Zinc biosensing with multiphoton excitation using carbonic anhydrase and improved fluorophores

Abstract

Previously, we had shown that the zinc-dependent binding of certain fluorescent aryl sulfonamide inhibitors could be used with apocarbonic anhydrase II to transduce the level of free zinc as a change in the fluorescence of the inhibitor. While inhibitors such as dansylamide, ABD-M, and ABD-N made possible quantitation of free zinc in the picomolar range with high selectivity, they have only modest absorbance which limits their utility. We describe here the synthesis and properties of two new probes, Dapoxyl sulfonamide and BTCS, and their use in zinc biosensing. Dapoxyl sulfonamide exhibits a dramatic increase and blue shift in its emission upon binding to holocarbonic anhydrase II, as well as a 20-fold increase in lifetime: it is thus well suited for quantitating free Zn(II) down to picomolar ranges. The anisotropy of BTCS increases fivefold upon binding to the holoprotein, making this probe well suited for anisotropy-based determination of zinc. BTCS and ABD-N are efficiently excited with two photon excitation using 1.5 ps pulses from a titanium sapphire laser, and exhibit the increased zinc-dependent anisotropy response anticipated on the basis of photoselection.

Genetically encoded ratiometric biosensors to measure intracellular exchangeable zinc in Escherichia coli

“…Dapoxyl sulfonamide was confirmed by comparison to known samples using thin layer silica chromatography (R f ∼0.8), liquid chromatography-mass spectrometry [LC-MS: m/z (M + H) single peak at 344.0] and fluorescence spectroscopy with an emission peak at 535 nm when excited at 350 nm in the presence of zinc-bound wild type carbonic anhydrase. 24 …”

“…This procedure also provides time for cells to adjust their zinc concentrations to accommodate the expressed zinc sensors. To verify that the signal increase was specifically caused by DPS binding to holo-CA, ethoxzolamide, a nonfluorescent inhibitor with a much higher affinity for holo-CA affinity (dissociation constant of 1.6 nm compared to 0.1 μM for DPS 24,35 ) was added to the DPS-treated cells. Upon addition of ethoxzolamide, the FRET channel emission decreased and the I FRET /I FP ratio returned to the baseline level (0.19 ± 0.03) [ Fig.…”

“…These inhibitors selectively bind to zinc-bound CAII and exhibit a large increase in quantum yield and a blueshift in the emission spectra, for example, dapoxyl sulfonamide (DPS) undergoes a >100-fold increase in quantum yield upon binding to holocarbonic anhydrase (CA). 24 A Förster resonance energy transfer (FRET)-based ratiometric zinc sensing scheme has previously been developed by covalently labeling CAII with a second fluorophore (AlexaFluor 594) that functions as a FRET acceptor to CA-bound DPS. 25 This sensor was delivered into PC-12 cells using a cell permeating peptide tag to measure the intracellular free zinc concentration in this mammalian cell line.…”

Protein-Based Biosensors with Polarization Transduction

“…An important potential advantage is that polarization requires no change ofexcitation or emission wavelength, making it particularly well-suited for laser-excited microscopies such as confocal microscopy and multiphoton excitation microscopy. While few reports have appeared describing multiphoton excited fluorescence anisotropy sensing (Thompson, Maliwal, & Zeng, 2000), this technique could potentially be incorporated into an imaging application as well. While filtration to avoid scattered light is important, it is a further advantage that one may use the entire emission band for anisotropy measurements instead of a narrow band for wavelength ratiometric measurements.…”

Carbonic Anhydrase and Heavy Metals

“…In particular, Cu 2+ , Co 2+ , Zn 2+ , Cd 2+ , and Ni 2+ have been determined at concentration down the picomolar range (Fierke and Thompson, 2001;Thompson and Jones, 1993;Mey et al, 2011) by changes in fluorescence emission (Thompson et al, 2000) and excitation wavelength ratios (Thompson et al, 2002), lifetimes (Thompson and Patchan, 1995), and anisotropy (polarization) (Elbaum et al, 1996;Thompson et al, 2000). The sensitivity, selectivity, analyte binding, kinetics and stability of the biosensors have been improved by subtle modification of the protein structure by directed mutagenesis (Kiefer et al, 1995;Hunt et al, 1999;DiTusa et al, 2001;McCall et al, 2004;Burton et al, 2000).…”