Chitosan-induced programmed cell death in plants

Chitosan, CN⁻, or H₂O₂ caused the death of epidermal cells (EC) in the epidermis of pea leaves that was detected by monitoring the destruction of cell nuclei; chitosan induced chromatin condensation and marginalization followed by the destruction of EC nuclei and subsequent internucleosomal DNA fragmentation. Chitosan did not affect stoma guard cells (GC). Anaerobic conditions prevented the chitosan-induced destruction of EC nuclei. The antioxidants nitroblue tetrazolium or mannitol suppressed the effects of chitosan, H₂O₂, or chitosan + H₂O₂ on EC. H₂O₂ formation in EC and GC mitochondria that was determined from 2′,7′-dichlorofluorescein fluorescence was inhibited by CN⁻ and the protonophoric uncoupler carbonyl cyanide m-chlorophenylhydrazone but was stimulated by these agents in GC chloroplasts. The alternative oxidase inhibitors propyl gallate and salicylhydroxamate prevented chitosan- but not CN⁻-induced destruction of EC nuclei; the plasma membrane NADPH oxidase inhibitors diphenylene iodonium and quinacrine abolished chitosan- but not CN⁻-induced destruction of EC nuclei. The mitochondrial protein synthesis inhibitor lincomycin removed the destructive effect of chitosan or H₂O₂ on EC nuclei. The effect of cycloheximide, an inhibitor of protein synthesis in the cytoplasm, was insignificant; however, it was enhanced if cycloheximide was added in combination with lincomycin. The autophagy inhibitor 3-methyladenine removed the chitosan effect but exerted no influence on the effect of H₂O₂ as an inducer of EC death. The internucleosome DNA fragmentation in conjunction with the data on the 3-methyladenine effect provides evidence that chitosan induces programmed cell death that follows a combined scenario including apoptosis and autophagy. Based on the results of an inhibitor assay, chitosan-induced EC death involves reactive oxygen species generated by the NADPH oxidase of the plasma membrane.     

Effect of Ca2+ on programmed death of guard and epidermal cells of pea leaves

The effect of Ca²⁺ on programmed death of guard cells (GC) and epidermal cells (EC) determined from destruction of the cell nucleus was investigated in epidermis of pea leaves. Ca²⁺ at concentrations of 1–100 μM increased and at a concentration of 1 mM prevented the CN—induced destruction of the nucleus in GC, disrupting the permeability barrier of GC plasma membrane for propidium iodide (PI). Ca²⁺ at concentrations of 0.1–1 mM enhanced drastically the number of EC nuclei stained by PI in epidermis treated with chitosan, an inducer of programmed cell death. The internucleosomal DNA fragmentation caused by CN^? was suppressed by 2 mM Ca²⁺ on 6 h incubation, but fragmentation was stimulated on more prolonged treatment (16 h). Presumably, the disruption of the permeability barrier of plasma membrane for PI is not a sign of necrosis in plant cells. Quinacrine and diphenylene iodonium at 50 μM concentration prevented GC death induced by CN^? or CN^? + 0.1 mM Ca²⁺ but had no influence on respiration and photosynthetic O₂ evolution in pea leaf slices. The generation of reactive oxygen species determined from 2′,7′-dichlorofluorescein fluorescence was promoted by Ca²⁺ in epidermal peels from pea leaves.     

Programmed cell death in plants: Protective effect of mitochondrial-targeted quinones

Ubiquinone or plastoquinone covalently linked to synthetic decyltriphenylphosphonium (DTPP⁺) or rhodamine cations prevent programmed cell death (PCD) in pea leaf epidermis induced by chitosan or CN⁻. PCD was monitored by recording the destruction of cell nuclei. CN⁻ induced the destruction of nuclei in both epidermal cells (EC) and guard cells (GC), whereas chitosan destroyed nuclei in EC not in GC. The half-maximum concentrations for the protective effects of the quinone derivatives were within the pico- and nanomolar range. The protective effect of the quinones was removed by a protonophoric uncoupler and reduced by tetraphenylphosphonium cations. CN⁻-Induced PCD was accelerated by the tested quinone derivatives at concentrations above 10⁻⁸–10⁻⁷ M. Unlike plastoquinone linked to the rhodamine cation (SkQR1), DTPP⁺ derivatives of quinones suppressed menadione-induced H₂O₂ generation in the cells. The CN⁻-induced destruction of GC nuclei was prevented by DTPP⁺ derivatives in the dark not in the light. SkQR1 inhibited this process both in the dark and in the light, and its effect in the light was similar to that of rhodamine 6G. The data on the protective effect of cationic quinone derivatives indicate that mitochondria are involved in PCD in plants.     

Programmed cell death in plants: Protective effect of phenolic compounds against chitosan and H<Subscript>2</Subscript>O<Subscript>2</Subscript>

Addition of chitosan or H₂O₂ caused destruction of nuclei of epidermal cells (EC) in the epidermis isolated from pea leaves. Phenol, a substrate of the apoplastic peroxidase-oxidase, in concentrations of 10⁻¹⁰–10⁻⁶ M prevented the destructive effect of chitosan. Phenolic compounds 2,4-dichlorophenol, catechol, and salicylic acid, phenolic uncouplers of oxidative phosphorylation pentachlorophenol and 2,4-dinitrophenol, and a non-phenolic uncoupler carbonyl cyanide m-chlorophenylhydrazone, but not tyrosine or guaiacol, displayed similar protective effects. A further increase in concentrations of the phenolic compounds abolished their protective effects against chitosan. Malate, a substrate of the apoplastic malate dehydrogenase, replenished the pool of apoplastic NADH that is a substrate of peroxidase-oxidase, prevented the chitosan-induced destruction of the EC nuclei, and removed the deleterious effect of the increased concentration of phenol (0.1 mM). Methylene Blue, benzoquinone, and N,N,N′,N′-tetramethyl-p-phenylenediamine (TMPD) capable of supporting the optimal catalytic action of peroxidase-oxidase cancelled the destructive effect of chitosan on the EC nuclei. The NADH-oxidizing combination of TMPD with ferricyanide promoted the chitosan-induced destruction of the nuclei. The data suggest that the apoplastic peroxidase-oxidase is involved in the antioxidant protection of EC against chitosan and H₂O₂.     

Participation of Chloroplasts in Plant Apoptosis

Mitochondria are known to participate in the initiation of programmed cell death (PCD) in animals and in plants. The role of chloroplasts in PCD is still unknown. We describe a new system to study PCD in plants; namely, leaf epidermal peels. The peel represents a monolayer consisting of cells of two types: phototrophic (guard cells) and chemotrophic (epidermal cells). The peels from pea (Pisum sativum L.) leaves were treated by cyanide as an inducer of PCD. We found an apoptosis-enhancing effect of illumination on chloroplast-containing guard cells, but not on chloroplastless epidermal cells. Antioxidants and anaerobiosis prevented the CN^–-induced apoptosis of cells of both types in the dark and in the light. On the other hand, methyl viologen and menadione known as ROS-generating reagents as well as the Hill reaction electron acceptors (BQ, DAD, TMPD, or DPIP) that are not oxidized spontaneously by O₂ were shown to prevent the CN^–-induced nucleus destruction in guard cells. Apoptosis of epidermal cells was potentiated by these reagents, and they had no influence on the CN^– effect. The light-dependent activation of CN^–-induced apoptosis of guard cells was suppressed by DCMU, stigmatellin or DNP-INT, by a protein kinase inhibitor staurosporine as well as by cysteine and serine protease inhibitors. The above data suggest that apoptosis of guard cells is initiated upon a combined action of two factors, i.e., ROS and reduced plastoquinone of the photosynthetic electron transfer chain. As to reduction of ubiquinone in the mitochondrial respiratory chain, it seems to be antiapoptotic for the guard cell.     

Involvement of chloroplasts in the programmed death of plant cells

The effect of cyanide, an apoptosis inducer, on pea leaf epidermal peels was investigated. Illumination stimulated the CN^–-induced destruction of guard cells (containing chloroplasts and mitochondria) but not of epidermal cells (containing mitochondria only). The process was prevented by antioxidants (-tocopherol, 2,5-di-tret-butyl-4-hydroxytoluene, and mannitol), by anaerobiosis, by the protein kinase C inhibitor staurosporine, and by cysteine and serine protease inhibitors. Electron acceptors (menadione, p-benzoquinone, diaminodurene, TMPD, DCPIP, and methyl viologen) suppressed CN^–-induced apoptosis of guard cells, but not epidermal cells. Methyl viologen had no influence on the removal of CN^–-induced nucleus destruction in guard cells under anaerobic conditions. The light activation of CN^–-induced apoptosis of guard cells was suppressed by DCMU (an inhibitor of the electron transfer in Photosystem II) and by DNP-INT (an antagonist of plastoquinol at the Q_o site of the chloroplast cytochrome b ₆ f complex). It is concluded that apoptosis initiation in guard cells depends on the simultaneous availability of two factors, ROS and reduced quinones of the electron transfer chain. The conditions for manifestation of programmed cell death in guard and epidermal cells of the pea leaf were significantly different.     

Role of chloroplast photosystems II and I in apoptosis of pea guard cells

We investigated the CN^–-induced apoptosis of guard cells in epidermal peels isolated from pea (Pisum sativum L.) leaves. This process was considerably stimulated by illumination and suppressed by the herbicides DCMU (an inhibitor of the electron transfer between quinones Q_A and Q_B in PS II) and methyl viologen (an electron acceptor from PS I). These data favor the conclusion drawn by us earlier that chloroplasts are involved in the apoptosis of guard cells. Pea mutants with impaired PS I (Chl-5), PS II (Chl-I), and PS II + PS I (Xa-17) were tested. Their lesions were confirmed by the ESR spectra of Signal I (oxidized PS I reaction centers) and Signal II (oxidized tyrosine residue Y_D in PS II). Destruction of nuclei (a symptom of apoptosis) and their consecutive disappearance in guard cells were brought about by CN^– in all the three mutants and in the normal pea plants. These results indicate that the light-induced enhancement of apoptosis of guard cells and its removal by DCMU are associated with PS II function. The effect of methyl viologen preventing CN^–-induced apoptosis in wild-type plants was removed or considerably decreased upon the impairment of the PS II and/or PS I activity.     

Programmed cell death in plants: Protective effect of tetraphenylphosphonium and tetramethylrhodamine cations used as transmembrane quinone carriers

Tetraphenylphosphonium (TPP⁺) and tetramethylrhodamine ethyl ester (TMRE⁺) cations used as transmembrane carriers of ubiquinone (MitoQ) and plastoquinone (SkQ, SkQR) in mitochondria prevented at nanomolar concentrations the chitosanor H₂O₂-induced destruction of the nucleus in epidermal cells of epidermis isolated from pea leaves. The protective effect of the cations was potentiated by palmitate. Penetrating anions of tetraphenylboron (TB⁻) and phenyl dicarbaundecaborane also displayed protective effects at micromolar concentrations; the effect of TB⁻ was potentiated by NH₄Cl. It is proposed that the protective effect of the penetrating cations and anions against chitosan is due to suppression of the generation of reactive oxygen species in mitochondria as a result of the protonophoric effect of the cations plus fatty acids and the anions plus NH₄⁺. Phenol was suitable as the electron donor for H₂O₂ reduction catalyzed by horseradish peroxidase, preventing the destruction of cell nuclei. The penetrating cations and anions, SkQ1, and SkQR1 did not maintain the peroxidase or peroxidase/oxidase reactions measured by their suitability as electron donors for H₂O₂ reduction or by the oxidation of exogenous NADH.     

Expression of photosynthesis gene-promoter fusions in leaf epidermal cells of transgenic tobacco plants

Fusions of the promoter regions of the pea plasto-cyanin, pea ferredoxin: NADP⁺ reductase and tobacco rbcS genes to the β-glucuronidase (GUS) reporter gene have been introduced into tobacco via Agro-bacterium-mediated transformation, and epidermal peels of the lower leaf surface of tissue-cultured and greenhouse-grown plants examined histochemically for GUS activity. For each of the constructs, GUS was detected in epidermal cells as well as in stomatal guard cells. Epidermal peels from plants in tissue culture stained more readily than those from greenhouse-grown plants. Light and electron microscopy clearly demonstrated the presence of chloroplasts in epidermal cells of tobacco leaves. These results provide further evidence for the correlation between the presence of chloroplasts and the expression of nuclear genes for photosynthesis components.     

Proton efflux through the chloroplast ATP synthase (CF0 · CF1) in the presence of sulfhydryl-modifying agents

The rate of photosynthetic electron transport measured in the absence of ADP and P_i is stimulated by low levels of Hg²⁺ or Ag⁺ (50% stimulation ≈ 3 Hg²⁺ or 6 Ag⁺/100 chlorophyll) to a plateau equal to the transport rate under normal phosphorylating conditions (i.e. +ADP, +P_i). Chloroplasts pretreated in the light under energizing conditions with N-ethylmaleimide show a similar stimulation of non-phosphorylating electron transport. The stimulations of non-phosphorylating electron transport by Hg²⁺, Ag⁺ and N-ethylmaleimide are reversed by the CF₁ inhibitor phlorizin, the CF₀ inhibitor triphenyltin chloride, and can be further stimulated by uncouplers such as methylamine. The Hg²⁺ and N-ethylmaleimide stimulations, but not the Ag⁺ stimulation, are completely reversed by low levels of ADP (2 μM), ATP (2 μM), and P_i (400 μM). Ag⁺, which is a potent inhibitor of ATP synthesis, has little or no effect upon phosphorylating electron transport (+ADP, +P_i). Concomitant with the stimulations of non-phosphorylating electron transport by Hg²⁺, Ag⁺ and ADP + P_i, there is a decrease in the level of membrane energization (as measured by atebrin fluorescence quenching) which is reversed when the CF₀ channel is blocked by triphenyltin. These results suggest that modification of critical CF₁ sulfhydryl residues by Hg²⁺, Ag⁺ or N-ethylmaleimide leads to the loss of intra-enzyme coupling between the transmembrane protontransferring and the ATP synthesis activities of the CF₀-CF₁ ATP synthase complex.     

Fluorescence Properties of Guard Cell Chloroplasts: EVIDENCE FOR LINEAR ELECTRON TRANSPORT AND LIGHT-HARVESTING PIGMENTS OF PHOTOSYSTEMS I AND II

The presence of chloroplasts in guard cells from leaf epidermis, coleoptile, flowers, and albino portions of variegated leaves was established by incident fluorescence microscopy, thus confirming the notion that guard cell chloroplasts are remarkably conserved. Room temperature emission spectra from a few chloroplasts in a single guard cell of Vicia faba showed one major peak at around 683 nanometers. Low-temperature (77 K) emission spectra from peels of albino portions of Chlorophytum comosum leaves and from mesophyll chloroplasts of green parts of the same leaves showed major peaks at around 687 and 733 nanometers, peaks usually attributed to photosystem II and photosystem I pigment systems, respectively. Spectra of peels of V. faba leaves showed similar peaks. However, fluorescence microscopy revealed that the Vicia peels, as well as those from Allium cepa and Tulipa sp., were contaminated with non-guard cell chloroplasts which were practically undetectable under bright field illumination. These observations pose restrictions on the use of epidermal peels as a source of isolated guard cell chloroplasts. Studies on the 3-(3,4-dichlorophenyl)-1,1-dimethylurea-sensitive variable fluorescence kinetics of uncontaminated epidermal peels of C. comosum indicated that guard cell chloroplasts operate a normal, photosystem II-dependent, linear electron transport. The above properties in combination with their reported inability to fix CO₂ photosynthetically may render the guard cell chloroplasts optimally suited to supply the reducing and high-energy phosphate equivalents needed to sustain active ion transport during stomatal opening in daylight.     

On the decarboxylation of α-keto acids by isolated chloroplasts

The mechanism of the decarboxylation of α-keto acids by isolated chloroplasts has been studied with the aid of superoxide dismutase and catalase. Using photosynthetic and enzymatic systems, which are known to catalyze peroxidic oxidations, we have been able to demonstrate that both the superoxide free radical ion and H₂O₂ are necessary for maximal rates of decarboxylation. In isolated chloroplasts, an auto-oxidizable electron acceptor as well as an electron donor for Photosystem I are absolute requirements for the decarboxylation. H₂O₂ seems to be the primary oxidant in the decarboxylation of pyruvate or glyoxylate by isolated chloroplasts. A secondary rate of decarboxylation is superimposed on the primary one, mediated by superoxide free radical ion. Mn²⁺ stimulates the decarboxylation probably via intermediarily-formed Mn³⁺ in a reaction, which is neither inhibited by catalase nor by superoxide dismutase. A decarboxylation of pyruvate or glyoxylate by isolated chloroplasts in the presence of NADP⁺ is initiated, as soon as the available NADP⁺ is fully reduced. In this case, the open-chain electron transport seems to switch from NADP⁺ to oxygen as the terminal electron acceptor.     

Elicitor-enhanced syringin production in suspension cultures of Saussurea medusa

Syringin production and related secondary metabolism enzyme activities in suspension cultures of Saussurea medusa treated with different elicitors (yeast extract, chitosan and Ag⁺) were investigated. All elicitors enhanced syringin production, and the optimal feeding protocol was the combined addition of 1.5% (v/v) yeast extract, 0.2 g l⁻¹ chitosan and 75 μM Ag⁺ at the 15th day of the cell culture. The highest syringin production reached 741.9 mg l⁻¹, which was 3.6−fold that of the control. The glucose−6-phosphate dehydrogenase (EC 1.1.1.49), phenylalanine ammonia lyase (EC 4.3.1.5) and peroxidase (EC 1.11.1.7) activities increased significantly after elicitor treatment. The maximum enzyme activities were obtained when the treatment time was 6 h.     

The influence of trace TiO2 on adsorption of Ag+-imprinted adsorbents made from chitosan and mycelium

The complex technology of molecular imprinting with a photocatalytic reaction introduces novel ways of treating industrial and living sewage. This paper deals with the effects of trace TiO₂ on Ag⁺-imprinted or non-imprinted adsorbents. NanoTiO₂ was added during the preparation of the adsorbents. The performance of these adsorbents was compared with other common adsorbents, such as activated carbon and chitosan. TiO₂ loading improved the adsorption ability for Ag⁺ of adsorbents. Adsorption equilibrium could be rapidly achieved at an initial Ag⁺ concentration of 200 mg/L under different light conditions (UV, visible light, and dark). After TiO₂ loading, the maximal adsorption capacity of Ag⁺-imprinted and non-imprinted adsorbents was 25.0% higher, at 155.0 and 134.3 mg/g, respectively, at the initial Ag⁺ concentration of 1,000 mg/L. In order to understand the binding state of Ag, Ti on the adsorbents surface, FTIR, XPS were measured. The FTIR analysis, before and after adding TiO₂, indicated that TiO₂ bound with adsorbents through hydrogen bonding. XPS analysis, before and after adsorption, indicated Ag⁺ was reduced to Ag⁰ on the adsorbent surface, leading to an increased adsorption of Ag⁺.     

Toxicity of biosynthetic silver nanoparticles on the growth,cell ultrastructure and physiological activities of barley plant

Silver nanoparticles (AgNPs) were biosynthesized using the cell-free filtrate of bacterium Proteus mirabilis, reacted with 1 mM of AgNO₃ solutions at 37 °C. The synthesis of AgNPs was monitored by UV–Vis spectroscopy and transmission electron microscopy (TEM) equipped with selected area electron diffraction (SAED). The results point to formation of spherical to cubical particles of AgNPs ranging in size from 5 to 35 nm with an average of 25 nm in diameter. The toxicity of Ag on barley (Hordeum vulgare L. cv. Gustoe) that was subjected to Ag⁺ as AgNO₃ and AgNPs was explored. The grain germination and seedling growth of barley decreased in the presence of 0.1 mM Ag⁺ and was inhibited at 1 mM Ag⁺. In contrast, our results indicated that the AgNPs at low concentration (0.1 mM) could be useful for barley grain germination and seedling growth. However, the higher concentrations of AgNPs (0.5 and 1 mM) reduced grain germination and exhibited a stronger reduction in the root length. A decline in the photosynthetic pigments and disorganization of chloroplast grana thylakoids in Ag⁺ and AgNPs-treated plants confirmed the leaf chlorosis. An increase of plastoglobuli within chloroplasts was observed in Ag⁺ and AgNPs-treated leaves. Ag⁺ caused dense aggregation of nuclear chromatin materials and degeneration of mitochondria. Ag⁺ and AgNPs increased contents of malondialdehyde, soluble proteins, total phenolic compounds and activity of guaiacol peroxidase in barley leaves; these results point to activation of plant defence mechanisms against oxidative stress in barley.     

The roles of weak light,oxygen and dithiothreitol in the photoreactivation of the oxygen evolving center of tris-washed and 2, 6-dichlorophenol indophenol-treated chloroplasts

The inactivated O₂-evolving center of Tris-washed chloroplasts was reactivated by DCPIP-treatment and photoreactivation in the presence of Mn²⁺, Ca²⁺, DTT and weak light. Many electron donors (Asc and reduced DCPIP, etc.) were found to be suitable substitutes for DTT. By studying the anaerobic inhibition of the reactivation, the electron acceptors O₂, NADP⁺, etc. were also found to be essential factors in photoreactivation. Weak light stimulated the chloroplast electron transport from the above-mentioned electron donors to the electron acceptor and effected the photoreactivation. More than 280 electrons were transported to NADP⁺ in the anaerobic photoreactivation of one unit of an O₂-evolving center with 400 Chl. Electron transport in the reactivation was inhibited by omitting DTT or Mn²⁺ ion, and by adding DCMU. The photoreactivated chloroplasts incorporated about 2 Mn by 400 Chl. Omission of DTT in the reactivation caused chloroplasts in the weak light to bind large amounts of excess Mn.Abbreviations Asc ascorbate - Chl chlorophyll - DCPIP 2, 6-dichlorophenol indophenol - DPC diphenyl carbazide - DTT dithiothreitol - Fd ferredoxin - STN a chloroplast preparation medium, containing 0.4 M sucrose, 0.05 M Tris-Cl and 0.01 M NaCl (pH 7.8 and 8.0) - TMPD tetramethyl-p-phenylenediamine     

The uptake of silver ions by Escherichia coli K12: toxic effects and interaction with copper ions

Summary Growth of Escherichia coli in chloridefree medium in batch culture is inhibited completely at concentrations of AgNO₃ greater than 2.5x10^-6 M. Incubation of non-growing cells in HEPES buffer (pH 7.4) at increasing levels of Ag⁺ results in the progressive saturation of two types of binding site. At one site, the Ag⁺ is not released by washing with 0.1 M nitric acid, and is probably intracellular. Silver bound to the second site is released by acid-washing, but not by buffer washing, and is assumed to be surface-bound. The amounts of Ag⁺ taken up from solution at the two sites is 1.6x10^-7 and 4.6x10^-7 mol (mg dry weight)^-1, respectively. Total accumulation of silver is 67 mg (g dry weight)^-1, similar to literature values found for silver-resistant bacteria. Binding of Ag⁺ at intracellular sites (observed at low [Ag⁺]) appears to be independent of pH. Addition of AgNO₃ to growing cells in mid-exponential phase of growth in concentrations that will inhibit growth results in substantially decreased accumulation of silver. Growth yield in chemostat culture is diminished in the presence of added Ag⁺, but this effect is moderated by added Cu²⁺, which may protect copper sites from Ag⁺ or compete with Ag⁺ for other sites at which Ag⁺ exerts toxic effects. Very small amounts of Cu²⁺ are found in cell samples from the chemostat compared to the substantial amounts of Ag⁺ taken up, but uptake of Cu²⁺ is decreased at higher [Ag⁺]/[Cu²⁺]ratios.     

Inhibition of photosynthesis by azide and cyanide and the role of oxygen in photosynthesis

Cyanide and azide inhibit photosynthesis and catalase activity of isolated, intact spinach (Spinacia oleracea) chloroplasts. When chloroplasts are illuminated in the presence of CN⁻ or N₃⁻, accumulation of H₂O₂ is observed, parallel to inhibition of photosynthesis. Photosynthetic O₂ evolution is inhibited to the same extent, under saturating light, whether CO₂ or phosphoglycerate is present as electron acceptor.     

Metal ion complexes of d-biotin in solution. Stability of the stereoselective thioether coordination

By spectrophotometry and ¹H nmr, several of the stability constants of the thioether complexes between Mg²⁺, Ca²⁺, Mn²⁺, Cu²⁺, Zn²⁺, Cd²⁺, or Ag⁺ and d-biotin (Bio), tetrahydrothiophene (Tht), and dimethyl sulfide (Dms) have been measured in 50% aqueous ethanol, 96% N,N-dimethylformamide (DMF), 98% d₆-dimethyl sulfoxide, or in D₂O. With decreasing concentration of water, the thioether interaction increases with the biologically important metal ions, whereas, e.g., Ag⁺ behaves in the opposite way. The stability of these complexes is, in general, quite small: for example, with d-biotin in 96% DMF (I = 1.0; 25°C) log K_M(Bio)^M = 0.03 and 1.64 for Cu²⁺ and Ag⁺, respectively; in D₂O (I = 0.5 for Ag⁺, all others 2–5; 27°C) log K_M(Bio)^M ? ?1.0, ?1.4, ?1.2, ?0.9, or 4.20 for Mg²⁺, Ca²⁺, Zn²⁺, Cd²⁺, or Ag⁺. In those cases where the difference log K_M(Tht)^M ? log K_M(Bio)^M can be calculated, it is in the order of 0.3 log units; this observation, as well as the chemical shifts measured, confirm the earlier suggestion that the interaction at the sulfur of biotin is stereoselective: the metal ion coordinates from “below” the tetrahydrothiophene ring of biotin to the sulfur atom, i.e., trans to the urea ring. It is emphasized that despite the low stability of these complexes with the biologically meaningful metal ions, the extent of the interaction is enough to create specific structures.     

Induction of helicity in polyuridylic acid and polyinosinic acid by silver ions

Silver ions binding to poly(U) and poly(I) produce highly ordered multistranded helices under conditions which would otherwise lead to random coils. Evidence for helicity comes from the hypochromicity and high ellipticity generated in the polymers by Ag⁺ binding, as well as from x-ray studies and from the cooperativity of the Ag⁺ complexing reaction. Continuous variation studies show that both polymers form 1:1 and 2:1 polymer–Ag⁺ complexes. Low pH favors the 1:1 complex with poly(U) and the 2:1 complex with poly(I); the reverse is true at high pH. Ag⁺ binding and proton-release experiments make it clear that at low pH, unprotonated electron-donor groups are complexed preferentially, but that at high pH, Ag⁺ readily displaces H⁺ from protonated groups. In poly(I) the unprotonated donor is N(7), leading at low pH to a 2:1 complex containing N(7)-Ag-N(7) bonds; at high pH, proton release from N(1) leads to a 1:1 complex containing N(1)-Ag-O bonds. In poly(U) there is no unprotonated donor; the low-pH 1:1 complex involves deprotonation of only one N(3) per bound Ag⁺, leading to N₃-Ag-O bonding, while high pH causes deprotonation of two N(3) per Ag⁺ and a 2:1 N(3)-Ag-N(3) complex. Thus silver ions react with the nucleotide bases in chemically predictable ways, and the formation of different Ag–nucleotide bonds leads to different multiple-helix structures.