Chromogenic Chemodosimeter Based on Capped Silica Particles to Detect Spermine and Spermidine

A new hybrid organic–inorganic material for sensing spermine (Spm) and spermidine (Spd) has been prepared and characterized. The material is based on MCM-41 particles functionalized with an N-hydroxysuccinimide derivative and loaded with Rhodamine 6G. The cargo is kept inside the porous material due to the formation of a double layer of organic matter. The inner layer is covalently bound to the silica particles, while the external layer is formed through hydrogen and hydrophobic interactions. The limits of detection determined by fluorimetric titration are 27 µM and 45 µM for Spm and Spd, respectively. The sensor remains silent in the presence of other biologically important amines and is able to detect Spm and Spd in both aqueous solution and cells.

Binding of a tetracationic meso-porphyrin to polyadenylic acid: a spectroscopic study

Binding of a tetracationic porphyrin (TMPyP4+) to poly(rA) has been studied in neutral buffered solution of low ionic strength in a wide range of molar phosphate-to-dye ratios (P/D) using absorption spectroscopy, polarized fluorescence and fluorimetric titration. Two competitive binding modes were identified: partial intercalation of porphyrin chromophores between adenine bases prevailing at P/D > 20 and its outside binding to poly(rA) backbone dominating at P/D < 6. Both of them were accompanied by enhancement of the porphyrin emission. Absence of the emission quenching near stoichiometric P/D ratios allowed us to assume that external binding occurs without the self-stacking of the porphyrin chromophores.

The labile zinc pool in plant cells

Zinc is the most abundant and important transition metal in plants; however, the dynamic aspects of zinc homeostasis in plant cells are poorly understood. In this study we explored the pool of labile exchangeable zinc complexes in plant cells, and the potential influence of changes in intracellular zinc availability on cellular physiology. Work was performed on cultivated cell extracts of Arabidopsis thaliana (L.) Heynh. and Thellungiella salsuginea (Pall.) O.E. Schulz grown under control (3.48 µM Zn2+), 10-fold Zn excess or Zn starvation conditions. The free and labile Zn contents in the extracts were then determined by fluorimetric titration. We observed for the first time that plant cells contain micromolar concentrations of labile zinc complexes that account for a low percentage of the total zinc content. Labile zinc is mainly protein bound. Zn starvation inhibits cell proliferation and leads to the disappearance of the labile zinc pool, whereas Zn excess drastically increases the labile zinc pool. Free Zn2+ is buffered at picomolar concentrations in the intracellular milieu, and the increase in free Zn2+ concentrations to low nanomolar values clearly modulates enzyme activity by direct reversible binding. Such increases in free Zn2+ can be achieved by the substantial influx of additional zinc or by the oxidation of zinc-binding thiols. The observed features of the labile zinc pool in plant cells suggest it has a role in intracellular zinc trafficking and zinc signalling.

Electronic absorption and fluorescence spectra of 2-phenyl-substituted benzothiazoles: study of excited-state proton transfer reactions

The absorption and fluorescence spectral properties of 2-(3′-hydroxyphenyl)-, 2-(4′-hydroxyphenyl)-, and 2-(3′,4′-dihydroxyphenyl)-, and 2-(4′-hydroxy-3′-methoxyphenyl), 2-(3′-hydroxy-4′-methoxyphenyl)-, and 2-(3′-methoxyphenyl)benzothiazoles have been studied in a number of solvents of varying polarity. The ionization constants (pKa) for various prototropic reactions of these molecules in both S0 and S1 states are determined. The effect of substitution on the spectral properties and on the pKa values are discussed. The molecules have been found to undergo biprotonic phototautomerism in dilute acid solutions. On the basis of the fluorimetric titration behaviour of the molecules (except 2-(3′-methoxyphenyl)benzothiazole), the existence of monocation–zwitterion equilibrium in the S1 state is proposed. PPP-SCF-MO-CI method has been used to calculate charge densities on the heteroatoms. Key words: spectra, proton transfer.

Separate binding sites for antimycin and mucidin in the respiratory chain of the bacterium Paracoccus denitrificans and their occurrence in other denitrificans bacteria

By means of the method of fluorimetric titration it has been shown that mucidin does not affect the attachment of antimycin to membranes from anaerobically grown Paracoccus denitrificans. The fluorimetric titration with antimycin can be used in the determination of the amount of the cytochrome bc1 complex in the membrane. In cells inhibited with antimycin, the oxidation of cytochromes c was accompanied by the reduction of cytochrome b; in the presence of mucidin this effect did not take place. The results, which indicated a difference in binding sites, were interpreted in terms of the Q-cycle [Mitchell (1976) J. Theor. Biol. 62, 327-367; Trumpower (1981) Biochim. Biophys. Acta 639, 129-155]. Comparable sensitivity towards antimycin and mucidin was shown by other typical denitrifying bacteria: Pseudomonas stutzeri and Alcaligenes xylosoidans, subspecies denitrificans.

Investigation of the binding of Ca2+, Mg2+, Mn2+ and K+ to the vitamin D-dependent Ca2+-binding protein from pig duodenum

The cation-binding properties of the vitamin D-dependent Ca2+-binding protein from pig duodenum were investigated, mainly by flow dialysis. The protein bound two Ca2+ ions with high affinity, and Mg2+, Mn2+ and K+ were all bound competitively with Ca2+ at both sites. The sites were distinguished by their different affinities for Mn2+, the one with the higher affinity being designated A (Kd 0.61 +/- 0.02 microM) and the other B (Kd 50 +/- 6 microM). Competitive binding studies allied to fluorimetric titration with Mg2+ showed that site A bound Ca2+, Mg2+ and K+ with Kd values of 4.7 +/- 0.8 nM, 94 +/- 18 microM and 1.6 +/- 0.3 mM respectively, and site B bound the same three cations with Kd values of 6.3 +/- 1.8 nM, 127 +/- 38 microM and 2.1 +/- 0.6 mM. For the binding of these cations, therefore, there was no significant difference between the two sites. In the presence of 1 mM-Mg2+ and 150 mM-K+, both sites bound Ca2+ with an apparent Kd of 0.5 microM. The cation-binding properties were discussed relative to those of parvalbumin, troponin C and the vitamin D-dependent Ca2+-binding protein from chick duodenum.