Turn-on, photostable, nontoxic and specific, iron(II) sensor.

The pressing need to develop a specific analytical sensor that can identify and quantify Fe(II) without a cytotoxic response was the major motivation drive in this work. The turn-on fluorescent sensor here described can successfully detect Fe(II) and discriminate this ion from other analytes that commonly act as interferents in biological media. Moreover, this reduced fluoresceinamine-based sensor has a high photostability and high dissociation constant, which is an indication that the complex obtained between reduced fluoresceinamine (RFL) and Fe(II) is highly stable. This fluorescence-based sensor has a binding mechanism of 1:1 and a positive cooperativity was found between analyte and sensor. The detection, quantification and sensitivity parameters of the sensor were determined: 21.6 ± 0.1 µM; 65.6 ± 0.1 µM and 48 ± 3 (×107) µM, respectively. To evaluate a possible cytotoxicity effect an erythrocyte assay was performed and the obtained data were evaluated considering CdTe Quantum Dots (QDs) passivated with mercaptoacetic acid has experimental control. According to the resulting data RFL is not cytotoxic even when used in high concentrations, 660 mM. On the other hand QDs are quite different. Indeed it was proven that these heavy metal-based nanoparticles are responsible for 40% erytrocytes hemolysis in concentrations of 600 mM.

Enzymatic synthesis of mono and dinucleoside polyphosphates.

BACKGROUND: Mono and dinucleoside polyphosphates (p(n)Ns and Np(n)Ns) exist in living organisms and induce diverse biological effects through interaction with intracellular and cytoplasmic membrane proteins. The source of these compounds is associated with secondary activities of a diverse group of enzymes. SCOPE OF REVIEW: Here we discuss the mechanisms that can promote their synthesis at a molecular level. Although all the enzymes described in this review are able to catalyse the in vitro synthesis of Np(n)Ns (and/or p(n)N), it is not clear which ones are responsible for their in vivo accumulation. MAJOR CONCLUSIONS: Despite the large amount of knowledge already available, important questions remain to be answered and a more complete understanding of p(n)Ns and Np(n)Ns synthesis mechanisms is required. With the possible exception of (GTP:GTP guanylyltransferase of Artemia), all enzymes able to catalyse the synthesis of p(n)Ns and Np(n)Ns are unspecific and the factors that can promote their synthesis relative to the canonical enzyme activities are unclear. GENERAL SIGNIFICANCE: The fact that p(n)Ns and Np(n)Ns syntheses are promiscuous activities of housekeeping enzymes does not reduce its physiological or pathological importance. Here we resume the current knowledge regarding their enzymatic synthesis and point the open questions on the field.

Pyrophosphate and tripolyphosphate affect firefly luciferase luminescence because they act as substrates and not as allosteric effectors.

The activating and stabilizing effects of inorganic pyrophosphate, tripolyphosphate and nucleoside triphosphates on firefly luciferase bioluminescence were studied. The results obtained show that those effects are a consequence of the luciferase-catalyzed splitting of dehydroluciferyl-adenylate, a powerful inhibitor formed as a side product in the course of the bioluminescence reaction. Inorganic pyrophosphate, tripolyphosphate, CTP and UTP antagonize the inhibitory effect of dehydroluciferyl-adenylate because they react with it giving rise to products that are, at least, less powerful inhibitors. Moreover, we demonstrate that the antagonizing effects depended on the rate of the splitting reactions being higher in the cases of inorganic pyrophosphate and tripolyphosphate and lower in the cases of CTP and UTP. In the case of inorganic pyrophosphate, the correlation between the rate of dehydroluciferyl-adenylate pyrophosphorolysis and the activating effect on bioluminescence only occurs for low concentrations because inorganic pyrophosphate is, simultaneously, an inhibitor of the bioluminescence reaction. Our results demonstrate that previous reports concerning the activating effects of several nucleotides (including some that do not react with dehydroluciferyl-adenylate) on bioluminescence were caused by the presence of inorganic pyrophosphate contamination in the preparations used.

Firefly luciferase produces hydrogen peroxide as a coproduct in dehydroluciferyl adenylate formation.

Firefly luciferase catalyzes the synthesis of H2O2 from the same substrates as the bioluminescence reaction: ATP and luciferin (D-LH2). About 80% of the enzyme-bound intermediate D-luciferyl adenylate (D-LH2-AMP) is oxidized into oxyluciferin, and a photon is emitted during this reaction. The enzyme pathway responsible for the generation of H2O2 is a side reaction in which D-LH2-AMP is oxidized into dehydroluciferyl adenylate (L-AMP). Like the bioluminescence reaction, the luciferase-catalyzed synthesis of H2O2 and L-AMP is a stereospecific process, involving only the natural D enantiomer. However, the intramolecular electron transfer postulated as essential to the light emission process is not involved in this side reaction.

Coenzyme A affects firefly luciferase luminescence because it acts as a substrate and not as an allosteric effector.

The effect of CoA on the characteristic light decay of the firefly luciferase catalysed bioluminescence reaction was studied. At least part of the light decay is due to the luciferase catalysed formation of dehydroluciferyl-adenylate (L-AMP), a by-product that results from oxidation of luciferyl-adenylate (LH2-AMP), and is a powerful inhibitor of the bioluminescence reaction (IC50 = 6 nm). We have shown that the CoA induced stabilization of light emission does not result from an allosteric effect but is due to the thiolytic reaction between CoA and L-AMP, which gives rise to dehydroluciferyl-CoA (L-CoA), a much less powerful inhibitor (IC50 = 5 microm). Moreover, the V(max) for L-CoA formation was determined as 160 min(-1), which is one order of magnitude higher than the V(max) of the bioluminescence reaction. Results obtained with CoA analogues also support the thiolytic reaction mechanism: CoA analogues without the thiol group (dethio-CoA and acetyl-CoA) do not react with L-AMP and do not antagonize its inhibitor effect; CoA and dephospho-CoA have free thiol groups, both react with L-AMP and both antagonize its effect. In the case of dephospho-CoA, it was shown that it reacts with L-AMP forming dehydroluciferyl-dephospho-CoA. Its slower reactivity towards L-AMP explains its lower potency as antagonist of the inhibitory effect of L-AMP on the light reaction. Moreover, our results support the conjecture that, in the bioluminescence reaction, the fraction of LH2-AMP that is oxidized into L-AMP, relative to other inhibitory products or intermediates, increases when the concentrations of the substrates ATP and luciferin increases.

Identification of luciferyl adenylate and luciferyl coenzyme a synthesized by firefly luciferase.

The firefly luciferase reaction intermediate luciferyl adenylate was detected by RP-HPLC analysis when the luciferase reaction was performed under a nitrogen atmosphere. Although this compound is always specified as an intermediate in the light-production reaction, this is the first report of its identification by HPLC in a luciferase assay medium. Under a low-oxygen atmosphere, luciferase can catalyze the synthesis of luciferyl coenzyme A from luciferin, ATP, and coenzyme A, but in air dehydroluciferyl coenzyme A was produced. The luciferase-catalyzed synthesis of these coenzyme A derivatives may be a consequence of the postulated recent evolutionary origin of firefly luciferases from an ancestral acyl-coenzyme A synthetase.

pH opposite effects on synthesis of dinucleoside polyphosphates and on oxidation reactions catalyzed by firefly luciferase.

Previous results have shown that an oxidizing product of firefly luciferin, dehydroluciferyl-adenylate, is the main intermediate in the process of synthesis of dinucleoside polyphosphates catalyzed by firefly luciferase (EC 1.13.12.7). However, we have found that the pH effects on the luciferase oxidizing processes and on the synthesis of dinucleoside polyphosphate are opposite: acidic assay media enhance the synthesis of dinucleoside polyphosphate and inhibit the oxidizing processes. The reason for this apparent contradiction lies on the activation effect of low pH on the adenylate transfer reaction from dehydroluciferyl-adenylate to the acceptor nucleotide.