Ternary Zn(II) Complexes of Fluorescent Zinc Probes Zinpyr-1 and Zinbo-5 with the Low Molecular Weight Component of Exchangeable Cellular Zinc Pool.

The intracellular exchangeable Zn(II) is usually measured with synthetic fluorescent zinc sensors. 4',5'-Bis[bis(2-pyridylmethyl)aminomethyl]-2',7'-dichlorofluorescein (Zinpyr-1) is a sensor containing the fluorescein platform and a duplicated chelating unit. Its advantages include brightness and a relatively high affinity for Zn(II), Kd = 0.7 nM. 2-(4,5-Dimethoxy-2-hydroxyphenyl)-4-(2-pyridylmethyl)aminomethylbenzoxazole (Zinbo-5) is a member of a growing family of ratiometric synthetic Zn(II) probes, offering a possibility to determine Zn(II) concentration independently of the sensor concentration. Cells, however, contain high, millimolar or nearly millimolar concentrations of low molecular weight ligands (LMWLs) capable of binding Zn(II) ions. Previously, we demonstrated that such LMWLs can perturb the performance of some fluorescent zinc sensors by competition and formation of ternary Zn(sensor) (LMWL) complexes. Here we tested Zinpyr-1 and Zinbo-5 in this respect. Despite structural differences, both sensors formed such ternary complexes. We determined their stability constants CKtern and performed numerical simulations of Zn(II) distributions at physiological concentrations of selected LMWLs. Glutamic acid was found to provide the strongest ternary complexes with either of the studied sensors. Zn(Zinpyr-1)(Glu) was an absolutely dominant Zn(II)/Zinpyr-1 species (more than 96% of the exchangeable Zn(II)), and Zn(Zinbo-5)(Glu) was the most abundant one (more than 40%) in these simulations. Our results indicate that under cellular conditions these sensors are able to report Zn(II) complexed to LMWLs rather than free Zn2+ ions. On the other hand, the specific affinity of Zn(Zinpyr-1) and Zn(Zinbo-5) for Glu creates interesting opportunities for determining glutamic acid in biological samples.

Ternary Zn(II) Complexes of FluoZin-3 and the Low Molecular Weight Component of the Exchangeable Cellular Zinc Pool.

Knowledge of the nature of exchangeable (labile) intracellular Zn(II) is increasingly important for biomedical research. The detection and quantitative determination of Zn(II) ions is usually performed by using Zn(II)-specific fluorescent sensors, among which 2-[2-[2-[2-[bis(carboxylatomethyl)amino]-5-methoxyphenoxy]ethoxy]-4-(2,7-difluoro-3-oxido-6-oxo-4a,9a-dihydroxanthen-9-yl)anilino]acetate (FluoZin-3) has been used most widely. Selectivity of this sensor for Zn(II) over other divalent cations was demonstrated, but possible interference in its performance by other compounds has not been investigated. Many potential low molecular weight ligands for Zn(II) ions (LMWLs) are abundant in the cell. In this study we demonstrate that FluoZin-3 is susceptible to competition for Zn(II) from LMWLs and also forms strong ternary complexes with some of them. We determined the set of conditional stability constants C Ktern for ternary Zn(FluoZin-3)(LMWL) complexes using fluorescence titrations and applied it to model the response of exchangeable zinc to FluoZin-3. We found that competition and formation of ternary complexes with LMWLs together strongly affect (net reduce) the Zn(FluoZin-3) fluorescence. This effect may cause a significant underestimation of exchangeable Zn(II). We also demonstrated a strong pH dependence of this effect. Reduced glutathione (GSH) emerged as the most important Zn(II) partner among the LMWLs, characterized with Ktern = 2.8 ± 0.2 × 106 M-1. Our experiments and calculations suggest that Zn(LMWL) complexes contribute to the exchangeable cellular zinc pool.

Transferrin as a drug carrier: Cytotoxicity, cellular uptake and transport kinetics of doxorubicin transferrin conjugate in the human leukemia cells.

Leukemias are one of most common malignancies worldwide. There is a substantial need for new chemotherapeutic drugs effective against this cancer. Doxorubicin (DOX), used for treatment of leukemias and solid tumors, is poorly efficacious when it is administered systemically at conventional doses. Therefore, several strategies have been developed to reduce the side effects of this anthracycline treatment. In this study we compared the effect of DOX and doxorubicin-transferrin conjugate (DOX-TRF) on human leukemia cell lines: chronic erythromyeloblastoid leukemia (K562), sensitive and resistant (K562/DOX) to doxorubicin, and acute lymphoblastic leukemia (CCRF-CEM). Experiments were also carried out on normal cells, peripheral blood mononuclear cells (PBMC). We analyzed the chemical structure of DOX-TRF conjugate by using mass spectroscopy. The in vitro growth-inhibition assay XTT, indicated that DOX-TRF is more cytotoxic for leukemia cells sensitive and resistant to doxorubicin and significantly less sensitive to normal cells compared to DOX alone. During the assessment of intracellular DOX-TRF accumulation it was confirmed that the tested malignant cells were able to retain the examined conjugate for longer periods of time than normal lymphocytes. Comparison of kinetic parameters showed that the rate of DOX-TRF efflux was also slower in the tested cells than free DOX. The results presented here should contribute to the understanding of the differences in antitumor activities of the DOX-TRF conjugate and free drug.

Oriented immobilization of His-tagged protein on a redox active thiol derivative of DPTA-Cu(II) layer deposited on a gold electrode--the base of electrochemical biosensors.

This paper concerns the development of an electrochemical biosensor for the determination of Aß(16-23') and A(ß1-40) peptides. The His-tagged V and VC1 domains of Receptor for Advanced Glycation end Products (RAGE) immobilized on a gold electrode surface were used as analytically active molecules. The immobilization of His6-RAGE domains consists of: (i) formation of a mixed layer of N-acetylcysteamine (NAC) and the thiol derivative of pentetic acid (DPTA); (ii) complexation of Cu(II) by DPTA; (iii) oriented immobilization of His6-RAGE domains via coordination bonds between Cu(II) sites from DPTA-Cu(II) complex and imidazole nitrogen atoms of a histidine tag. Each modification step was controlled by cyclic voltammetry (CV), Osteryoung square-wave voltammetry (OSWV), and atomic force microscopy (AFM). The applicability of the proposed biosensor was tested in the presence of human plasma, which had no influence on its performance. The detection limits for Aß(1-40) determination were 1.06 nM and 0.80 nM, in the presence of buffer and human plasma, respectively. These values reach the concentration level of Aß(1-40) which is relevant for determination of its soluble form in human plasma, as well as in brain. This indicates the promising future application of biosensor presented for early diagnosis of neurodegenerative diseases.