Estimation of pseudopeptide metal ion complexation tendencies by special MS methods,HPCE and circular dichroism

Summary To obtain more insight into catalytic mechanisms of metallo enzymes and specific metal complexation by proteins we use linear and cyclic pseudopeptides as mimetics. Knowledge about tendencies of complex formation of different ligands with selected transition metal ions is an indispensable prerequisite for the development of homo-and hetero-dinuclear metallo enzyme mimetics. Three pseudotripeptide ligands were investigated with respect to formation tendency and properties of complexes with the transition metal ions Cu²⁺, Zn²⁺ Ni²⁺, Co²⁺ and Mn²⁺. To study complexation tendencies we applied different methods. One of the important prerequisites for the application in a secreening of series of peptide ligands is the necessity for a minimal amount of substance. We used and compared certain masspectrometric methods for the estimation of a rank order of complexation of certain transition metal ions. We also applied spectrophotometric titration, circular dichroism measurements, capillary electrophoresis and pH-rate profile of catalytic activity in the attempt to evaluate complex formation tendencies. Except for the spectrophotometric pH-titration and the pH-profile of catalytic activity all methods, were applicable, but each method has its advantages and disadvantages depending on the separation effect of the ligand from the metal complex, and depending on the spectroscopic properties of ligand and complex. The results regarding complex formation are compared to each other. Comparison of pairs by MALDI-TOF-and ESI-MS allows an estimation of the rank order of complexation tendency of one ligand with different metal ions and requires the least amount of substance. The other investigated methods provided additional information on structural properties of the formed complexes; however either they required too much pseudopeptide ligand or were not applicable for all transition metal ions used in this study.     

Enhancement of the luminescence properties of Sr3(PO4)2:Dy3+,Li+ white‐light‐emitting phosphors by charge compensator Li+ co‐doping

Sr₃(PO₄)₂:Dy³⁺,Li⁺ phosphors were prepared using a simple high temperature solid method for luminescence enhancement. The structures of the as‐prepared samples agreed well with the standard phase of Sr₃(PO₄)₂, even when Dy³⁺ and Li⁺ were introduced. Under ultraviolet excitation at 350 nm, the Sr₃(PO₄)₂:Dy³⁺ sample exhibited two emission peaks at 483 nm and 580 nm, which were due to the ⁴F_9/2 → ⁶H_15/2 and ⁴F_9/2 → ⁶H_13/2 transitions of Dy³⁺ ions, respectively. A white light was fabricated using these two emissions from the Sr₃(PO₄)₂:Dy³⁺ phosphors. The luminescence properties of Sr₃(PO₄)₂:Dy³⁺,Li⁺ phosphors, including emission intensity and decay time, were improved remarkably with the addition of Li⁺ as the charge compensator, which would promote their application in near‐ultraviolet excited white‐light‐emitting diodes.     

Interactive effect of ammonia and crowding stress on ion-regulation and expression of immune-related genes in juvenile turbot (Scophthalmus maximus)

The present study was to investigate the interactive effect of ammonia and crowding stress on ion-regulation and expression of immune-related genes in juvenile turbot (Scophthalmus maximus). The fish were exposed to four total ammonia nitrogen (0, 5, 20, 40 mg/L TAN) and two stocking density. After 96 h of exposure, blood, gill, and liver samples were collected to measure biochemical parameters and mRNA levels of immune-related genes. The results showed that co-exposure to high TAN (20 and 40 mg/L) and high density significantly increased plasma sodium (Na⁺), gill Na⁺/K⁺-ATPase activity and mRNA levels. Following individual and combined exposure to high TAN and high density, the heat shock protein 70 (HSP 70), HSP 90, tumor necrosis factor-α (TNF-α), and interleukin-1β (IL-1β) genes expression were obviously higher than their control. Conversely, the lysozyme (LZM) and hepcidin mRNA levels were down-regulated in liver of fish exposed to high TAN alone and combination of high TAN-density. Moreover, glutathione S-transferase (GST) mRNA levels significantly increased in treatments with individual high TAN and high density, but decreased in high TAN-density co-exposed fish. Overall, ion homeostasis and immune status were adversely influenced in high exposed turbot under high density.     

Nitrite and nitroso compounds can serve as specific catalase inhibitors

Objective: We present evidence that nitrite and nitrosothiols, nitrosoamines and non-heme dinitrosyl iron complexes can reversibly inhibit catalase with equal effectiveness.

Methods: Catalase activity was evaluated by the permanganatometric and calorimetric assays.

Results: This inhibition is not the result of chemical transformations of these compounds to a single inhibitor, as well as it is not the result of NO release from these substances (as NO traps have no effect on the extent of inhibition). It was found that chloride and bromide in concentration above 80 mM and thiocyanate in concentration above 20 μM enhance catalase inhibition by nitrite and the nitroso compounds more than 100 times. The inhibition degree in this case is comparable with that induced by azide.

Discussion: We propose that the direct catalase inhibitor is a positively charged NO-group. This group acquires a positive charge in the active center of enzyme by interaction of nitrite or nitroso compounds with some enzyme groups. Halides and thiocyanate protect the NO⁺ group from hydration and thus increase its inhibition effect. It is probable that a comparatively low chloride concentration in many cells is the main factor to protect catalase from inhibition by nitrite and nitroso compounds.     

Oxidative Inactivation of Brain Alkaline Phosphatase Responsible for Hydrolysis of Phosphocholine

Abstract : Alkaline phosphatase, one of the enzymes responsible for the conversion of phosphocholine into choline, was purified from bovine brain membrane, where the phosphatase is bound as glycosylphosphatidylinositollinked protein, and subjected to oxidative inactivation. The phosphatase activity, based on the hydrolysis of p-nitrophenyl phosphate and phosphocholine, decreased slightly after the exposure to H₂O₂. Inclusion of Cu²⁺ in the incubation with 1 mM H₂O₂ led to a rapid decrease of activity in a time- and concentration-dependent manner. In comparison, the H₂O₂/Cu²⁺ system was much more effective than the H₂O₂/Fe²⁺ system in inactivating brain phosphatase. In a further study, it was observed that the hydroxy radical scavengers mannitol, ethanol, or benzoate failed to prevent against H₂O₂/Cu²⁺-induced inactivation of the phosphatase, excluding the involvement of extraneous hydroxy radicals in metalcatalyzed oxidation. In addition, it was found that both substrates, p-nitrophenyl phosphate and phosphocholine, and an inhibitor, phosphate ion, at their saturating concentrations exhibited a remarkable, although incomplete, protection against the inactivating action of H₂O₂/Cu²⁺. A similar protection was also expressed by divalent metal ions such as Mg²⁺ or Mn²⁺. Separately, it was found that H₂O₂/Fe²⁺-induced inactivation was prevented by p-nitrophenyl phosphate or Mg²⁺ but not phosphate ions. Thus, it is implied that phosphocholine-hydrolyzing alkaline phosphatase in brain membrane might be one of enzymes susceptible to metal-catalyzed oxidation.     

The Effect of Cadmium on Photosystem II in Spinach Chloroplasts

Cadmium ions, as an environmental pollution factor, significantly inhibited the photosynthesis especially, photosystem Ⅱ activity in isolated spinach chloroplasts. The presence of 5 mmol/l Cd2+ inhibited the O2-evolution to 53%. Cd2+ reduced the activity of photoreduction of DCIP and the variable fluorescence of chloroplasts and PSⅡ preparation. The inhibited DCIP photoreduction activity could only be restored slightly by the addition of an artificial electron donor of PSII, DPC, and the inhibited variable fluorescence could not be obviously recovered by the addition of NH2OH, another artificial electron donor of PSⅡ. It is considered that, besides the oxidizing side of PSI1, Cd2+ could also inhibit directly the PSⅡ reaction center. The inhibitory effect of Cd2+ on the whole chain electron transport (H2O→MV) was more serious than on O2-evolution (H2O→DCMU). It is suggested that the oxidizing side of PSⅡ is not the only site for Cd2+ action. There may be another site inhibited by Cd2+ in the electron transport chain between PSⅠ and PSⅡ.     

Further characterization of tunicate and tunichrome electrochemiluminescence

Electrophoresis, size exclusion chromatography, fluorescence, and electrochemiluminescence (ECL) data obtained from the cell-rich perivisceral fluid (‘blood’) of the tunicate, Molgula occidentalis, suggests that a yellowish protein or protein subunit of <6.5 kDa is probably responsible for the low level intrinsic ECL reported previously. Variable potential ECL scans and spectrofluorometric analyses directly indicated that chlorophylls from ingested phytoplankton were not significant contaminants of M. occidentalis blood samples. Chlorophylls were also examined indirectly from monocultures of dinoflagellate and diatom species to determine their ECL levels. While ECL was observed in these cultures, high concentrations (10⁶ organisms/mL) of dinoflagellates or diatoms were needed to produce ECL levels comparable to those observed for M. occidentalis blood. Additional work with two synthetic tunichrome isomers indicated 10-fold increases in ECL when the ‘2,3,4-tunichrome’ was reacted with Tl⁺ in 1:1 and 1:2 metal ion to ligand molar ratios. The ‘3,4,5-tunichrome’ isomer exhibited a titration curve with Hg²⁺ suggestive of the existence of at least two Hg²⁺ binding sites and a 9- to 10-fold increase in ECL output. © 1997 John Wiley & Sons, Ltd.     

Preliminary studies of the enhanced electrochemiluminescence of 2,3-diaminonaphthalene in the presence of specific metal ions

Electrochemiluminescence (ECL) of 200 ppm 2,3-diaminonaphthalene (2,3-DAN) was studied alone and in conjunction with 100 ppm of 34 different metal and non-metal ions and revealed three relatively intense ECL responses from interactions of 2,3-DAN with Au⁺, Fe⁺³ and V⁺⁵. ECL responses from Cr⁺⁶ or Ru⁺³ with 2,3-DAN were less intense, but noteworthy, as was the coloured fluorescent product of the non-metal ion Se⁺⁴ interaction with 2,3-DAN. Several intense 2,3-DAN–metal ion ECL reactions were studied in greater detail and revealed various titration curves with ionic detection limits in the low ppm range, using a fixed level (200 ppm) of 2,3-DAN. © 1998 John Wiley & Sons, Ltd.     

Limnological Characteristics of 70 Lakes Spanning Arctic Treeline from Coronation Gulf to Great Slave Lake in the Central Northwest Territories,Canada

Latitudinal transects across subpolar ecozones display striking changes in lakewater chemistry reflecting steep gradients in vegetation, climate, and other variables. This paper explores the relationships among chemical and physical lakewater characteristics of 70 lakes spanning arctic treeline in Canada's Central Northwest Territories. Principal components analysis (PCA) was used to examine trends and relationships among environmental variables and these 70 sites. In general, lakes in this data set were dilute, slightly acidic to slightly alkaline, and nutrient-poor. However, a strong trend toward more concentrated lakewater conditions in densely forested areas was observed relative to tundra regions. Interrelationships among measured limnological variables appear to be strongly influenced by catchment characteristics associated with proximity of sites to treeline.     

The self-assembly and liquid crystal formation of d(GpGpApGpG)

d(GpGpApGpG) dissolved in water forms liquid crystalline phases of the cholesteric and hexagonal type. The building blocks of the phases are columnar four-stranded aggregates composed of G quartets. The diameter of the columnar aggregates is larger and the melting temperature is lower than for homoguanylic derivatives, indicating a distorsion and loss of stability due to the presence of adenines, which do not form a hydrogen-bonded quartet. The present study shows that, as often observed for single crystals, it is possible also in the liquid crystalline phase to construct long columnar aggregates composed of shorter segments of the blunt-end type in a “closed architecture.” © 1997 John Wiley & Sons, Inc. Biopoly 42: 561–574, 1997