A cuvette-compatible Zn2+ sensing tool for conventional spectrofluorometers prepared by copolymerization of macrocyclic fluoroionophores on quartz glass surface.

We report here the development of a surface-modified quartz glass sheet, which affords an opportunity for converting conventional spectrofluorometers to ion-selective optochemical sensors by placing it diagonally into a photometric cuvette. Moreover, we describe a generalizable technique, which allows the usage of any polymerizable ionophores for developing multiple-use fluorescent chemosensors of various selectivity. A fluorescent bis(acridino)-crown ether containing allyl groups was photocatalytically copolymerized with a methacrylate-acrylamide-based monomer mixture to obtain an ion-selective sensor membrane layer on the surface of the cuvette-compatible glass sheet. This glass membrane-based direct optode enabled the analysis of Zn2+above a lower limit of detection of 2.2 × 10-7mol·l-1with an excellent reusability. Limiting factors, like pH and competing ionic or organic agents were thoroughly investigated. Moreover, spiked river-water samples were measured to demonstrate applicability. The proposed sensor placed in any conventional spectrofluorometer provides an innovative method for perturbation-free analysis of Zn2+for all the chemists in need of a fast, easy-to-use, portable and regenerable analyzer without the requirement of an analyte-specific instrumentation.

Beyond Chelation: EDTA Tightly Binds Taq DNA Polymerase, MutT and dUTPase and Directly Inhibits dNTPase Activity.

EDTA is commonly used as an efficient chelator of metal ion enzyme cofactors. It is highly soluble, optically inactive and does not interfere with most chemicals used in standard buffers making EDTA a common choice to generate metal-free conditions for biochemical and biophysical investigations. However, the controversy in the literature on metal-free enzyme activities achieved using EDTA or by other means called our attention to a putative effect of EDTA beyond chelation. Here, we show that EDTA competes for the nucleotide binding site of the nucleotide hydrolase dUTPase by developing an interaction network within the active site similar to that of the substrate. To achieve these findings, we applied kinetics and molecular docking techniques using two different dUTPases. Furthermore, we directly measured the binding of EDTA to dUTPases and to two other dNTPases, the Taq polymerase and MutT using isothermal titration calorimetry. EDTA binding proved to be exothermic and mainly enthalpy driven with a submicromolar dissociation constant considerably lower than that of the enzyme:substrate or the Mg:EDTA complexes. Control proteins, including an ATPase, did not interact with EDTA. Our findings indicate that EDTA may act as a selective inhibitor against dNTP hydrolyzing enzymes and urge the rethinking of the utilization of EDTA in enzymatic experiments.