Kinetics of formation of metallic silver and binding of silver ions by tissue components.

The effect of time on the formation of metallic silver by tissue reducing groups follows a curve which can be divided into three main parts. In the first, which may last for several hours, the reaction is very slow, and only an undetectably small amount of metallic silver is produced. In the second period the speed of the reaction first increases in a progressive manner and then begins to decrease gradually; during the third period the speed approaches zero asymptotically. Binding of the silver ions by the tissue commences initially at its fastest rate; the level then decreases steadily to zero within about a quarter of an hour. There is no direct relationship between the amount of silver ion bound to the tissue and the formation of metallic silver. The latter cannot take place by way of direct (non-catalysed) reaction. The following mechanism is proposed for the process: Transfer of electrons from the reducing molecules to the silver ions is mediated at first by certain tissue sites (catalytic points) and then also by the steadily increasing total surface area of the metallic silver grains (autocatalysis). On the basis of this mechanism, several anomalies of both the argentaffin and argyrophil reactions are explained.     

Factors affecting the formation of metallic silver and the binding of silver ions by tissue components

Summary The rate of formation of metallic silver has a maximum when plotted as a function of pH. The site of this maximum on a pH scale differs noticeably for various tissue elements. By contrast, the amount of silver ions bound to the tissue is a monotonously increasing function of the pH. A temperature rise decreases the length of the induction period and increases the gradient of the ascending section of the kinetic curve representing the formation of metallic silver. It also increases the maximum amount of silver ions bound to the tissue. An increase in the concentration (activity) of the silver ions in the impregnating bath has the same effect. Chemical composition and concentration of the complexing agent, as well as special ions in the impregnating bath to which earlier some definitive role has been attributed in the silver staining methods, proved to be ineffective when both pH and activity of silver ions were kept constant. Illumination of the reaction was also ineffective. The kinetic curves obtained in nonaqueous but polar media (e.g., acetone) exhibited the same qualitative characteristics as those obtained in aqueous solutions. No reaction between silver ions and tissue was observed in apolar solvents.     

In vivo liberation of silver ions from metallic silver surfaces

In vivo liberation of electrically charged silver atoms/silver ions from metallic silver pellets, silver grids and silver threads placed in the brain, skin and abdominal cavity was proved by way of the histochemical technique autometallography (AMG). A bio-film or “dissolution membrane” inserted between the metallic surface and macrophages was recognized on the surface of the implanted silver after a short period of time. Bio-released silver ions bound in silver–sulphur nanocrystals were traced within the first 24 h in the “dissolution membrane” and the “dissolucytotic” macrophages. In animals that had survived 10 days or more, silver nanocrystals were detected both extra- and intracellularly in places far away from the implant including regional lymph nodes, liver, kidneys and the central nervous system (CNS). The accumulated silver was always confined to lysosome-like organelles. Dissolucytotic silver was extracellularly related to collagen fibrils and fibres in connective tissue and basement membranes. Our study demonstrates that (1) the number of bio-released silver ions depends on the size of the surface of the implanted silver, (2) the spread of silver ions throughout the body takes place primarily not only through the vascular system, but also by retrograde axonal transport. It is concluded that implantation of silver or silver-plated devices is not recommendable.     

Effect of silver ions on ethylene biosynthesis by tomato fruit tissue

Mature-green tomato fruit (Lycopersicon esculentum Mill.) were treated asymmetrically with 2 millimolar silver thiosulfate (STS) through a cut portion of the peduncle while still attached to the plant. One-half of the fruit received silver and remained green while the other half ripened normally and was silver-free (less than 0.01 parts per billion). Harvested mature-green fruit were also treated with STS through the cut pedicel. Green tissue from silver-treated fruit had levels of 1-aminocyclopropane-1-carboxylic acid (ACC, the immediate ethylene precursor) slightly less or similar to that of turning or red-ripe tissue from the same fruit, and similar to that of mature-green tissue from control fruit. Ethylene production was higher in green tissue from silver-treated fruit than from either red tissue from the same fruit, or mature-green tissue from control fruit. By inhibiting ACC synthesis with aminoethoxyvinyl glycine, and by applying ACC ± silver to excised disks of pericarp tissue from control or silver-treated tomatoes, we showed that short-term silver treatment did not affect the biological conversion of ACC to ethylene, while long-term treatment stimulated both the conversion of ACC to ethylene and the synthesis of ACC.     

The histidine kinase CusS senses silver ions through direct binding by its sensor domain

The Cus system of Escherichia coli aids in protection of cells from high concentrations of Ag(I) and Cu(I). The histidine kinase CusS of the CusRS two-component system functions as a Ag(I)/Cu(I)-responsive sensor kinase and is essential for induction of the genes encoding the CusCFBA efflux pump. In this study, we have examined the molecular features of the sensor domain of CusS in order to understand how a metal-responsive histidine kinase senses specific metal ions. We find that the predicted periplasmic sensor domain of CusS directly interacts with Ag(I) ions and undergoes a conformational change upon metal binding. Metal binding also enhances the tendency of the domain to dimerize. These findings suggest a model for activation of the histidine kinase through metal binding events in the periplasmic sensor domain.     

Ultrastructural localization and chemical binding of silver ions in human organotypic skin cultures

Organotypic cultures of human breast skin incubated with silver bandage or treated with silver sulfadiazine accumulated silver in epithelial cells and in macrophages, fibroblasts and collagen fibrils and fibres of underlying connective tissue. Ultrastructurally, the accumulated silver was found in lysosome-like vesicles of the different cells and evenly spread along collagen structures. Apoptotic nuclei were present but few. Autometallographic amplification of 2D-PAGE gels revealed that glutathione S-transferase and glutathion detoxify silver ions in the epidermal cell by binding them in silver–sulphur nanocrystals. Thus, the cytotoxic effect of silver ions seems to be muted by silver ions by being: (1) taken up by undamaged cells, neutralised by glutathione (GSH) and accumulated in lysosomal vesicles, (2) bound extracellularly to SH-groups of the collagen fibres.     

Kinetics of conformational changes induced by the binding of various metal ions to bovine alpha-lactalbumin.

The kinetic effects of the binding of various metal ions (Ca(2+), Cd(2+), Co(2+), Mg(2+), Mn(2+), Sr(2+) and Zn(2+)) to apo bovine alpha-lactalbumin has been monitored by means of stopped-flow fluorescence spectroscopy. Our results show that the measured rate constant for the binding of metal ions to the Ca(2+)-site increases with increasing binding constant. This is, however, not the case for metal ions binding to the Zn(2+)-site. The binding experiments performed at different temperatures allowed us to calculate the activation energy for the transition from the metal-free to the metal-loaded state of the protein. These values do not depend on the nature of the metal ion but are correlated with the type of binding site. As a result, we were able to demonstrate that Mg(2+), a metal ion which was thought to bind to the Ca(2+)-site, shows the same binding characteristics as Co(2+) and Zn(2+) and therefore most likely interacts with the residues belonging to the Zn(2+)-binding site.     

Inhibition of Cav3.1 channel by silver ions

We have investigated the effects of AgCl and AgNO3 on the Cav3.1 calcium channels stably expressed in the HEK 293 cells. Ca2+ was used as a charge carrier. Both forms of Ag+ blocked the Cav3.1 channel and negatively shifted the I-V relations in a concentration-dependent manner. The inhibition of current amplitude by AgCl was voltage-dependent and increased with increasing amplitude of the depolarizing pulse. Furthermore, AgCl but not AgNO3 accelerated the kinetics of current activation. No effect on current inactivation or steady-state inactivation of the channel was observed for AgCl or AgNO3.     

Oxidation catalyzed by H+ ions improves the silver intensification of 3,3'-diaminobenzidine staining by strongly suppressing tissue argyrophilia

We found that a 15-min treatment of tissue sections with 2 M/l H2O2 dissolved in 6 M/l sulphuric acid strongly suppresses the argyrophilia of tissue elements, thus facilitating selective silver intensification in histochemical procedures. When applied inserted between the 3,3'-diaminobenzidine reaction and physical development, this treatment allows the selective and sensitive visualization of very low levels of peroxidase activity.     

Biosorption of silver ions by processed Aspergillus niger biomass

Summary An alkali treated A. niger biomass was found to efficiently sequester silver ions from dilute as well as concentrated solutions (2.5–1000 ppm Ag⁺), with an ability to bind it to a level of upto 10% of dry weight. Biosorption of silver ions was not influenced by pH between 5–7. The bound Ag⁺ could be fully desorbed by dilute HNO₃ and the biosorbent regenerated by washing with Ca²⁺/Mg²⁺ solution. This biosorbent is unique in that the mechanism of metal ion sorption has been found to be exclusively by stoichiometric exchange with Ca²⁺ and Mg²⁺ of the biosorbent.     

Kinetics of conformational changes in Nereis sarcoplasmic calcium-binding protein upon binding of divalent ions

The sarcoplasmic calcium-binding protein (SCP) of the sandworm Nereis possesses three Ca2(+)-Mg2+ sites but no Ca2(+)-specific site. Binding of Mg2+, but not of Ca2+, displays a marked positive cooperativity. The apparent cooperativity of Ca2+ binding in the presence of Mg2+ results from the allostery in Mg2+ dissociation. Binding of the first Ca2+ or Mg2+ induces all the conformational change, monitored by Trp fluorescence. In displacement reactions the conformational changes occur in the step SCP.Mg3----SCP.Ca1Mg2. Stopped-flow experiments indicate that Trp fluorescence changes upon Ca2(+)-binding are instantaneous whereas Mg2(+)-binding involves a fast pre-equilibrium (Keq = 28 M-1), followed by two slow consecutive conformational changes with k1 = 13.5 s-1 and k2 = 0.21 s-1. The fluorescence change after dissociation of Ca2+ from SCP is monophasic with k = 0.02 s-1; that after Mg2+ dissociation is biphasic with k1 = 0.8 s-1 and k2 = 0.1 s-1. Trp life time measurements also indicate that Ca2(+)- and Mg2(+)-induced conformational changes are completely different. Displacement of bound Ca2+ by Mg2+ can be described by two consecutive reactions in which the first (without fluorescence change) corresponds to the dissociation of the last Ca2+ (k1 = 2.4 s-1) and the second (k2 = 0.45 s-1) to the final conformational change observed upon direct Mg2+ binding. Displacement of bound Mg2+ by Ca2+ follows the kinetic scheme of simple competition; the conformational rate constant approaches asymptotically (up to the limit of 129 s-1) the dissociation rate of Mg2+ as the concentration of Ca2+ increases. In summary, after fast dissociation of Ca2+ or Mg2+, Nereis SCP slowly converts to the metal-free configuration, but in Ca2(+)-Mg2+ exchange reactions, the conformational changes are nearly as fast as the cation dissociation reactions.     

Tolerance ofAzotobacter for metallic and non-metallic ions

The effect of 51 different inorganic salts on the growth of threeAzotobacter strains was studied by means of a diffusion technique in which 10^–4 mole of each salt was deposited in the centre of a solid medium in a petri dish previously streaked with anAzotobacter suspension.After 1 or 2 days, sterile zones appeared around the point of deposition of the salt, ranging from 5 to 100 mm in diameter. Besides inhibitory effects on growth, effects on slime formation and pigmentation were noted.High toxicity was found for the cations of Cs, Ba, Co, Ni, Cu, Au, Zn, Hg and Tl, and for KIO₃, Na₂CrO₄, Sb₂O₃SbOCl, Na₂SeO₃, Na₂TeO₃ and Na₂B₄O₇. Chlorides or sulfates of Na, K, Mg, Ca and Sr were well tolerated. The cations of Li, Rb, Be, Cr, Cd, Al, In, Sn, Pb, and UO₂(NO₃)₂, VOSO₄, NaVO₃, Na₂WO₄, NaBiO₃ and Na₂HAsO₄ had an intermediate effect.The results are discussed against the background of the limitations inherent to the method used. They are further compared with those of a previous study on the tolerance ofChlorella for inorganic ions (den Dooren de Jong, 1965).The author is indebted to the Director of the Academic Hospital Dijkzigt, Rotterdam, and to Prof. Dr. H. Esseveld, Head of the Central Bacteriological Laboratory, Rotterdam for providing facilities for the performance of this study.