In situ measurement of free zinc in an ischemia model and cell culture using a ratiometric fluorescence-based biosensor

Zeng, Huihui; Bozym, Rebecca A.; Rosenthal, Robert; Fiskum, Gary; Cotto-Cumba, Cynthia; Westerberg, Nissa; Fierke, Carol A.; Stoddard, Andrea K.; Cramer, Michele; Frederickson, Christopher J.; Thompson, Richard B.

doi:10.1117/12.614168

Cited by 7 publications

(4 citation statements)

References 30 publications

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“…Additionally, iron is the most abundant metal in HDAC8-His, immediately following the metal affinity column (Table ); the bound nickel is likely derived from the metal affinity column. In bacteria and in mammalian cells, where HDAC8 is naturally expressed, the concentration of labile iron is estimated to be orders of magnitude greater than that of labile zinc (nM to μM vs fM to pM), and this intracellular iron is primarily present as Fe(II) ( − ). Assuming that the identity of the catalytic metal bound to HDAC8 is under thermodynamic control, these results suggest that HDAC8 may use Fe(II) as a metal cofactor in vivo rather than Zn(II).…”

Section: Discussionmentioning

confidence: 99%

Catalytic Activity and Inhibition of Human Histone Deacetylase 8 Is Dependent on the Identity of the Active Site Metal Ion

2006

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Histone deacetylases play a key role in regulating transcription and other cellular processes by catalyzing the hydrolysis of epsilon-acetyl-lysine residues. For this reason, inhibitors of histone deacetylases are potential targets for the treatment of cancer. A subset of these enzymes has previously been shown to require divalent metal ions for catalysis. Here we demonstrate that histone deacetylase 8 (HDAC8) is catalytically active with a number of divalent metal ions in a 1:1 stoichiometry with the following order of specific activity: Co(II) > Fe(II) > Zn(II) > Ni(II). The identity of the catalytic metal ion influences both the affinity of the HDAC inhibitor suberoylanilide hydroxamic acid (SAHA) and the Michaelis constant, with Fe(II)- and Co(II)-HDAC8 having K(M) values that are over 5-fold lower than that of Zn(II)-HDAC8. These data suggest that Fe(II), rather than Zn(II), may be the in vivo catalytic metal. In further support of this hypothesis, recombinant HDAC8 purified from E. coli contains 8-fold more iron than zinc before dialysis, and the HDAC8 activity in cell lysates is oxygen-sensitive. Identification of the in vivo metal ion of HDAC8 is essential for understanding the biological function and regulation of HDAC8 and for the development of improved inhibitors of this class of enzymes.

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Section: Discussionmentioning

confidence: 99%

Catalytic Activity and Inhibition of Human Histone Deacetylase 8 Is Dependent on the Identity of the Active Site Metal Ion

2006

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“…Most indicators are fluorescent ligands that change their emission properties upon complexation. Ratiometric fluorescent indicators have been developed for Zn(II) and Ca(II); [18][19][20][21][22][23][24][25][26][27] however, fluorescence methods are often less amenable for metal ions like Cu(II) that quench fluorescence. 28,29 We are developing ratiometric fluorescent indicators that overcome the quenching properties of these metal ions using the thermal phase transition of poly(N-isopropylacrylamide) (polyNIPAM).…”

Section: Introductionmentioning

confidence: 99%

Intermolecular approach to metal ion indicators based on polymer phase transitions coupled to fluorescence resonance energy transfer

Yao

Jones

et al. 2012

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An approach to ratiometric fluorescence detection of quenching metal ions was devised by copolymerizing N-isopropylacrylamide with small percentages of bipyridine and amine monomers. The copolymer was divided into two portions. The amine group on one portion was functionalized with AlexaFluor555 (donor fluorophore) and the other with AlexaFluor647 (acceptor fluorophore). The indicator consists of a mixture of these two portions. Aggregation above the lower critical solution temperature (LCST) of this copolymer brings about a large increase in fluorescence resonance energy transfer (FRET). Addition of Cu(II) and other complexing metal ions to the aggregated copolymer introduces charge onto the backbone, causing the copolymer to redissolve with a resulting decrease in FRET. The ratio of acceptor to donor fluorescence varies with Cu(II) concentration. A plot of intensity ratio vs. pCu is sigmoidal with a log K(f) of 6.1 for the Cu(II)-bipyridine complex. The data are consistent with the formation of a 1 : 1 complex. The copolymer responds to higher concentrations of other transition metal ions. The selectivity for Cu(II) is consistent with the literature values for 1 : 1 formation constants for bipyridine with metal ions.

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“…For instance, the wild type zinc ion binding site exhibits picomolar affinity in the example shown in Figure 3, but a protein variant can be substituted having different affinity (44) or selectivity (B. McCranor, et al, submitted), or metal binding kinetics (42) depending upon the particular circumstances and application. Our distinguished colleagues have demonstrated an infrared ratiometric fluorescent zinc sensor (45) with high affinity and selectivity, but changing its affinity, selectivity, or kinetics requires resynthesizing the molecule.…”

Section: Resultsmentioning

confidence: 99%

Long Wavelength Fluorescence Ratiometric Zinc Biosensor

et al. 2013

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A protein-based emission ratiometric fluorescence biosensor is described that exhibits sensitivity to free zinc ion solutions down to picomolar concentrations. Ratiometric measurements are widely used to assure accurate quantitation, and emission ratios are preferred for laser scanning microscopes such as confocal fluorescence microscopes. The relatively long emission wavelengths used are well suited to studies in tissues and other matrices which exhibit significant fluorescence background, and the apo-carbonic anhydrase moiety recognizes zinc ion with high and controllable specificity.

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In situ measurement of free zinc in an ischemia model and cell culture using a ratiometric fluorescence-based biosensor

Cited by 7 publications

References 30 publications

Catalytic Activity and Inhibition of Human Histone Deacetylase 8 Is Dependent on the Identity of the Active Site Metal Ion

Catalytic Activity and Inhibition of Human Histone Deacetylase 8 Is Dependent on the Identity of the Active Site Metal Ion

Intermolecular approach to metal ion indicators based on polymer phase transitions coupled to fluorescence resonance energy transfer

Long Wavelength Fluorescence Ratiometric Zinc Biosensor

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