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In the current study, the effect of metal ions in combination with buffers (citrate, acetate, pH 4.5) on the stability of aqueous solutions of oxytocin was investigated. Both monovalent metal ions (Na+ and K+) and divalent metal ions (Ca2+, Mg2+, and Zn2+) were tested all as chloride salts. The effect of combinations of buffers and metal ions on the stability of aqueous oxytocin solutions was determined by RP-HPLC and HP-SEC after 4 weeks of storage at either 4°C or 55°C. Addition of sodium or potassium ions to acetate- or citrate-buffered solutions did not increase stability, nor did the addition of divalent metal ions to acetate buffer. However, the stability of aqueous oxytocin in aqueous formulations was improved in the presence of 5 and 10 mM citrate buffer in combination with at least 2 mM CaCl2, MgCl2, or ZnCl2 and depended on the divalent metal ion concentration. Isothermal titration calorimetric measurements were predictive for the stabilization effects observed during the stability study. Formulations in citrate buffer that had an improved stability displayed a strong interaction between oxytocin and Ca2+, Mg2+, or Zn2+, while formulations in acetate buffer did not. In conclusion, our study shows that divalent metal ions in combination with citrate buffer strongly improved the stability of oxytocin in aqueous solutions.

The decrease in the sensitivity of electrospray ionization mass spectrometry caused by the presence of metal salts, such as sodium chloride, in the sample matrix is well known and is particularly problematic for biological samples. We report here that addition of high levels of ammonium acetate can improve analyte signal in aqueous electrospray solutions and counteracts the signal suppression caused by sodium chloride. A ~3-fold improvement in S/N is obtained by adding 8 M ammonium acetate to aqueous solutions of cytochrome c without added sodium chloride. No organic solvents or acids are added into the electrospray solutions. The signal-to-noise ratios of cytochrome c and ubiquitin (10−5 M) ions formed from aqueous solutions containing 2.0 × 10−2 M sodium chloride are improved by factors of ~7 and 11, respectively, by adding 7 M ammonium acetate to the solution. We propose that this effect is a result of the precipitation of Na+ and Cl− from solution within the evaporating electrospray droplets prior to the formation of gas-phase protein ions. This method is potentially useful for improving the abundance of protein ions formed from solutions in which the molecules have a nativelike conformation and is particularly advantageous for such solutions that have high levels of sodium.

The effects of commonly used therapeutic doses of hydrochlorothiazide and probenecid, given singly and in combination, on the urinary excretion of monovalent and divalent ions and on acid-base equilibrium were studied in four patients with idiopathic hypercalciuria.

Probenecid had no effect on the urinary excretion of monovalent ions but resulted in a sustained increase in the urinary excretion of calcium, magnesium and citrate and a temporary increase in the urinary excretion of ammonium, in addition to its well-known effects on uric acid metabolism. A temporary fall in serum phosphorus levels was also observed.

Probenecid also modified the response to hydrochlorothiazide in that the urinary excretion of calcium, magnesium and citrate was greater during combined therapy than when hydrochlorothiazide was administered alone. Probenecid prevented or abolished the increase in serum uric acid levels associated with the use of thiazide but did not modify the effects of hydrochlorothiazide on the urinary excretion of sodium, chloride, potassiu, phosphorus, ammonium, titratable acid and bicarbonate.

Background

The use of a combination of different drugs in postoperative analgesia extends the time of analgesia, makes it more efficient and allows the use of lower drug doses, which leads to less risk of side effects and drug dependence. The aim of this study was to develop and validate an HPLC method to determine the stability of fentanyl citrate and bupivacaine hydrochloride mixtures in standard infusion solutions of 0.9% sodium chloride and 5% glucose.

Methods

After optimisation, the HPLC method parameters were as follows: LiChrospher 100 CN, 250×4 mm (10 µm) column; mobile phase: mixture of acetonitrile and phosphate buffer at pH 2.8 (3:7, V/V) with addition of 0.08 g/l potassium chloride; flow rate: 1.5 ml/min; column temperature: 30°C; spectrophotometric detection at 210 nm. Development of the method involved checking the impact of acetonitrile and KCl concentrations in the mobile phase and choosing the internal standard. Method validation included determining the specificity of the method, its accuracy, linearity, precision, repeatability, limits of detection and quantification.

Results

The retention times of bupivacaine hydrochloride, fentanyl citrate and procaine hydrochloride, used as an internal standard, were approximately 10 min, 15 min and 5 min, respectively. Method validation confirmed its selectivity, accuracy and precision. The average values of the variation and accuracy coefficients were 0.70% and 99.02% for bupivacaine hydrochloride, and 1.76% and 104.53% for fentanyl citrate. The intermediate precision values were 1.25% for bupivacaine hydrochloride and 1.52% for fentanyl citrate.

The divalent ion requirements of rabbit platelet injury by endotoxin have been defined by the use of various anticoagulant solutions and have been compared to the divalent ion requirements of platelet injury produced by addition of antigen to immune platelet-rich plasma. The endotoxin-platelet interaction takes place in citrated blood. Platelet damage by antigen is inhibited by citrate, but preincubation of antigen and immune platelet-poor plasma in the absence of citrate results in a substance, presumably antigen-antibody complement complex, which then does injure platelets in the presence of citrate. Neither endotoxin nor preincubated antigen injures platelets in the presence of sodium EDTA in concentrations sufficient to interact with all divalent cations present in plasma. These observations have been interpreted by viewing the platelet-endotoxin interaction as a consequence of platelet phagocytosis of endotoxin, a reaction not requiring complement but requiring definite small concentrations of divalent cations. The interaction of antigen and platelets is regarded as a two phase reaction, the first requiring the participation of complement and concentrations of divalent cation larger than those provided in citrated plasma, the second requiring smaller concentrations of divalent cation, no further participation of complement, and active in citrated plasma. This second phase is regarded as representing platelet phagocytosis of immune complexes.

The hydration of the alkali metal ions in aqueous solution has been studied by large angle X-ray scattering (LAXS) and double difference infrared spectroscopy (DDIR). The structures of the dimethyl sulfoxide solvated alkali metal ions in solution have been determined to support the studies in aqueous solution. The results of the LAXS and DDIR measurements show that the sodium, potassium, rubidium and cesium ions all are weakly hydrated with only a single shell of water molecules. The smaller lithium ion is more strongly hydrated, most probably with a second hydration shell present. The influence of the rubidium and cesium ions on the water structure was found to be very weak, and it was not possible to quantify this effect in a reliable way due to insufficient separation of the O–D stretching bands of partially deuterated water bound to these metal ions and the O–D stretching bands of the bulk water. Aqueous solutions of sodium, potassium and cesium iodide and cesium and lithium hydroxide have been studied by LAXS and M–O bond distances have been determined fairly accurately except for lithium. However, the number of water molecules binding to the alkali metal ions is very difficult to determine from the LAXS measurements as the number of distances and the temperature factor are strongly correlated. A thorough analysis of M–O bond distances in solid alkali metal compounds with ligands binding through oxygen has been made from available structure databases. There is relatively strong correlation between M–O bond distances and coordination numbers also for the alkali metal ions even though the M–O interactions are weak and the number of complexes of potassium, rubidium and cesium with well-defined coordination geometry is very small. The mean M–O bond distance in the hydrated sodium, potassium, rubidium and cesium ions in aqueous solution have been determined to be 2.43(2), 2.81(1), 2.98(1) and 3.07(1) Å, which corresponds to six-, seven-, eight- and eight-coordination. These coordination numbers are supported by the linear relationship of the hydration enthalpies and the M–O bond distances. This correlation indicates that the hydrated lithium ion is four-coordinate in aqueous solution. New ionic radii are proposed for four- and six-coordinate lithium(I), 0.60 and 0.79 Å, respectively, as well as for five- and six-coordinate sodium(I), 1.02 and 1.07 Å, respectively. The ionic radii for six- and seven-coordinate K+, 1.38 and 1.46 Å, respectively, and eight-coordinate Rb+ and Cs+, 1.64 and 1.73 Å, respectively, are confirmed from previous studies. The M–O bond distances in dimethyl sulfoxide solvated sodium, potassium, rubidium and cesium ions in solution are very similar to those observed in aqueous solution.

The hydration of alkali metal ions has been studied by large angle X-ray scattering, LAXS, and double difference infrared spectroscopy. The obtained M−O bond distances from LAXS have been compared to relevant crystal structures, conclusions about hydration numbers in aqueous solution have been made, and new ionic radii have been proposed. Hydration numbers of six, seven, eight and eight are proposed for the sodium, potassium, rubidium and cesium ions in aqueous solution.

When two strains of phage T5 (heat-susceptible form T5st+ and its heat-resistant mutant T5st) were placed in solutions containing various high concentrations of chelating agents (sodium citrate and ethylenediaminetetraacetic acid) at room temperature, they could be effectively inactivated by rapid dilution in distilled water of relatively low temperatures (2 to 37 C). This phenomenon has been termed “chelating agent shock” (CAS). The susceptibility of phage T5 to CAS increased with an increase in the concentration of chelating agents and with an increase in temperature of the water used for rapid dilution. Under any given condition, T5st+ was much more sensitive to CAS than was T5st. Phage T5 was protected against inactivation by the addition of monovalent or divalent metal salts, but not by the addition of nonionic solutes, to the shocking water prior to CAS treatment. This finding is compatible with the view that cations combined with the phage protein are removed by the chelating agent, although no metal ion has been identified in the phage protein. Alternatively, since the chelating agents used are polyanions, they may bind relatively tightly to the protein subunits in the head of T5, thereby distorting the structure of the phage head. Rapid dilution of these distorted particles could lead to loss of phage DNA. No evidence for recovery of phage activity could be obtained by the addition of metal salts to the inactivated phage after CAS. The morphological properties of phage inactivated by CAS are similar to those of heat-inactivated T5 phage. Electron micrographs showed that most of the phage particles consisted of empty head membranes; some of the particles had lost their tails. Both heritable and nonheritable resistance to heat was accompanied by resistance to CAS in phage T5. The sensitive element detected by each test seemed to be the same.

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The autolytic N-acetylmuramidase present in Lactobacillus acidophilus strain 63 AM Gasser has an optimal pH between 5 and 6 when lysing intact cells or isolated cell walls. Cellular lysis at pH 5 is two to four times more rapid in citrate buffer of 0.01 M and 0.5 M or higher than in 0.1 M acetate buffer. It seems that sulfhydryl groups are required for both cell and wall autolysis. Heavy metal ions and p-chloro-mercuribenzoate, at low concentrations, are powerful inhibitors. Ethylenediaminetetraacetic acid stimulates cellular but not wall autolysis in acetate buffer to the level obtained in citrate buffer. The possible involvement of sulfhydryl groups in a mechanism of control of cellular autolytic activity is discussed. The autolytic enzyme, although unstable in solution at 37 C, can be extracted from walls by the use of solutions of bovine serum albumin (100 μg/ml) in 0.01 N NaOH. Soluble enzyme extracted from walls rebinds on to sodium decylsulfate-treated walls, but three times as much of the wall material is required to completely re-adsorb the activity.

1. Purified citrate-extracted ichthyocol obtained from carp swim bladders has been further characterized with respect to its content of certain amino acids and carbohydrate substances. 2. The degree of solubilization or dispersion of ichthyocol by solutions of certain salts maintained in the range of neutral pH and at a temperature of 0–2°C. has been determined. 3. While a number of salts of monovalent cations had no significant solubilizing effects on ichthyocol, ammonium chloride in a concentration of 1 M did cause solution of the protein. 4. Sodium thiosulfate in a range of concentrations caused the solubilization of ichthyocol but was most effective in an intermediate concentration of 0.25 M. 5. Several salts of divalent cations, in particular the chlorides of calcium, magnesium, and barium, and magnesium thiosulfate in concentrations ranging from 0.3 to 1 M caused the immediate and complete solubilization of the ichthyocol. 6. Solutions of ichthyocol in calcium chloride, magnesium chloride, and sodium thiosulfate buffered or adjusted to pH 7.0, were studied with respect to intrinsic viscosity of the protein, optical rotation, ultracentrifugal sedimentation, and reconstitution into fibers. It was found in each case that the original characteristics of the collagen, as determined previously in acid solution, were maintained when the protein was dissolved in salt solutions of neutral pH. No evidence of denaturation or gelatinization could be found when ichthyocol was solubilized under the stated conditions. 7. Collagen in neutral solution with sodium thiosulfate, calcium chloride, or magnesium chloride was not attacked by trypsin as determined viscometrically at 20.0°C., but was rapidly degraded by a purified bacterial collagenase.

The mannoprotein which is a major component of the cell wall of Saccharomyces cerevisiae is an effective bioemulsifier. Mannoprotein emulsifier was extracted in a high yield from whole cells of fresh bakers' yeast by two methods, by autoclaving in neutral citrate buffer and by digestion with Zymolase (Miles Laboratories; Toronto, Ontario, Canada), a beta-1,3-glucanase. Heat-extracted emulsifier was purified by ultrafiltration and contained approximately 44% carbohydrate (mannose) and 17% protein. Treatment of the emulsifier with protease eliminated emulsification. Kerosene-in-water emulsions were stabilized over a broad range of conditions, from pH 2 to 11, with up to 5% sodium chloride or up to 50% ethanol in the aqueous phase. In the presence of a low concentration of various solutes, emulsions were stable to three cycles of freezing and thawing. An emulsifying agent was extracted from each species or strain of yeast tested, including 13 species of genera other than Saccharomyces. Spent yeast from the manufacture of beer and wine was demonstrated to be a possible source for the large-scale production of this bioemulsifier.

Summary

The effect of the simultaneous presence of mono- and divalent cations on the thermodynamics of polyelectrolyte solutions is not fully understood. In physiological conditions, combinations of these ions affect structure formation in biopolymer systems. It is known that divalent counterions form a tight sheath around the polymer backbone, while monovalent ions are distributed in a diffuse cloud. Dynamic light scattering measurements of the collective diffusion coefficient D and the osmotic compressibility of semi-dilute hyaluronan solutions containing different ratios of sodium and calcium ions are compared with simple polyelectrolyte models. Scaling relationships are derived in terms of polymer concentration and ionic strength J of the added salt. Differences in the effects of sodium and calcium ions are expressed only through J.

Dead dried Chlorella vulgaris was studied in terms of its performance in binding divalent copper, cadmium, and lead ions from their aqueous or 50% v/v methanol, ethanol, and acetone solutions. The percentage uptake of cadmium ions exhibited a general decrease with decrease in dielectric constant values, while that of copper and lead ions showed a general decrease with increase in donor numbers. Uptake percentage becomes less sensitive to solvent properties the larger the atomic radius of the biosorbed ion, and uptake of copper was the most affected. FT-IR analyses revealed stability of the biomass in mixed solvents and a shift in vibrations of amide(I) and (II), carboxylate, glucose ring, and metal oxygen upon metal binding in all media. ΔνCOO values (59–69 cm−1) confirmed bidentate metal coordination to carboxylate ligands. The value of νasCOO increased slightly upon Cu, Cd, and Pb biosorption from aqueous solutions indicating lowering of symmetry, while a general decrease was noticed in mixed solvents pointing to the opposite. M–O stretching frequencies increased unexpectedly with increase in atomic mass as a result of solvent effect on the nature of binding sites. Lowering polarity of the solvent permits variations in metal-alga bonds strengths; the smaller the metal ion, the more affected.

Citrate uptake in Bacillus subtilis is stimulated by a wide range of divalent metal ions. The metal ions were separated into two groups based on the expression pattern of the uptake system. The two groups correlated with the metal ion specificity of two homologous B. subtilis secondary citrate transporters, CitM and CitH, upon expression in Escherichia coli. CitM transported citrate in complex with Mg2+, Ni2+, Mn2+, Co2+, and Zn2+ but not in complex with Ca2+, Ba2+, and Sr2+. CitH transported citrate in complex with Ca2+, Ba2+, and Sr2+ but not in complex with Mg2+, Ni2+, Mn2+, Co2+, and Zn2+. Both transporters did not transport free citrate. Nevertheless, free citrate uptake could be demonstrated in B. subtilis, indicating the expression of at least a third citrate transporter, whose identity is not known. For both the CitM and CitH transporters it was demonstrated that the metal ion promoted citrate uptake and, vice versa, that citrate promoted uptake of the metal ion, indicating that the complex is the transported species. The results indicate that CitM and CitH are secondary transporters that transport complexes of divalent metal ions and citrate but with a complementary metal ion specificity. The potential physiological function of the two transporters is discussed.

Studies of taste receptor cells, chorda tympani (CT) neurons, and brainstem neurons show stimulus interactions in the form of inhibition or enhancement of the effectiveness of sucrose when mixed with acids or citrate salts, respectively. To investigate further the effects of acids and the trivalent citrate anion on sucrose responses in hamsters (Mesocricetus auratus), we recorded multifiber CT responses to 100 mM sucrose; a concentration series of HCl, citric acid, acetic acid, sodium citrate (with and without amiloride added), potassium citrate, and all binary combinations of acids and salts with 100 mM sucrose. Compared with response additivity, sucrose responses were increasingly suppressed in acid + sucrose mixtures with increases in titratable acidity, but HCl and citric acid were more effective suppressors than acetic acid. Citrate salts suppressed sucrose responses and baseline CT neural activity to a similar degree. Citrate salts also elicited prolonged, concentration-dependent, water-rinse responses. The specific loss in sucrose effectiveness as a CT stimulus with increasing titratable acidity was confirmed; however, no increase in sucrose effectiveness was found with the addition of citrate. Further study is needed to define the chemical basis for effects of acids and salts in taste mixtures.

Interactions between divalent metal ions and biomolecules are common both in solution and in the gas phase. Here, the intrinsic effect of divalent alkaline earth metal ions (Be, Mg, Ca, Sr, Ba) on the structure of glycine in the absence of solvent is examined. Results from both density functional and Moller–Plesset theories indicate that for all metal ions except beryllium, the salt-bridge form of the ion, in which glycine is a zwitterion, is between 5 and 12 kcal/mol more stable than the charge-solvated structure in which glycine is in its neutral form. For beryllium, the charge-solvated structure is 5–8 kcal/mol more stable than the salt-bridge structure. Thus, there is a dramatic change in the structure of glycine with increased metal cation size. Using a Hartree–Fock-based partitioning method, the interaction between the metal ion and glycine is separated into electrostatic, charge transfer and deformation components. The charge transfer interactions are more important for stabilizing the charge-solvated structure of glycine with beryllium relative to magnesium. In contrast, the difference in stability between the charge-solvated and salt-bridge structure for magnesium is mostly due to electrostatic interactions that favor formation of the salt-bridge structure. These results indicate that divalent metal ions dramatically influence the structure of this simplest amino acid in the gas phase.

A microplate technique was developed to determine the conditions under which pure cultures of algae removed heavy metals from aqueous solutions. Variables investigated included algal species and strain, culture age (11 and 44 days), metal (mercury, lead, cadmium, and zinc), pH, effects of different buffer solutions, and time of exposure. Plastic, U-bottomed microtiter plates were used in conjunction with heavy metal radionuclides to determine concentration factors for metal-alga combinations. The technique developed was rapid, statistically reliable, and economical of materials and cells. Results (expressed as concentration factors) were in reasonably good agreement with literature values. All species of algae studied removed mercury from solution. Green algae proved better at accumulating cadmium than did blue-green algae. No alga studied removed zinc, perhaps because cells were maintained in the dark during the labeling period. Chlamydomonas sp. proved superior in ability to remove lead from solution.

Ecological risk assessment can be enhanced with predictive models for metal toxicity. Modelings of published data were done under the simplifying assumption that intermetal trends in toxicity reflect relative metal-ligand complex stabilities. This idea has been invoked successfully since 1904 but has yet to be applied widely in quantitative ecotoxicology. Intermetal trends in toxicity were successfully modeled with ion characteristics reflecting metal binding to ligands for a wide range of effects. Most models were useful for predictive purposes based on an F-ratio criterion and cross-validation, but anomalous predictions did occur if speciation was ignored. In general, models for metals with the same valence (i.e., divalent metals) were better than those combining mono-, di-, and trivalent metals. The softness parameter (sigma p) and the absolute value of the log of the first hydrolysis constant ([symbol: see text] log KOH [symbol: see text]) were especially useful in model construction. Also, delta E0 contributed substantially to several of the two-variable models. In contrast, quantitative attempts to predict metal interactions in binary mixtures based on metal-ligand complex stabilities were not successful.

Pasteurella multocida is a pathogen of veterinary and medical importance. Here, we report the 1.85 Å resolution crystal structure of the class C acid phosphatase from this organism (denoted rPmCCAP). The structure shows that rPmCCAP exhibits the same haloacid dehalogenase fold and dimeric assembly as the class C enzyme from Haemophilus influenzae. Formation of the dimer in solution is demonstrated using analytical ultracentrifugation. The active site is devoid of a magnesium ion due to the presence of citrate in the crystallization buffer. Absence of the metal ion minimally perturbs the active site structure, which suggests that the main role of the ion is to balance the negative charge of the substrate rather than stabilize the active site structure. The crystal lattice displays unusual crystal packing involving the C-terminal polyhistidine tag mimicking the substrate. Steady-state kinetic constants are determined for the substrates NMN, 5´-AMP, 3´-AMP, 2´-AMP, and p-nitrophenyl phosphate. The highest catalytic efficiency is observed with NMN. The production of polyclonal anti-rPmCCAP antibodies is demonstrated, and these antibodies are shown to cross-react with the H. influenzae class C phosphatase. The antibodies are used to detect PmCCAP in clinical P. multocida and Mannheimia haemolytica strains cultured from infected animals.

The divalent transition-metal cations Fe, Co, and Ni were used to test the hypothesis that Mn ions pass through calcium channels because Mn ions have a relatively low energy of hydration. The test ions were applied to the bath and comparisons were made of their effects on Ca or Mn spikes elicited from myoepithelial cells of the proventriculus of the polychaete worm Syllis spongiphila. Control experiments showed that (a) results obtained using deoxygenated solutions (required to stabilize Fe2+ ions) could be compared with those using solutions containing oxygen, and (b) the test cations did not measurably affect the electrical coupling between cells. Ca spikes were reversibly abolished by the test cations in the order of effectiveness: Fe (16.1 mM +/- 1.0, SE; n = 15) = Co (14.6 mM +/- 0.8; n = 27) less than Ni (8.3 mM +/- 0.7; n = 16). The test cations diminished Mn spikes by decreasing maximum rates of rise (Fe = Co less than Ni) and overshoot amplitudes (Fe less than Co less than Ni). The test cations also increased the current intensity required for Ca (Fe = Co less than Ni) or Mn spike initiation (Fe less than Co less than Ni). Since the energies of hydration of Fe, Co, and Ni increase stepwise from that of Mn, and the effectiveness of these ions in diminishing Ca and Mn spikes increased in the order Fe less than or equal to Co less than Ni, these data support the hypothesis that Mn ions pass through Ca channels because they shed waters of hydration relatively easily. An additional observation was that, at below-blocking concentrations, the test cations caused decreased duration of Mn spikes and increased duration of Ca spikes.

Upon the addition of protons to an aqueous solution, a component of the H+ load will be bound i.e. buffered. In an aqueous solution containing a triprotic acid, H+ can be bound to three different states of the acid as well as to OH- ions that are derived from the auto-ionization of H2O. In quantifying the buffering process of a triprotic acid, one must define the partitioning of H+ among the three states of the acid and also the OH- ions in solution in order to predict the equilibrium pH value. However, previous quantitative approaches that model triprotic acid titration behaviour and used to predict the equilibrium pH rely on the mathematical convenience of electroneutrality/charge balance considerations. This fact has caused confusion in the literature, and has led to the assumption that charge balance/electroneutrality is a causal factor in modulating proton buffering (Stewart formulation). However, as we have previously shown, although charge balance can be used mathematically as a convenient tool in deriving various formulae, electroneutrality per se is not a fundamental physicochemical parameter that is mechanistically involved in the underlying buffering and proton transfer reactions. The lack of distinction between a mathematical tool, and a fundamental physicochemical parameter is in part a reason for the current debate regarding the Stewart formulation of acid-base analysis. We therefore posed the following question: Is it possible to generate an equation that defines and predicts the buffering of a triprotic acid that is based only on H+ partitioning without incorporating electroneutrality in the derivation? Towards this goal, we derived our new equation utilizing: 1) partitioning of H+ buffering; 2) conservation of mass; and 3) acid-base equilibria. In validating this model, we compared the predicted equilibrium pH with the measured pH of an aqueous solution consisting of Na2HPO4 to which HCl was added. The measured pH values were in excellent agreement with the predictions of our equation. Our results provide further important evidence that one can mathematically model the chemistry of acid-base phenomenology without relying on electroneutrality (Stewart formulation) considerations.

Electrospray ionization (ESI) mass spectrometry (MS) is a powerful method for analyzing the active forms of macromolecular complexes of biomolecules. However, these solutions often contain high concentrations of salts and/or detergents that adversely effect ESI performance by making ion formation less reproducible, causing severe adduction or ion suppression. Many methods for separating complexes from nonvolatile additives are routinely used with ESI-MS, but these methods may not be appropriate for complexes that require such stabilizers for activity. Here, the effects of buffer loading using concentrations of ammonium acetate ranging from 0.22 to 1.41 M on the ESI mass spectra of a solution containing a domain truncation mutant of a σ54 activator from Aquifex aeolicus were studied. This 44.9 kDa protein requires the presence of millimolar concentrations of Mg2+, BeF3−, and ADP, (at ∼60 °C) to assemble into an active homo-hexamer. Addition of ammonium acetate can improve signal stability and reproducibility, and can significantly lower adduction and background signals. However, at higher concentrations, the relative ion abundance of the hexamer is diminished, while that of the constituent monomer is enhanced. These results are consistent with loss of enzymatic activity as measured by ATP hydrolysis and indicate that the high concentration of ammonium acetate interferes with assembly of the hexamer. This shows that buffer loading with ammonium acetate is effective for obtaining ESI signal for complexes that require high concentrations of essential salts, but can interfere with formation of, and/or destabilize complexes by disrupting crucial electrostatic interactions at high concentration.

The periodic distribution of known and suspected carcinogenic metal ions is described, and the chemical behavior of various types of metal ions is explained in terms of the general theory of hard and soft acids and bases. The chelate effect is elucidated, and the relatively high stability of metal chelates in very dilute solutions is discussed. The concepts employed for the chelate effect are extended to explain the high stabilities of macrocyclic and cryptate complexes. Procedures for the use of equilibrium data to determine the speciation of metal ions and complexes under varying solution conditions are described. Methods for assessing the interferences by hydrogen ion, competing metal ions, hydrolysis, and precipitation are explained, and are applied to systems containing iron(III) chelates of fourteen chelating agents designed for effective binding of the ferric ion. The donor groups available for the building up of multidentate ligands are presented, and the ways in which they may be combined to achieve high affinity and selectivity for certain types of metal ions are explained.

The relationship between formation of active in-line attack conformations and monovalent (Na+) and divalent (Mg2+) metal ion binding in the hammerhead ribozyme has been explored with molecular dynamics simulations. To stabilize repulsions between negatively charged groups, different requirements of threshold occupancy of metal ions were observed in the reactant and activated precursor states both in the presence or absence of a Mg2+ in the active site. Specific bridging coordination patterns of the ions are correlated with the formation of active in-line attack conformations and can be accommodated in both cases. Furthermore, simulation results suggest that the hammerhead ribozyme folds to form an electronegative recruiting pocket that attracts high local concentrations of positive charge. The present simulations help to reconcile experiments that probe the metal ion sensitivity of hammerhead ribozyme catalysis and support the supposition that Mg2+, in addition to stabilizing active conformations, plays a specific chemical role in catalysis.

Splittstoesser, D. F. (Cornell University, Geneva, N.Y.), and D. F. Farkas. Effect of cations on activation of Bacillus popilliae spores. J. Bacteriol. 92: 995–1001. 1966.—Cations affected the rate at which dormant Bacillus popilliae spores were heat-activated. Maximal rates were achieved in calcium solution at pH 7 or in tris(hydroxymethyl)aminomethane buffer at pH 9 to 10. A combination of calcium plus an alkaline pH, however, retarded activation. The rates also were markedly reduced by potassium and hydrogen ions but were not affected by sodium, cesium, or lithium. Divalent cations may be required for activation since ethylenediaminetetraacetic acid also inhibited, and potassium was shown to be competing with calcium for some active site.

Various levels of protection against x-irradiation damage in bacteriophage T1 may be obtained by the addition of inorganic salts to the aqueous virus suspensions during irradiation. The highest survival values are obtained with the nitrite salts, and their protective power is attributed primarily to their function as reducing agents. The nitrate ion shows greater protection than the corresponding sulfate or chloride ions. This may be due in part to the lower energy level of the nitrate ion, by reason of resonance. Since greater expenditure of incident energy is required to raise the ion from the ground state, the energy thus dissipated may be ineffective in the inactivation of virus particles. The ammonium salts exhibit protection of a different order of magnitude from that of the metallic salts. It is postulated that NH4+ protects in a threefold way: (a) dehydration, (b) reduction, in which the ammonia is oxidized to nitrite and the nitrite to nitrate, and (c) stabilization of the virus protein. Metallic salts likewise protect, but a point of maximum protection is reached in lower concentrations than in the case of the ammonium salts. After this maximum protection is reached, there is a rapid decline in survival with increased concentration. This prevents protection of the order of magnitude that can be obtained with the ammonium salts. It is postulated that a specific cationic interaction with the phage may be responsible for the decreased protection. Bacteriophage is protected during x-irradiation by an alkaline pH, in the case of NH4OH. This protection could not be produced with NaOH, presumably because of the greater hydrolysis of the protein components of the virus particle in solutions of NaOH, whereas NH4OH stabilizes the protein.