Tris(2-pyridylmethyl)amine (TPA) as a membrane-permeable chelator for interception of biological mobile zinc

Abstract

We report the characterization of tris(2-pyridylmethyl)amine (TPA) as a membrane-permeable zinc chelator for intercepting biological mobile zinc. Compared to N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN), TPA chelates zinc with faster kinetics in cuvettes, live cells, and brain slices. TPA also is generally less toxic than TPEN in cell culture. Mechanistic analysis indicates that these improvements arise from both the electronic and steric properties of TPA including weaker metal-binding affinity, lower pKa, and smaller size. These results demonstrate that TPA chelation is a valuable addition to the methodologies available for investigating mobile zinc in biology.

Fig. 1

Addition of 100 µM TPA (blue) or TPEN (red) to a 1.0 µM solution of Zn₂(ZP1) in pH 7.0 buffer containing 50 mM PIPES and 100 mM KCl at 25 °C resulted in decrease of fluorescence over time. Fluorescence emission at 527 nm was recorded and was normalized to unbound ZP1 levels.

Fig. 2

Representative stopped flow data for zinc reactions with TPA (top) and TPEN (bottom) in pH 7.0 buffers containing 50 mM PIPES and 100 mM KCl. The fraction of metal-bound chelator is plotted versus time. Black dots were derived from the measured absorbance at 268 nm, and red curves correspond to global fitting results using second-order kinetics. Data correspond to mixing 35 µM chelator with 40, 50, 62.5, 75, 100 µM ZnCl₂ (post-mixing concentrations).

Fig. 3

Eyring plots of (A) zinc removal from Zn₂(ZP1) by chelators, and (B) chelators reacting with zinc ions in pH 7.0 buffers. Blue: TPA; red: TPEN. In A, solid and empty diamonds correspond to removal of the first and second zinc ions, respectively.

Fig. 4

(A) Fluorescence images of ZP1-treated HeLa cells. Left to right: DIC images, ZP1 signals, ZP1 fluorescence 10 min after addition of 20 µM zinc pyrithione, and after addition of 100 µM chelator (top: TPA, bottom: TPEN) recorded at 2, 5, and 10 min. Scale bar = 25 µm. (B) The intracellular ZP1 signals were quantitated and normalized to the levels before zinc addition. Signals from 8–12 cells in four culture dishes were averaged. Error bars represent standard errors of the mean.

Fig. 5

(A) Selected images of acute hippocampal slices recorded with two-photon microscopy after treatment with blank ACSF media (control) or with 100 µM TPEN or TPA. Chelators were introduced at t = 0 min. Scale bar = 200 µm. (B) Normalized fluorescence intensities vs. time. n = 3 (control), 4 (TPEN) and 4 (TPA). Error bars represent standard errors of the mean.

Fig. 6

Survival percentage of HeLa cells after treatment with TPA or TPEN for 1 h (A) or 24 h (B), as quantified with MTT assay. Error bars represent standard errors of the mean.

Scheme 1

Structures of membrane-impermeable and membrane-permeable zinc chelators.

Scheme 2

Chelators quench zinc-induced fluorescence of ZP1 by removing zinc ions sequentially from the complex.