High Concentrations Of Monovalent Cations Research Articles

Complexation and precipitation of scale-forming cations in oilfield produced water with polyelectrolytes

Divalent cations such as Ba2+, Sr2+, Ca2+, and Mg2+, commonly found in oil/gas produced water at high concentrations, adversely affect oil and gas production by forming scales in pipes, tanks, process equipment such as oil-water separators, and treatment equipment such as filters and reverse osmosis membranes. If the water is reinjected, e.g., for water flooding, scales may also form on the well screens and in the producing formation. Water soluble polyelectrolytes have been used to inhibit scale formation in oil and gas production and to eliminate cationic metals from water via membrane separation. However, the underlying interactions between scale-forming cations and various polyelectrolytes are not well understood. To this end, two commercially available anionic polyelectrolytes with different repeating functional groups, poly(acrylic acid) (PAA) and poly(sodium 4-styrenesulfonate) (PSS), were used to evaluate complexation and precipitation of Ba2+, Sr2+, Ca2+, and Mg2+ in binary systems, multi-ion mixtures, and produced water. The apparent dissociation constant (pKa) of PAA was determined by titration to be 6.7, indicating a high affinity of PAA for H+. The strength of the complexes formed between PAA and scale-forming cations (binding strength) decreased in the order Ba2+ > Ca2+ > Sr2+ > Mg2+ at pH 5. Higher pH values led to a higher affinity of PAA for divalent cations, which is attributable to the increase in binding sites for cations as the PAA more completely ionizes. By contrast, the affinity of PSS decreases in the order Ba2+ > Sr2+ > Ca2+ > Mg2+, with no significant pH effect. The formation and precipitation behavior of polyelectrolyte complexes (PECs) was investigated by reacting cations with the sodium salt of PAA (sodium polyacrylate) or with PSS. Only Ba2+ formed precipitates with PSS in single-cation solutions, although small amounts of Sr2+ were also removed by PSS from a mixed cation solution. However, all of the scale-forming cations formed PEC precipitates with PAA in both binary and mixed-ion solutions, depending on the cation concentration and molar ratio of PAA to divalent cations. The maximum amount of precipitation occurred at a molar ratio of PAA to M2+ of 1.5–1.7, while overdosing of PAA caused dispersion of the precipitates. High concentrations of monovalent cations inhibited precipitation of divalent cations with PAA. Up to 73% of scale-forming cations were removed from field-collected produced water samples through repeated (4 times) addition of PAA at a molar concentration ratio of 1. The release of scale-forming cations from PAA-M2+ precipitates, thereby regenerating PAA, was achieved by adding HCl.

Influence of Monovalent Cations on the Efficiency of Ferrous Ion Oxidation, Total Iron Precipitation, and Adsorptive Removal of Cr(VI) and As(III) in Simulated Acid Mine Drainage with Inoculation of Acidithiobacillus ferrooxidans

Acid mine drainage is highly acidic and contains large quantities of Fe and heavy metal elements. Thus, it is important to promote the transformation of Fe into secondary iron minerals that exhibit strong heavy-metal removal abilities. Using simulated acid mine drainage, this work analyzes the influence of monovalent cations (K+, NH4+, and Na+) on the Fe2+ oxidation and total Fe deposition efficiencies, as well as the phases of secondary iron minerals in an Acidithiobacillus ferrooxidans system. It also compares the Cr(VI) (K2Cr2O7) and As(III) (As2O3) removal efficiencies of different schwertmannites. The results indicated that high concentrations of monovalent cations (NH4+ ≥ 320 mmol/L, and Na+ ≥ 1600 mmol/L) inhibited the biological oxidation of Fe2+. Moreover, the mineralizing abilities of the three cations differed (K+ > NH4+ > Na+), with cumulative Fe deposition efficiencies of 58.7%, 28.1%, and 18.6%, respectively [n(M) = 53.3 mmol/L, cultivation time = 96 h]. Additionally, at initial Cr(VI) and As(III) concentrations of 10 and 1 mg/L, respectively, the Cr(VI) and As(III) removal efficiencies exhibited by schwertmannites acquired by the three mineralization systems differed [n(Na) = 53.3 > n(NH4) = 53.3 > n(K) = 0.8 mmol/L]. Overall, the analytical results suggested that the removal efficiency of toxic elements was mainly influenced by the apparent structure, particle size, and specific surface area of schwertmannite.

Comparison of Enzymatic Traits between Native and Recombinant Glycine Sarcosine N-Methyltransferase from Methanohalophilus portucalensis FDF1T.

The halophilic methanoarchaeon Methanohalophilus portucalensis FDF1T possesses the ability to synthesize the osmolyte betaine from its precursor, glycine, in response to extracellular salt stress through a three-step transmethylation process. Analysis of recombinant glycine sarcosine N-methyltransferase (rGSMT) and recombinant sarcosine dimethylglycine N-methyltransferase (rSDMT) from Escherichia coli indicated that betaine synthesis is rate-limited by rGSMT and is constitutively activated by rSDMT. Therefore, it is of interest to purify native GSMT from Methanohalophilus portucalensis to further compare its enzymatic characteristics and kinetics with rGSMT. In this study, native GSMT was purified through DEAE ion exchange and gel filtration chromatography with 95% purity. The enzymatic characteristics of GSMT and rGSMT showed similar trends of activities that were activated by high concentrations of monovalent cations. Both were feedback-inhibited by the end product, betaine, and competitively inhibited by S-adenosylhomocysteine (SAH). Native GSMT was 2-fold more sensitive to SAH than rGSMT. Notably, comparison of the kinetic parameters illustrated that the turnover rate of glycine methylation of GSMT was promoted by potassium ions, whereas rGSMT was activated by increasing protein-glycine binding affinity. These results suggest that GSMT and rGSMT may have different levels of post-translational modifications. Our preliminary mass spectrometry evidence indicated that there was no detectable phosphosite on GSMT after the complicated purification processes, whereas purified rGSMT still possessed 23.1% of its initial phosphorylation level. We believe that a phosphorylation-mediated modification may be involved in the regulation of this energy consuming betaine synthesis pathway during the stress response in halophilic methanoarchaea.

DNA A-tracts are not curved in solutions containing high concentrations of monovalent cations.

The intrinsic curvature of seven 98 bp DNA molecules containing up to four centrally located A6-tracts has been measured by gel and capillary electrophoresis as a function of the number and arrangement of the A-tracts. At low cation concentrations, the electrophoretic mobility observed in polyacrylamide gels and in free solution decreases progressively with the increasing number of phased A-tracts, as expected for DNA molecules with increasingly curved backbone structures. Anomalously slow electrophoretic mobilities are also observed for DNA molecules containing two pairs of phased A-tracts that are out of phase with each other, suggesting that out-of-phase distortions of the helix backbone do not cancel each other out. The mobility decreases observed for the A-tract samples are due to curvature, not cation binding in the A-tract minor groove, because identical free solution mobilities are observed for a molecule with four out-of-phase A-tracts and one with no A-tracts. Surprisingly, the curvature of DNA A-tracts is gradually lost when the monovalent cation concentration is increased to ∼200 mM, regardless of whether the cation is a hydrophilic ion like Na+, NH4+, or Tris+ or a hydrophobic ion like tetrabutylammonium. The decrease in A-tract curvature with increasing ionic strength, along with the known decrease in A-tract curvature with increasing temperature, suggests that DNA A-tracts are not significantly curved under physiological conditions.

Active-Site Monovalent Cations Revealed in a 1.55-Å-Resolution Hammerhead Ribozyme Structure

We have obtained a 1.55-Å crystal structure of a hammerhead ribozyme derived from Schistosoma mansoni under conditions that permit detailed observations of Na+ ion binding in the ribozyme's active site. At least two such Na+ ions are observed. The first Na+ ion binds to the N7 of G10.1 and the adjacent A9 phosphate in a manner identical with that previously observed for divalent cations. A second Na+ ion binds to the Hoogsteen face of G12, the general base in the hammerhead cleavage reaction, thereby potentially dissipating the negative charge of the catalytically active enolate form of the nucleotide base. A potential but more ambiguous third site bridges the A9 and scissile phosphates in a manner consistent with that of previous predictions. Hammerhead ribozymes have been observed to be active in the presence of high concentrations of monovalent cations, including Na+, but the mechanism by which monovalent cations substitute for divalent cations in hammerhead catalysis remains unclear. Our results enable us to suggest that Na+ directly and specifically substitutes for divalent cations in the hammerhead active site. The detailed geometry of the pre-catalytic active-site complex is also revealed with a new level of precision, thanks to the quality of the electron density maps obtained from what is currently the highest-resolution ribozyme structure in the Protein Data Bank.

Behavior of a hammerhead ribozyme in aqueous solution at medium to high temperatures

The "RNA world" hypothesis proposes that--early in the evolution of life--RNA molecules played important roles both in information storage and in enzymatic functions. However, this hypothesis seems to be inconsistent with the concept that life may have emerged under hydrothermal conditions since RNA molecules are considered to be labile under such extreme conditions. Presently, the possibility that the last common ancestor of the present organisms was a hyperthermophilic organism which is important to support the hypothesis of the hydrothermal origin of life has been subject of strong discussions. Consequently, it is of importance to study the behavior of RNA molecules under hydrothermal conditions from the viewpoints of stability, catalytic functions, and storage of genetic information of RNA molecules and determination of the upper limit of temperature where life could have emerged. In the present work, self-cleavage of a natural hammerhead ribozyme was examined at temperatures 10-200 °C. Self-cleavage was investigated in the presence of Mg(2+), which facilitates and accelerates this reaction. Self-cleavage of the hammerhead ribozyme was clearly observed at temperatures up to 60 °C, but at higher temperatures self-cleavage occurs together with hydrolysis and with increasing temperature hydrolysis becomes dominant. The influence of the amount of Mg(2+) on the reaction rate was also investigated. In addition, we discovered that the reaction proceeds in the presence of high concentrations of monovalent cations (Na(+) or K(+)), although very slowly. Furthermore, at high temperatures (above 60 °C), monovalent cations protect the ribozyme against degradation.

Effect of Feeding Patterns on the Performance of Activated Sludge Systems

Excessive amounts of monovalent cations are known to cause deterioration in settling and dewatering properties of activated sludge. In this study, variations in the feeding pattern to a sequencing batch reactor (SBR) were evaluated to determine if the feed pattern could influence effluent quality and sludge characteristics under high monovalent cation concentrations. Data showed that deflocculation caused by high concentrations of sodium could be mitigated by using a feed cycle where the influent to the SBR was provided over a period of 1 minute. In contrast, when the feed was provided over 4 hours, deterioration in settling and effluent water quality was observed, as reflected by an increase in effluent suspended solids, effluent chemical oxygen demand, and capillary suction time.

Slowing Down a Hammerhead Ribozyme Allows Glimpses of It in Action

Slowing Down a Hammerhead Ribozyme Allows Glimpses of It in Action

Functional expression of the voltage-gated neuronal mammalian potassium channel rat ether à go-go1 in yeast

It has been shown previously that heterologous expression of inwardly rectifying potassium channels (K+-channels) from plants and mammals in K+-transport defective yeast mutants can restore the ability of growth in media with low [K+]. In this study, the functional expression of an outward rectifying mammalian K+-channel in yeast is presented for the first time. The outward-rectifying mammalian neuronal K+-channel rat ether à go-go channel 1 (rEAG1, Kv 10.1) was expressed in yeast (Saccharomyces cerevisiae) strains lacking the endogenous K+-uptake systems and/or alkali-metal-cation efflux systems. It was found that a truncated channel version, lacking almost the complete intracellular N-terminus (rEAG1 Delta 190) but not the full-length rEAG1, partially complemented the growth defect of K+-uptake mutant cells (trk1,2 Delta tok1 Delta) in media containing low K+ concentrations. The expression of rEAG1 Delta 190 in a strain lacking the cation efflux systems (nha1 Delta ena1-4 Delta) increased the sensitivity to high monovalent cation concentrations. Both phenotypes were observed, when rEAG1 Delta 190 was expressed in a trk1,2 Delta and nha1, ena1-4 Delta mutant strain. In the presence of K+-channel blockers (Cs+, Ba2+ and quinidine), the growth advantage of rEAG1 Delta 190 expressing trk1,2 tok1 Delta cells disappeared, indicating its dependence on functional rEAG1 channels. The results demonstrate that S. cerevisiae is a suitable expression system even for voltage-gated outward-rectifying mammalian K+-channels.

Low specificity of metal ion binding in the metal ion core of a folded RNA

The structure and activity of nucleic acids depend on their interactions with metal ions. Fundamental to these interactions is the degree of specificity observed between the metal ions and nucleic acids, and a complete description of nucleic acid folding requires that we understand the nature of the interactions with metal ions, including specificity. The prior demonstration that high concentrations of monovalent cations prevent nonspecific association of divalent ions with nucleic acids provides a novel and powerful means to examine site-specific metal ion binding isolated from complicating effects of the ion atmosphere. Using these high monovalent cation solution conditions we have monitored the affinity of a series of divalent metal ions for two site-specific metal ion binding sites in the P4-P6 domain of the Tetrahymena group I intron ribozyme. The metal ion core of this highly structured RNA binds two divalent metal ions under these conditions. Despite multiple metal ion-RNA interactions observed in the X-ray crystallographic structure of P4-P6 RNA at the metal ion binding sites, these sites exhibit low specificity among Mn(2+), Mg(2+), Ca(2+), Ni(2+), and Zn(2+). Nevertheless, the largest divalent metal ions tested, Sr(2+) and Ba(2+), were excluded from binding, exhibiting affinities at least two orders of magnitude weaker than observed for the other metal ions. Thus, a picture emerges of two metal ion binding sites, each with a high tolerance for metal ions with different properties but also with limits to accommodation. These limits presumably arise from steric or electrostatic features of the metal ion binding sites.

HDV Ribozyme Activity in Monovalent Cations

Activity of the two ribozymes from hepatitis delta virus in monovalent salts was examined and compared to activity in Mg2+. Both ribozymes self-cleaved in high concentrations of monovalent cations, and an active site cytosine was required for cleavage activity under those conditions. Cleavage rates were 30-50-fold higher for reactions in LiCl than for reactions in NaCl or NH4Cl, and a thio effect indicated that chemistry was rate-determining for cleavage of the HDV genomic ribozyme in LiCl. Still, in LiCl, there was a more than 100-fold increase in the rate when MgCl2 was included in the reaction. However, the pH-rate profiles for the reactions in LiCl with and without MgCl2 were both bell-shaped with the pH optima in the neutral range. These findings support the idea that monovalent cations can partially substitute for divalent metal ions in the HDV ribozymes, although a divalent metal ion is more effective in supporting catalysis. The absence of a dramatic change in the general shape of pH-rate profiles in LiCl, relative to the profile for reactions including Mg2+, is in contrast to earlier data for the reactions in NaCl and limits our interpretation of the specific role played by the divalent metal ion in the catalytic mechanism.

Mg2+-Induced Compaction of Single RNA Molecules Monitored by Tethered Particle Microscopy

We have applied tethered particle microscopy (TPM) as a single molecule analysis tool to studies of the conformational dynamics of poly-uridine(U) messenger (m)RNA and 16S ribosomal (r)RNA molecules. Using stroboscopic total internal reflection illumination and rigorous selection criteria to distinguish from nonspecific tethering, we have tracked the nanometer-scale Brownian motion of RNA-tethered fluorescent microspheres in all three dimensions at pH 7.5, 22°C, in 10mM or 100mM NaCl in the absence or presence of 10mM MgCl2. The addition of Mg2+ to low-ionic strength buffer results in significant compaction and stiffening of poly(U) mRNA, but not of 16S rRNA. Furthermore, the motion of poly(U)-tethered microspheres is more heterogeneous than that of 16S rRNA-tethered microspheres. Analysis of in-plane bead motion suggests that poly(U) RNA, but less so 16S rRNA, can be modeled both in the presence and absence of Mg2+ by a statistical Gaussian polymer model. We attribute these differences to the Mg2+-induced compaction of the relatively weakly structured and structurally disperse poly(U) mRNA, in contrast to Mg2+-induced reinforcement of existing secondary and tertiary structure contacts in the highly structured 16S rRNA. Both effects are nonspecific, however, as they are dampened in the presence of higher concentrations of monovalent cations.

Soil organic matter and its lignin component in surface horizons of salt-affected soils of the Argentinian Pampa

Salt-affected soils differ in their chemical properties to all other soils. Sodicity and salinity may affect the soil organic matter component of these soils. In a field experiment, we investigated organic matter decomposition in nonsaline nonsodic Aquic Argiudoll, a nonsaline sodic Typic Natraquoll, nonsaline nonsodic Petrocalcic Paleudoll and a saline sodic Typic Natralboll in the Pampa Deprimida, Argentina. The objectives were to identify the degree of stabilization of organic matter by association with mineral particles in these soils and to follow in particular the fate of lignin in these soils. We measured organic carbon, total nitrogen and the extent of lignin alteration with soil depth and in various particle size fractions. The salt-affected soils contained much less organic carbon and nitrogen in their mollic epipedons than the nonsaline nonsodic soils, and bioturbation into deeper layers was restrained. In the salt-affected soils most of the organic matter was in sand-sized particles. Retarded degradation of plant residues was indicated by the pattern of lignin-derived phenols, suggesting less alteration of lignin in the salt-affected soils than in the nonsaline nonsodic soils. We suggest that this results from the effects of high pH, high sodicity, and high salinity on the microorganisms and their enzymatic activities. The high pH and high concentrations of monovalent cations decreased formation of solid organo-mineral complexes. We conclude that in the salt-affected soils oxidatively altered organic compounds are susceptible to losses in dissolved or colloidal forms, because these compounds are not stabilized against leaching and mineralization by chemical bonding to soil minerals.

Effects of water chemistries and properties of membrane on the performance and fouling—a model development study

The performance of nanofiltration (NF) and reverse osmosis (RO) membranes involves complex interactions between water chemistries and properties of the membrane as previously observed by other researchers; however, little has been done on comparing the effects of different parameters at once. In this study, a factorial design was employed to integrate four independent factors, i.e., membrane type, dissolved organic carbon (DOC) composition, divalent cation and monovalent cation, to investigate their influences on specific flux decline and fouling. Quantitative linear models were also built to predict the specific flux decline and fouling formation. The model developed to evaluate the specific flux decline displayed a high prediction capability with an adjusted R 2 of 0.93, and with membrane properties and DOC composition exhibiting the highest influences. It was found that mechanical sieving was identified as the most important rejection mechanism and biofouling contributed more to the flux decline than abiotic fouling did. Membrane biofouling was found to be strongly affected by monovalent cation concentration and membrane properties, such as roughness and zeta potential. High membrane roughness and zeta potential (i.e. more negative membranes) along with high monovalent cation concentrations in the feed water were observed to contribute to biofouling control. Finally, the humic acid composition was identified as the main abiotic foulant, which was enhanced by high initial specific flux values.

Characterization of Bacteriophage T3 DNA Ligase

DNA ligases of bacteriophage T4 and T7 have been widely used in molecular biology for decades, but little is known about bacteriophage T3 DNA ligase. Here is the first report on the cloning, expression and biochemical characterization of bacteriophage T3 DNA ligase. The polyhistidine-tagged recombinant T3 DNA ligase was shown to be an ATP-dependent enzyme. The enzymatic activity was not affected by high concentration of monovalent cations up to 1 M, whereas 2 mM ATP could inhibit its activity by 50%. Under optimal conditions (pH 8.0, 0.5 mM ATP, 5 mM DTT, 1 mM Mg(2+) and 300 mM Na(+)), 1 fmol of T3 DNA ligase could achieve 90% ligation of 450 fmol of cohesive dsDNA fragments in 30 min. T3 DNA ligase was shown to be over 5-fold more efficient than T4 DNA ligase for ligation of cohesive DNA fragments, but less active for blunt-ended DNA fragments. Phylogenetic analysis showed that T3 DNA ligase is more closely related to T7 DNA ligase than to T4 DNA ligase.

Kappa-Carrageenan gel as agent to sequester paralytic shellfish poison.

The action of k-carrageenan gel to sequester paralytic shellfish poison (PSP) was tested and characterized. When an extract from a Philippine strain of Pyrodinium bahamense var. compressum was used as PSP solution, the PSP-sequestering property of kappa-carrageenan gel was found to be dependent on gel surface area, interaction time, and polysaccharide concentration. The interaction was also found to be affected by high concentrations of monovalent cations. The characteristics of kappa-carrageenan as a PSP-sequestering agent all point to cation exchange as its mechanism of action. It is also proposed that the polysaccharide gel can be utilized as an agent to alleviate PSP intoxication.

Chiral Discotic Columnar Germs of Nucleosome Core Particles

In concentrated solution and in the presence of high concentrations of monovalent cations, nucleosome core particles order into a discotic columnar mesophase. This phase is limited to finite-sized hexagonal germs that further divide into six coiled branches, following an iterative process. We show how the structure of the germs comes from the competition between hexagonal packing and chirality with a combination of dendritic facetting and double-twist configurations. Geometrical considerations lead us to suspect that the chirality of the eukaryotic chromosomes may originate from the same competition.

Evaluating the hydraulic conductivity of GCLs permeated with non-standard liquids

Fundamental factors and testing considerations affecting the evaluation of the hydraulic conductivity of geosynthetic clay liners (GCLs) permeated with non-standard liquids (i.e., liquids other than water) are discussed and supported with test data. Both aggregate-size distribution and montmorillonite content of the bentonite portion of the GCL have a potential effect on the hydraulic conductivity of GCLs. Other factors affecting the hydraulic conductivity of GCLs permeated with non-standard liquids include thickness of the adsorbed layer, prehydration of the GCL, void ratio of the GCL, and test duration. Results show that non-standard liquids containing both high concentrations of monovalent cations (e.g., 0.6 M NaCl) as well as low concentrations of divalent cations (e.g. 0.0125 M CaCl 2) can cause significant increases (⩾1 order of magnitude) in hydraulic conductivity provided the test is performed sufficiently long to allow for exchange of adsorbed cations. Results also indicate that termination of hydraulic conductivity tests involving prehydrated GCLs before chemical equilibrium is established may result in measured hydraulic conductivities that do not represent equilibrium and may be unconservatively low. Finally, control of average effective stress is more important than control of hydraulic gradient when evaluating the hydraulic conductivity of GCLs.

Purification and Catalytic Activities of the Two Domains of the Allene Oxide Synthase-Lipoxygenase Fusion Protein of the CoralPlexaura homomalla

The conversion of fatty acid hydroperoxides to allene epoxides is catalyzed by a cytochrome P450 in plants and, in coral, by a 43-kDa catalase-related hemoprotein fused to the lipoxygenase that synthesizes the 8R-hydroperoxyeicosatetraenoic acid (8R-HPETE) substrate. We have expressed the separate lipoxygenase and allene oxide synthase (AOS) domains of the coral protein in Escherichia coli (BL21 cells) and purified the proteins; this system gives high expression (1.5 and 0.3 micromol/liter, respectively) of catalytically active enzymes. Both domains show fast reaction kinetics. Catalytic activity of the lipoxygenase domain is stimulated 5-fold by high concentrations of monovalent cations (500 mM Na(+), Li(+), or K(+)), and an additional 5-fold by 10 mM Ca(2+). The resulting rates of reaction are approximately 300 turnovers/s, 1-2 orders of magnitude faster than mammalian lipoxygenases. This makes the coral lipoxygenase well suited for partnership with the AOS domain, which shows maximum rates of approximately 1400 turnovers/s in the conversion of 8R-HPETE to the allene oxide. Some unusual catalytic activities of the two domains are described. The lipoxygenase domain converts 20.3omega6 partly to the bis-allylic hydroperoxide (10-hydroperoxyeicosa-8,11,14-trienoic acid). Metabolism of the preferred substrate of the AOS domain, 8R-HPETE, is inhibited by the enantiomer 8S-HPETE. Although the AOS domain has homology to catalase in primary structure, it is completely lacking in catalatic action on H(2)O(2); catalase itself, as expected from its preference for small hydroperoxides, is ineffective in allene oxide synthesis from 8R-HPETE.

The hammerhead, hairpin and VS ribozymes are catalytically proficient in monovalent cations alone

Background: The catalytic activity of RNA enzymes is thought to require divalent metal ions, which are believed to facilitate RNA folding and to play a direct chemical role in the reaction. Results: We have found that the hammerhead, hairpin and VS ribozymes do not require divalent metal ions, their mimics such as [Co(NH 3) 6] 3+, or even monovalent metal ions for efficient self-cleavage. The HDV ribozyme, however, does appear to require divalent metal ions for self-cleavage. For the hammerhead, hairpin and VS ribozymes, very high concentrations of monovalent cations support RNA-cleavage rates similar to or exceeding those observed in standard concentrations of Mg 2+. Analysis of all reaction components by inductively coupled plasma-optical emission spectrophotometry (ICPOES) and the use of a variety of chelating agents effectively eliminate the possibility of contaminating divalent and trivalent metal ions in the reactions. For the hairpin ribozyme, fluorescence resonance energy transfer experiments demonstrate that high concentrations of monovalent cations support folding into the catalytically proficient tertiary structure. Conclusions: These results directly demonstrate that metal ions are not obligatory chemical participants in the reactions catalysed by the hammerhead, hairpin, and VS ribozymes. They permit us to suggest that the folded structure of the RNA itself contributes more to the catalytic function than was previously recognised, and that the presence of a relatively dense positive charge, rather than divalent metal ions, is the general fundamental requirement. Whether this charge is required for catalysis per se or simply for RNA folding remains to be determined.