysine binding. At physiological pH the poly(Lys⁵⁰, Tyr⁵⁰)–DNA complex is soluble until there is one lysine and one tyrosine per nucleotide in the complex. The same ratio of amino acid residues to nucleotide has also been observed in copolypeptide-bound regions of the complex. The addition of more poly(Lys⁵⁰, Tyr⁵⁰) to DNA yields a constant melting temperature, T_m′, for bound base pairs at 90°C which is close to that of polylysine-bound DNA under the same condition. The melting temperature, T_m, of free base pairs at about 60°C on the other hand, is increased by 10°C as more copolypeptide is bound to DNA. As the temperature is raised, both absorption and CD spectra of the complexes with high coverage are changed, suggesting structural alteration, perhaps deprotonation, on bound tyrosine. The results in this report also suggest that intercalation of tyrosine in DNA is unlikely to be the mode of binding.     

Interaction of spermine and DNA

The effect of spermine upon the denaturation temperature (T_m) of DNA's of various base compositions has been found to depend upon both the base composition of the DNA and the pH of the solution. Measurement of the hydrogen ion titration curve of spermine as a function of temperature reveals that the net charge of the spermine molecule is undergoing a rapid change with temperature in the range of temperatures at which DNA denatures. Since the value of T_m depends upon base composition, the correlation of the effect of spermine upon T_m with the base composition of the DNA used may be explainable in terms of the changing degree of ionization of spermine. The binding of spermine to native DNA has also been studied by dialysis equilibrium. There is no significant variation either in the number of strongly binding sites or strength of binding with base composition. It is concluded that there is no evidence of correlation between the binding of spermine and the base composition of DNA.     

Thermal Denaturation of β-Glucosidase B from <Emphasis Type="Italic">Paenibacillus polymyxa</Emphasis> Proceeds Through a Lumry-Eyring Mechanism

β-glucosidase B (BglB), 1,4-β-d-glucanohydrolase, is an enzyme with various technological applications for which some thermostable mutants have been obtained. Because BglB denatures irreversibly with heating, the stabilities of these mutants are assessed kinetically. It, therefore, becomes relevant to determine whether the measured rate constants reflect one or several elementary kinetic steps. We have analyzed the kinetics of heat denaturation of BglB from Paenibacillus polymyxa under various conditions by following the loss of secondary structure and enzymatic activity. The denaturation is accompanied by aggregation and an initial reversible step at low temperatures. At T ≥ T _m , the process follows a two-state irreversible mechanism for which the kinetics does not depend on the enzyme concentration. This behavior can be explained by a Lumry-Eyring model in which the difference between the rates of the irreversible and the renaturation steps increases with temperature. Accordingly, at high scan rates (≥1 °C min⁻¹) or temperatures (T ≥ T _m ), the measurable activation energy involves only the elementary step of denaturation.     

In vitro aggregation behavior of a non-amyloidogenic λ light chain dimer deriving from U266 multiple myeloma cells

Excessive production of monoclonal light chains due to multiple myeloma can induce aggregation-related disorders, such as light chain amyloidosis (AL) and light chain deposition diseases (LCDD). In this work, we produce a non-amyloidogenic IgE λ light chain dimer from human mammalian cells U266, which originated from a patient suffering from multiple myeloma, and we investigate the effect of several physicochemical parameters on the in vitro stability of this protein. The dimer is stable in physiological conditions and aggregation is observed only when strong denaturating conditions are applied (acidic pH with salt at large concentration or heating at melting temperature T_m at pH 7.4). The produced aggregates are spherical, amorphous oligomers. Despite the larger β-sheet content of such oligomers with respect to the native state, they do not bind Congo Red or ThT. The impossibility to obtain fibrils from the light chain dimer suggests that the occurrence of amyloidosis in patients requires the presence of the light chain fragment in the monomer form, while dimer can form only amorphous oligomers or amorphous deposits. No aggregation is observed after denaturant addition at pH 7.4 or at pH 2.0 with low salt concentration, indicating that not a generic unfolding but specific conformational changes are necessary to trigger aggregation. A specific anion effect in increasing the aggregation rate at pH 2.0 is observed according to the following order: SO₄ ⁻≫Cl⁻>H₂PO₄ ⁻, confirming the peculiar role of sulfate in promoting protein aggregation. It is found that, at least for the investigated case, the mechanism of the sulfate effect is related to protein secondary structure changes induced by anion binding.     

Changes in Conformation,Stability and Condensation of DNA by Univalent and Divalent Cations in Methanol-Water Mixtures

Abstract

Circular dichroism spectroscopy, absorption spectroscopy, measurements of T_m values, sedimentation analysis and electron microscopy were used to study properties of calf thymus DNA in methanol-water mixtures as a function of monovalent cation (Na⁺ or Cs⁺) concentration and also in the presence of divalent cations Ca²⁺, Mg²⁺, and Mn²⁺. In the absence of divalent cations only slight conformational changes occured and no condensation and/or aggregation could be detected. The T_m values depend on the amount of methanol and on the nature and concentration of cations. In methanol-water mixtures higher thermal stability was observed in solutions containing Cs⁺ ions. Up to 40% (v/v) methanol the addition of divalent ions leads to DNA stabilization. At methanol concentration higher than 50% the presence of divalent cations causes DNA condensation and denaturation even at room temperature. The denaturation is reversible with respect to EDTA addition indicating that no separation of complementary strands occured and the resulting form of DNA is probably similar to the P form. DNA destacking appears to be a direct consequence of stronger cation binding by the condensed DNA in methanol-water mixtures.     

Helix-coil transition in nucleoprotein. Effect of ionic strength on thermal denaturation of polylysine-DNA complexes

Thermal denaturation of direct-mixed and reconstituted polylysine–DNA complexes in 2.5 × 10^?4 M EDTA, pH 8.0 and various concentrations of NaCl has been studied. For both complexes, increasing ionic strength of the solution raises T_m, the melting temperature of free base pairs. The linear dependence of T_m on log Na⁺ indicates that the concept of electrostatic shielding on phosphate lattice of an infinitely long pure DNA by Na⁺ can be applied to short free DNA segments in a nucleoprotein. For a direct-mixed polylysine–DNA complex, the melting temperature of bound base pairs T_m′ remains constant at various ionic strengths. On the other hand, the T_m′ in a reconstituted polylysine–DNA complex is shifted to lower temperature at higher ionic strength. This phenomenon occurs for reconstituted complex with long polylysine of one thousand residues or short polylysine of one hundred residues. It is shown that such a decrease of T_m′ is not due to a reduction of coupling melting between free and bound regions in a complex when the ionic strength is raised. It is also not due to intermolecular or intramolecular change from a reconstituted to a direct-mixed complex. It is suggested that this phenomenon is due to structural change on polylysine-bound regions by ionic strength. It is suggested further that Na⁺ may replace water molecules and bind polylysine-bound regions in a reconstituted complex. Such a dehydration effect destabilizes these regions and lowers T_m′. This explanation is supported by circular dichroism (CD) results.     

Reversible and non-reversible thermal denaturation of lysozyme with varying pH at low ionic strength

DSC analysis has been used to quantify the reversibility of unfolding following thermal denaturation of lysozyme. Since the temperature at which protein unfolding occurs, T_m, varies with different solution conditions, the effect on the melting temperature and the degree of refolding after thermal denaturation in low ionic strength sodium phosphate buffers (5–1000 mM) over a range of pH (5–9) in the presence/absence of disaccharides is examined. This study compares the enthalpies of unfolding during successive heating cycles to quantify reversibility following thermal denaturation. The disaccharides, trehalose and maltose were used to assess if the disaccharide induced increase in T_m is reflected in the reversibility of thermally induced denaturation. There was extensive overlap between the T_m values where non-reversible and reversible thermal denaturation occurred. Indeed, for pH 6, at the highest and lowest T_m, no refolding was observed whereas refolding was observed for intermediate values, but with similar T_m values having different proportions of refolded protein. We established a method to measure the degree of reversible unfolding following thermal denaturation and hence indirectly, the degree to which protein is lost to irreversible aggregation, and show that solution conditions which increase melt transition temperatures do not automatically confer an increase in reversibility. This type of analysis may prove useful in assessing the stability of proteins in both the biopharmaceutical and food industries.     

Helix–coil transition and conformational studies of protamine–DNA complexes

Protamine–DNA complexes prepared by the method of direct and slow mixing in 2.5 × 10^?4M EDTA, pH 8.0, have been studied by thermal denaturation and circular dichroism. The complexes show biphasic melting with T_m at about 50 °C corresponding to the melting of free DNA regions and T_m′ at about 92 °C corresponding to the melting of protamine-bound regions. In protamine-bound regions there are 1.38 amino acid residues per nucleotide, indicating a nearly completely charge neutralization. T_m is increased but T_m′ is not when the ionic strength of the buffer is raised. This also supports a full charge neutralization in protamine-bound regions. The circular dichroism of the complexes can be decomposed into two components, Δ_ε0 of free DNA regions in B-form conformation and Δ_εb of protamine-bound regions in a characteristic conformation neither that of B- nor C-form but somewhere between them.     

Proton fluxes associated with erythrocyte membrane anion exchange

Summary Transient extracellular pH changes accompany the exchange of chloride for sulfate across the erythrocyte membrane. The direction of the extracellular pH change during chloride efflux and sulfate influx depends on experimental conditions. When bicarbonate is present, the extracellular pH drops sharply at the outset of the anion exchange and tends to follow the partial ionic equilibrium described by Wilbrandt (W. Wilbrandt, 1942.Pfluegers Arch. 246:291). When bicarbonate is absent, however, the anion exchange causes the pH to rise, indicating that protons are cotransported with sulfate during chloridesulfate exchange. The pH rise can be reversed by the addition of HCO ₃ ^– (4 m) or 2,4-dinitrophenol (90 m). This demonstrates that the proton-sulfate cotransport can drive proton transport uphill. The stoichiometry of the transport is that one chloríde exchanges for one sulfate plus one proton. These results support the titratable carrier model proposed by Gunn (Gunn, R.B. 1972.In: Oxygen Affinity of Hemoglobin and Red Cell Acid-Base Status. M. Roth and P. Astrup, editors. p. 823. Munksgaard, Copenhagen) for erythrocyte membrane anion exchange.     

ISOLATION,PURIFICATION, AND CHARACTERIZATION OF THERMOPHILIC T80 ISOENZYME OF XYLOSE ISOMERASE FROM THE XEROPHYTE Cereus pterogonus

A thermostable isoenzyme (T₈₀) of xylose isomerase from the eukaryote xerophyte Cereus pterogonus was purified to homogeneity by precipitation with ammonium sulfate and column chromatography on Dowex-1 ion exchange, with Sephadex G-100 gel filtration, resulting in an approximately 25.55-fold increase in specific activity and a final yield of approximately 17.9%. Certain physiochemical and kinetic properties (K_m and V_max) of the T₈₀ xylose isomerase isoenzyme were investigated. The molecular mass of the purified T₈₀ isoenzyme was 68 kD determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Polyclonal antibodies against the purified T₈₀ isoenzyme recognized a single polypeptide band on Western blots. The activation energy required for the thermal denaturation of the isoenzyme was determined to be 61.84 KJ mol^?1. The use of differential scanning calorimetry established the melting temperature of the CPXI isoenzyme to be 80°C, but when studied with added metal ions, melting temperature increases to more than the normal. Fluorescence spectroscopy of T₈₀ isoenzymes yielded an emission peak with λ_em at 320 nm and 340 nm, respectively, confirming the presence of Trp residue in these proteins. Electron paramagnetic resonance (EPR) analysis at liquid nitrogen temperature established the presence of Mn²⁺ and Co²⁺ associated with each isoenzyme. These enzyme species exhibited different thermal and pH stabilities compared to their mesophilic counterparts and offered greater efficiency in functioning as a potential alternate catalytic converter of glucose in the production of high-fructose corn syrup (HFCS) for the sweetener industry and for ethanol production.     

On the course of thermal denaturation of deoxyribonucleic acids after γ-irradiation

Summary The changes which are caused by action of-irradiation onDNAs of various origin were followed by spectrophotometric method at differing thermal denaturation curves. It was found that all measured bacterialDNAs as well asDNA isolated from calf thymus, irradiated by exposures higher that 5×10⁴ R, produced significantly decreasedT _m values with concomittantly decreased hyperchromic effect and changed transition intervals obtained in 10^–2 M sodium phosphate, 10^–3 M EDTA medium at pH 7. It was also observed that higher-irradiation exposures caused the loss of renaturation ability ofEscherichia coli DNA.Abbreviations used DNA deoxyribonucleic acid - G guanine - C cytosine - E ₂₆₀ extinction (absorbancy) measured at 260m - T _m the temperature corresponding to the midpoint of the absorbance rise - 2/3 the transition interval of the denaturation curve corresponding to the temperature interval between 17–83% of the total absorbancy increment of the denaturation curve - SSC standard saline citrate buffer (0.15 M NaCl, 0.015 M sodium citrate, pH 7) - PE 10^–2 M sodium phosphate buffer [molarity related to (PO₄)] - PE 10^–3 M EDTA, pH 7. 1.000 ml prepared as follows: 0.608 g NaH₂PO₄.2 H₂O, 0.218 g Na₂HPO₄.12 H₂O, 0.372 g disodium salt of EDTA, 1.0 ml 1 M NaOH. Concentration of Na ions — 0.02 M.     

Carboxylation and mineralization ofm-cresol by a sulfate-reducing bacterial enrichment

An enrichment culture that anaerobically degradedm-cresol under sulfate-reducing conditions was obtained from an anoxic aquifer.m-Cresol removal by the culture was greatest when sulfate or thiosulfate served as electron acceptors; sulfite, nitrate, and CO₂ were poor substitutes for sulfate. A¹⁴C-labeled carboxylated intermediate was detected when the culture was given¹⁴C-labeled bicarbonate and nonlabeledm-cresol or nonlabeled bicarbonate and¹⁴C-labeledm-cresol. Metabolism of the carboxylated intermediate yielded¹⁴C-acetate, which was eventually converted to¹⁴CO₂. Trace quantities of methylbenzoic acid were also detected as a putativem-cresol intermediate. The importance of this dehydroxylated intermediate in the anaerobic degradation ofm-cresol is unclear, since an amendment of 2-methylbenzoic acid was not degraded by the culture. The stoichiometry of electron acceptor consumption and carbon mass balances confirm thatm-cresol was mineralized by the culture.     

Recognition of silver nanoparticles surface‐adsorbed citrate anions by macrocyclic polyammonium cations: a spectrophotometric approach to study aggregation kinetics and evaluation of association constant

In this report, we have studied the recognition of citrate anions adsorbed on the surface of silver nanoparticles (cit‐Ag‐NPs), by macrocyclic polyammonium cations (MCPACs): Me₆[14]ane‐N₄H₈⁴⁺ (Tet‐A/Tet‐B cations) and [32]ane‐N₈H₁₆⁸⁺, which are well reputed anion recognizers and are treated as to mimic of biological polyamines. The study was monitored on ultraviolet–visible spectroscopy by performing a titration of the aqueous dispersion of the cit‐Ag‐NPs by the aqueous solution of MCPACs. The ultraviolet–visible time‐scan plots over the reduction of the absorption band of surface plasmon resonance of cit‐Ag‐NPs at 390 nm are well fitted with fourth‐order polynomial equation and are employed to determine the initial aggregation rate constants. It has been stated that the aggregation is the result in electrostatic attraction followed by H‐bond formation between the surface‐adsorbed citrate anions and added MCPACs. The atomic force microscopy results have evidenced aggregation of cit‐Ag‐NPs in presence of MCPACs. The evaluated H‐bonded association constant (K_asso) using Benesi–Hildebrand method reveals that [32]ane‐N₈H₁₆⁸⁺ cations form stronger association complex, as expected, with the citrate anions than the Me₆[14]ane‐N₄H₈⁴⁺ cations. The study would thus provide the insight of molecular interactions involved in nanoparticle surface‐adsorbed anions with biological polyamines. Copyright © 2016 John Wiley & Sons, Ltd.     

Involvement of the antiplatelet activity of magnesium sulfate in suppression of protein kinase C and the Na<Superscript>+</Superscript>/H<Superscript>+</Superscript> exchanger

Magnesium sulfate is widely used to prevent seizures in pregnant women with hypertension. The aim of this study was to examine the inhibitory mechanisms of magnesium sulfate in platelet aggregation in vitro. In this study, magnesium sulfate concentration-dependently (0.6–3.0 mM) inhibited platelet aggregation in human platelets stimulated by agonists. Magnesium sulfate (1.5 and 3.0 mM) also concentration-dependently inhibited phosphoinositide breakdown and intracellular Ca⁺² mobilization in human platelets stimulated by thrombin. Rapid phosphorylation of a platelet protein of M_r 47,000 (P47), a marker of protein kinase C activation, was triggered by phorbol-12-13-dibutyrate (PDBu, 50 nM). This phosphorylation was markedly inhibited by magnesium sulfate (3.0 mM). Magnesium sulfate (1.5 and 3.0 mM) further inhibited PDBu-stimulated platelet aggregation in human platelets. The thrombin-evoked increase in pHi was markedly inhibited in the presence of magnesium sulfate (3.0 mM). In conclusion, these results indicate that the antiplatelet activity of magnesium sulfate may be involved in the following two pathways: (1) Magnesium sulfate may inhibit the activation of protein kinase C, followed by inhibition of phosphoinositide breakdown and intracellular Ca⁺² mobilization, thereby leading to inhibition of the phosphorylation of P47. (2) On the other hand, magnesium sulfate inhibits the Na⁺/H⁺ exchanger, leading to reduced intracellular Ca⁺² mobilization, and ultimately to inhibition of platelet aggregation and the ATP-release reaction.     

Ligand binding analysis and screening by chemical denaturation shift

The identification of small molecule ligands is an important first step in drug development, especially drugs that target proteins with no intrinsic activity. Toward this goal, it is important to have access to technologies that are able to measure binding affinities for a large number of potential ligands in a fast and accurate way. Because ligand binding stabilizes the protein structure in a manner dependent on concentration and binding affinity, the magnitude of the protein stabilization effect elicited by binding can be used to identify and characterize ligands. For example, the shift in protein denaturation temperature (T_m shift) has become a popular approach to identify potential ligands. However, T_m shifts cannot be readily transformed into binding affinities, and the ligand rank order obtained at denaturation temperatures (?60 °C) does not necessarily coincide with the rank order at physiological temperature. An alternative approach is the use of chemical denaturation, which can be implemented at any temperature. Chemical denaturation shifts allow accurate determination of binding affinities with a surprisingly wide dynamic range (high micromolar to sub nanomolar) and in situations where binding changes the cooperativity of the unfolding transition. In this article, we develop the basic analytical equations and provide several experimental examples.     

Monitoring Cl<Superscript>−</Superscript> Movement in Single Cells Exposed to Hypotonic Solution

Self-referencing ion - selective electrodes (ISEs), made with Chloride Ionophore I-Cocktail A (Fluka), were positioned 1–3 μm from human embryonic kidney cells (tsA201a) and used to record chloride flux during a sustained hyposmotic challenge. The ISE response was close to Nernstian when comparing potentials (V_N) measured in 100 and 10 mM NaCl (ΔV_N = 57 ± 2 mV), but was slightly greater than ideal when comparing 1 and 10 mm NaCl (ΔV_N = 70 ± 3 mV). The response was also linear in the presence of 1 mm glutamate, gluconate, or acetate, 10 μm tamoxifen, or 0.1, 1, or 10 mm HEPES at pH 7.0. The ISE was ∼3 orders of magnitude more selective for Cl⁻ over glutamate or gluconate but less than 2 orders of magnitude move selective for Cl⁻ over bicarbonate, acetate, citrate or thiosulfate. As a result this ISE is best described as an anion sensor. The ISE was ‘poisoned’ by 50 μm 5−nitro-2-(3phenylpropyl-amino)-benzoic acid (NPPB), but not by tamoxifen. An outward anion efflux was recorded from cells challenged with hypotonic (250 ± 5 mOsm) solution. The increase in efflux peaked 7–8 min before decreasing, consistent with regulatory volume decreases observed in separate experiments using a similar osmotic protocol. This anion efflux was blocked by 10 μm tamoxifen. These results establish the feasibility of using the modulation of electrochemical, anion-selective, electrodes to monitor anions and, in this case, chloride movement during volume regulatory events. The approach provides a real-time measure of anion movement during regulated volume decrease at the single-cell level.     

Denaturation of RNA with dimethyl sulfoxide

The denaturation of single-stranded and double-stranded RNA's in solutions with varying proportions of dimethyl sulfoxide has been followed by changes in absorbancy, optical rotation, and—with a double-stranded form of bacteriophage of MS2 RNA— infectivity for bacterial spheroplasts. By these criteria the RNA's studied, including the synthetic polynucleotide rG:rC, are completely denatured at room temperature in high concentrations of this solvent. In lower concentrations, the T_m of the RNA preparation is decreased only slightly as the dimethyl sulfoxide concentration is raised until a critical concentration is reached. The T_m falls sharply with small further increases in dimethyl sulfoxide concentration. Sedimentation studies can be conducted directly in these media. The determination of sedimentation velocity in 99% dimethyl sulfoxide containing 0.001M EDTA provides a reliable estimate of RNA molecular weights.