Structural and spectroscopic insight into the metal binding properties of the: O -aminophenol- N, N, O -triacetic acid (APTRA) chelator: Implications for design of metal indicators

Article abstract of DOI:10.1039/c6dt01557c

The o-aminophenol-N,N,O-triacetic acid (APTRA) chelator is employed extensively as a metal-recognition moiety in fluorescent indicators for biological free Mg²⁺, as well as in low-affinity indicators for the detection of high levels of cellular Ca²⁺. Despite its widespread use in sensor design, the limited metal selectivity of this chelating moiety can lead to binding of competing cations that complicate the fluorescence-based detection of metals of interest in complex samples. Reported herein are the structural characterization of APTRA complexes with various biologically relevant cations, and the thermodynamic analysis of complex formation with Mg²⁺, Ca²⁺ and Zn²⁺. Our results indicate that the low affinity of APTRA for Mg²⁺, which makes it a suitable metal-recognition moiety for sensitive analysis of typical millimolar levels of this metal in cells, stems from a much higher enthalpic cost of Mg²⁺ binding compared to that of other cations. The results are discussed in the context of indicator design, highlighting the aspects that may aid the future development of fluorescent sensors with enhanced metal selectivity profiles.

Full text of DOI:10.1039/c6dt01557c

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PAPER
Structural and spectroscopic insight into the
metal binding properties of the o-aminophenol-
N,N,O-triacetic acid (APTRA) chelator: implications
for design of metal indicators†
Cite this: DOI: 10.1039/c6dt01557c
Michael Brady, Sebastian D. Piombo, Chunhua Hu and Daniela Buccella*
The o-aminophenol-N,N,O-triacetic acid (APTRA) chelator is employed extensively as a metal-reco-
gnition moiety in fluorescent indicators for biological free Mg2+, as well as in low-affinity indicators for
the detection of high levels of cellular Ca2+. Despite its widespread use in sensor design, the limited metal
selectivity of this chelating moiety can lead to binding of competing cations that complicate the fluor-
escence-based detection of metals of interest in complex samples. Reported herein are the structural
characterization of APTRA complexes with various biologically relevant cations, and the thermodynamic
analysis of complex formation with Mg2+, Ca2+ and Zn2+. Our results indicate that the low affinity of
APTRA for Mg2+, which makes it a suitable metal-recognition moiety for sensitive analysis of typical milli-
molar levels of this metal in cells, stems from a much higher enthalpic cost of Mg2+ binding compared to
that of other cations. The results are discussed in the context of indicator design, highlighting the aspects
that may aid the future development of fluorescent sensors with enhanced metal selectivity profiles.
Received 22nd April 2016,
Accepted 5th July 2016
DOI: 10.1039/c6dt01557c
www.rsc.org/dalton
Introduction
The development of colorimetric and fluorescent metal-
responsive indicators has had a great impact in the study of
the cell biology of metals, enabling the visualization of metal
cations in the cellular milieu by non-destructive microscopy
techniques. Typical indicators for metal ions combine a metal
chelator with a fluorophore or chromophore that transduces a
metal binding event into an optical readout.1 The o-amino-
phenol-N,N,O-triacetic acid (APTRA) chelator2–4 (Fig. 1) has
been employed extensively as a metal-recognition moiety in
fluorescent indicators designed for the study of the physiologi-
cal levels of Mg2+, e.g. FURAPTRA or Mag-Fura-2,5 as well as in
Fig. 1 APTRA recognition unit and an example of an APTRA-based flu-
‘low affinity’ fluorescent indicators for the detection of high orescent sensor for Mg2+ detection.
levels of Ca2+.6–8,9 Less often, fluorescent indicators based on
this chelator have found use in the detection of high levels of
free Zn2+, particularly in biological systems that are rich in this
based on this metal-recognition moiety requires some previous
metal cation such as neurons.10–12 Because of the limited
selectivity of APTRA among these physiologically relevant di-
valent cations, accurate determination of Mg2+ with indicators
knowledge of the levels of competing ions in the system, and
the use of Ca2+- and Zn2+-specific chelators for their sequestra-
tion. Whereas this practice may be suitable for the study of Mg2+
in the presence of low concentrations of competing ions, it is
not of general applicability to metal-rich cell types and tissues.
On the other hand, the use of APTRA-based indicators for the
detection of less abundant Ca2+ and Zn2+ in biological samples
often involves the assumption that competing Mg2+ levels
remain constant in the system under study, a premise that is not
entirely justified and is likely to introduce important errors.
Department of Chemistry, New York University, New York, NY 10003, USA.
E-mail: dbuccella@nyu.edu
†Electronic supplementary information (ESI) available: Supporting figures, crys-
tallographic tables and refinement details, ITC data and detailed data fitting pro-
cedures. CCDC 1474449–1474452. For ESI and crystallographic data in CIF or
other electronic format see DOI: 10.1039/c6dt01557c
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In the context of cellular imaging, indicators that offer a tate fashion, with the metal cation approximately in the plane
highly selective optical response for the cation of interest are of the aminophenol moiety adopting a pseudo octahedral
most desirable. Significantly, the highest sensitivity13 in cation geometry. The sixth coordination site is occupied by a
detection is achieved with indicators comprising metal- bridging carboxylate of an adjacent complex, which leads to
binding units that form complexes with dissociation constants the formation of extended chains of [(APTRA)Mg]− units in the
(Kd) roughly within one order of magnitude of the average con- solid state (Fig. S1, ESI†). The Zn2+ complex is nearly
centration of the metal to be detected. Typical concentrations isomorphous to the Mg2+ analogue. The Ca2+ complex, on the
of chelatable metal cations in biological samples vary greatly other hand, crystallizes as a dimeric species of the formula
by metal; for example, the intracellular concentration of labile [Ca(OH2)3]2{[(APTRA)Ca(OH2)]2(μ-H2O)2}Cl2 (Fig. 2 and S2†). In
Zn2+ is estimated to fall below nanomolar levels,14,15 whereas the structure, two aqua ligands bridge two Ca2+ ions chelated
that of Mg2+ is typically found in the low millimolar range16,17 each by one pentadentate APTRA ligand. The marked struc-
and K+ averages to around 150 mM.18 Thus, attaining a selective tural differences between the eight-coordinate Ca2+ complex
optical response for a target metal cation in a complex, metal and the six-coordinate Zn2+ and Mg2+ counterparts suggest
rich samples such as the cellular milieu requires careful con- that chelators with greater denticity than APTRA might be
sideration of both the relative affinities of the chelator for exploited to achieve greater selectivity for Ca2+ over smaller
various metal cations (binding selectivity) and the concen- divalent cations, by taking advantage of the ability of the
trations of such cations in the sample under study.4 We report larger cation to accommodate coordination numbers greater
herein the structural characterization of APTRA complexes with than six. The design of bis(o-aminophenoxy)ethane-N,N,N,N-
various biologically relevant cations, and the thermodynamic tetraacetic acid (BAPTA chelator) by Tsien19 is the most promi-
analysis of complex formation with Mg2+, Ca2+ and Zn2+. The nent example of this logic.
results of these studies are discussed in the context of indicator
In the absence of other added metal ions, the K+ complex of
design, highlighting the aspects that may aid the development APTRA can be crystallized by diffusion of acetonitrile into an
of new generations of chelators and fluorescent sensors with aqueous solution of the K3APTRA salt at pH 7, as depicted in
enhanced metal selectivity profiles for imaging applications.
Fig. 3. The APTRA motif has also been shown to bind various
first row transition metals. For example, a complex with Fe3+
has been prepared in situ, whereas complexes of Mn2+ and Ni2+
have been isolated, and the Co2+ counterpart has been structu-
rally characterized by Hualin and Peiyi;20,21 the latter shows a
geometry in the solid state that resembles that of the Mg2+ and
Zn2+ complexes reported herein. Because the coordination of
open shell transition metal cations often leads to quenching of
fluorescence emission by organic indicators, and/or has not
been reported to cause significant interference in the context of
bioimaging applications with APTRA-based sensors, the
binding of these metals is not discussed further in this report.
Results and discussion
Structural characterization of APTRA complexes of
magnesium, calcium and zinc
The complexes of APTRA with Mg2+, Ca2+ and Zn2+ were pre-
pared by treating aqueous solutions of M3APTRA (M = K, Na)
salts at neutral pH with an excess of the corresponding metal
chloride. Crystalline samples were obtained from liquid
diffusion of an organic solvent into the aqueous solution of
the complex (Fig. 2 and Table S1†). Despite the addition of
excess Me4NCl to the mixture, all the metal APTRA complexes
crystallized as anions with a metal cation as the counterion.
Study of the solution coordination properties of APTRA by
absorption spectroscopy
The Mg2+ complex of APTRA crystallizes as [Mg The apparent binding constants reported for fluorescent indi-
(OH2)6][(APTRA)Mg]2. The chelator coordinates in a pentaden- cators are often determined from the fluorescence titration
Fig. 2 Crystal structures of the complexes of APTRA with Mg2+, Ca2+ and Zn2+ (hydrogen atoms in all the structures and bridging Ca(OH2)3 moieties
in the calcium complex were omitted for clarity). The zinc chelate is one of two chelates present in the asymmetric unit. Thermal ellipsoids shown at
50% probability.
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Fig. 3 Crystal structure of (H2APTRA)K (hydrogens omitted for clarity).
Thermal ellipsoids shown at 50% probability.
data, which are influenced by both ground state and excited
state equilibria and may vary significantly depending on the
choice of fluorophore and experimental conditions. To enable
comparisons on an even ground, we sought to characterize
the metal binding properties of the plain APTRA chelating
unit by spectrophotometric titrations conducted in aqueous
buffer at pH 7.0 (Fig. 4). The samples of K3APTRA for spectro-
scopic studies were obtained by quantitative hydrolysis of
the ester form, which is stable for long-term storage. The de-
esterified, free acid form of the chelator was found to undergo
partial decomposition upon standing in aqueous solution,
particularly at reduced pH.22 Accordingly, stock solutions in
aqueous buffer at neutral pH were stored frozen for short
periods of time and thawed immediately before use. Further-
more, potentiometric pH titrations were not performed.
Fig. 4 Absorption spectra (left) and binding isotherms (right) of 50 μM
solutions of APTRA, treated with increasing concentrations of (A) MgCl2;
(B) CaCl2; or (C) Zn2+ from a Zn2+/EGTA buffered system. All the experi-
ments were conducted in 50 mM PIPES, 100 mM KCl buffer at pH 7.0,
25 °C. The reported apparent binding constants correspond to averages
of three independent titrations. [M2+
[M2+] = free metal concentration.
] = total metal concentration;
t
Addition of Ca2+ or Mg2+ to a solution of the chelator led to
a decrease in the intensity of the bands at 254 and 287 nm,
and the appearance of a new maximum at 269 nm with a spectrophotometric studies involving competition with an EGTA
shoulder at 275 nm. The same changes were observed in the buffer. For all three metal cations the chelation by APTRA is
presence of increasing concentrations of Zn2+, which were mainly entropically driven and, as is commonly observed with
adjusted with a Zn–EGTA buffered system. Similar spectral hard cations, the binding is endothermic to various extents.23
changes have been reported for the structurally-related BAPTA The structural similarities between the Mg2+ and Zn2+ com-
chelator upon treatment with Ca2+,19 indicating a decrease in plexes observed in the solid state suggest that differences in the
the delocalization of the electron density of the nitrogen and entropy of binding for these two cations in solution may not
oxygen atoms into the aminophenol as a result of their coordi- stem from reorganization of the organic ligand, but likely from
nation to the metal. This observation is consistent with the differences in the hydration shell of the metal.
solid-state structures determined by X-ray diffraction.
Among the three metal cations studied, the binding of
All the binding isotherms could be fitted satisfactorily with Mg2+ carries the greatest enthalpic cost, leading to significant
a 1 : 1 ligand-to-metal binding model (Fig. 4), affording values enthalpy–entropy compensation and an overall weaker appar-
for apparent binding constants summarized in Table 1. Van’t ent binding at neutral pH. Remarkably, this feature offers an
Hoff analyses of the apparent binding constants for Ca2+ and advantage in the design of metal indicators with high sensi-
Mg2+ over the range from 15 to 45 °C (Fig. S3†) were employed tivity for biological free Mg2+, given the typical sub-millimolar
to determine the enthalpic and entropic contributions to concentrations of this cation in cells and other typical samples
metal binding, summarized in Table 1. The enthalpies of (vide supra). The lower enthalpic cost of binding other metals,
complex formation were further confirmed through isothermal however, leads to low KMg,M selectivity ratios that result in
titration calorimetry (ITC, Fig. S4†). In the case of Zn2+ possible interferences by Zn2+ and Ca2+ in the analysis of
binding, a direct study of the metal–ligand interaction by ITC Mg2+. These become an important concern in systems in
was preferred over the more complex variable temperature which concentrations of chelatable Zn2+ and Ca2+ are high and
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Table 1 Thermodynamic parameters for the formation of APTRA complexes with Mg2+, Ca2+ and Zn2+ at neutral pHa
ΔH (kcal mol−1
Spectroscopy
)
ΔS (cal mol−1 K−1
)
K′ (M−1
)
ITC
Spectroscopy
ITC
44
24.7 0.6
50.3 0.9
Mg2+
Ca2+
Zn2+
(5.6 0.3) × 102
(1.02 0.07) × 105
(7 1) × 107
8.6 0.4
−0.3 0.4
N.D.b
9.3 0.8
0.6 0.1
4.7 0.3
41
22
1
1
3
N.D.b
a Measurements performed in 50 mM PIPES, 100 mM KCl, pH 7.0 at 25 °C. b N.D. = not determined.
may fall within the range in which the indicators are most sen- cellular milieu. We report herein the structural characteri-
sitive to them. In particular, samples that exceed typical sub- zation of the Mg2+, Ca2+ and Zn2+ complexes of APTRA, a
nanomolar free Zn2+ resting levels14,15 or sub-micromolar Ca2+ cation recognition motif commonly employed in the design of
concentrations24 either transiently (e.g. in signaling events) or Mg2+ and low-affinity Ca2+ indicators. The structures of the
permanently (metal-rich tissues or cells; some cellular orga- complexes in the solid state highlight the differences in
nelles) must be analyzed with great care.
coordination number and geometry adopted by Ca2+ compared
Despite efforts over the years, progress in the design of to Mg2+ and Zn2+, supporting the notion that chelators with
selective indicators for the study of Mg2+ under physiological greater denticity are better choices for selective binding of the
conditions has been limited.25,26 For the detection of sub- larger Ca2+ over the smaller divalent cations. Thermodynamic
millimolar concentrations of chelatable or ‘free’ Mg2+ present analysis of metal binding indicates that the low affinity of
in typical biological matrices, weak binding such as that APTRA for Mg2+ stems from the higher enthalpic cost of
exhibited by APTRA is ideal. Attaining the desired selectivity binding of Mg2+ compared to other cations, which leads to a
thus requires changes geared toward destabilizing the binding sizeable enthalpy–entropy compensation. This feature is
of competing cations rather than strengthening the inter- advantageous for the design of weak chelators that are better
actions with Mg2+. Decreasing the denticity of the chelator by tuned for the detection of high concentrations of Mg2+ typi-
removing one or more metal–carboxylate interactions may cally found in biological systems. Selectivity, however, remains
provide a useful strategy: as the smaller entropic gain is then an unresolved issue, which opens the door to likely interfer-
compensated by a smaller enthalpic penalty for Mg2+ binding, ences by Zn2+ and Ca2+ in the detection of physiological levels
the overall impact on Mg2+ complexation is expected to be of Mg2+ (and vice versa) that are often ignored but deserve
lower than that on the binding of cations that are more entro- attention. To achieve both sensitive and selective detection of
pically driven, i.e. with lower enthalpic contributions. In this Mg2+, the binding of other cations to the metal-recognition
regard, bidentate β-dicarbonyl chelators such as those reported unit must be destabilized with respect to the binding of the
by Levy27 and the groups of Suzuki and Oka28–31 show Mg2+ target Mg2+. Our analysis of the relative entropic and enthalpic
affinities in a similar range to APTRA, yet bind Ca2+ and Zn2+ contributions to metal coordination by APTRA suggests that
much more weakly than the pentadentate chelator. Indicators this goal may be accomplished by decreasing the denticity of
based on this metal-recognition motif are thus better suited the chelators employed for Mg2+ recognition in future indi-
for selective detection of Mg2+ and magnesium-containing cator design.
species, showing negligible interference from other physiologi-
cally relevant cations. Their low denticity, however, makes
them prone to form ternary complexes with endogenous intra-
cellular Mg2+ chelators32 and renders them unable to dis-
tinguish ‘free’ from ‘bound’ forms of the metal in a way akin
to that of APTRA.5h As such, low denticity chelators offer
limited information on magnesium cellular speciation. A com-
promise through the design of intermediate denticity chelators
may provide an answer to this conundrum and enable the
development of better tools for cellular imaging applications.
Experimental
General materials and methods
The methyl ester of APTRA was prepared according to a pub-
lished procedure.7 Piperazine-N,N-bis(2-ethanesulfonic acid)
(PIPES), 99.999% KCl, 99.999% MgCl2, 99.999% ZnCl2, 99.99%
CaCl2, and high-purity 25% HCl, 45% KOH, and 50% NaOH
were purchased from Sigma Aldrich. Other solvents and
reagents were purchased from various vendors and used as
received. All aqueous solutions were prepared with deionized
water with a resistivity of ≥18.2 MΩ. Spectroscopic measure-
ments were conducted at pH 7.0 in aqueous buffer containing
Conclusions
The design of metal indicators for applications in bioimaging 50 mM PIPES, 100 mM KCl. Buffer was treated with the
demands the development of chelators with high metal selecti- CHELEX resin (Bio-Rad) according to the manufacturer proto-
vity profiles that can afford sensitive detection of the target col to remove trace metal ions. NMR spectroscopic data were
metal cation, with low interference from other ions in the obtained on Bruker AVANCE-400 or 500 NMR spectrometers.
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Absorbance spectra were collected on a Cary 100 UV-Vis small additions of 1 M aqueous HCl. The solution was filtered
spectrophotometer using 1 cm quartz cuvettes. The tempera- and treated with 1.0 M aqueous MgCl2 (200 μL, 0.20 mmol).
ture during analysis was maintained within 0.02 °C by using Colorless crystals (plates) of [Mg(OH2)6][(APTRA)Mg]2·(diox-
a Quantum Northwest TC225 Temperature Controller. HPLC ane)·6(H2O) were obtained at 4 °C by liquid diffusion of
analysis was employed to verify the purity of the chelator, and dioxane into the aqueous solution of the complex.
was conducted on an Agilent 1260 Infinity HPLC with diode
Crystallization of [Zn(OH2)6][(APTRA)Zn]2
array UV-Vis and fluorescence detection, using a Zorbax
Extend C18 column (4.6 × 50 mm, 1.8 μm) with a gradient of A solution of K3APTRA·6KOH (0.10 mmol) in water (250 μL)
MeOH (25 to 75% over 20 min) and water (+0.1% TFA).
was treated with Me4NCl (2 equiv.) and brought to pH 7 with
small additions of 1 M HCl. The solution was filtered and
treated with 1.0 M aqueous ZnCl2 (200 μL, 0.20 mmol). Color-
Preparation of APTRA solutions for spectroscopy
APTRA methyl ester (16.91 mg, 0.05201 mmol) was suspended less crystals (plates) of [Zn(OH2)6][(APTRA)Zn]2·(dioxane)·6
in MeOH (514 μL) and treated with 1.0 M aqueous KOH (H2O) were obtained at 4 °C by liquid diffusion of dioxane into
(514 μL). The resulting mixture was allowed to stand at room the aqueous solution of the complex.
temperature for 3 h, after which all solids had dissolved. The
Single crystal X-ray structure determination
reaction mixture was then diluted to 5 mL with 50 mM PIPES
buffer, 100 mM KCl, pH 7.0 and split into aliquots that were Single crystals suitable for X-ray analysis were coated with
stored at −20 °C and thawed immediately prior to use. Quanti- immersion oil and mounted on MiTeGen micromounts for
tative hydrolysis of the ester was verified by HPLC analysis analysis. Diffraction data were collected at 100 K on a Bruker
(Fig. S6†).
SMART APEXII CCD X-ray diffractometer equipped with graph-
ite-monochromated Mo-Kα radiation (λ = 0.71073 Å). Struc-
tures were solved by direct methods and difference Fourier
map techniques, and were refined by full-matrix least-squares
Preparation of M3APTRA (M = Na, K) salts for crystallization
experiments
APTRA methyl ester (400 mg, 1.23 mmol) and MOH (M = K or procedures on F2 with SHELX.33 Non-hydrogen atoms were
Na, 9 equiv.) were dissolved in a 1 : 1 mixture of MeOH and refined with anisotropic displacement parameters. Hydrogen
water (25 mL) and stirred at room temperature for 3 h. The atoms on carbons were placed in idealized positions (C–H =
solvent was removed by rotary evaporation and the residue 0.95–1.00 Å) and included as riding with Uiso(H) = 1.2 or 1.5
was further dried in vacuo overnight to yield M3APTRA·6MOH Ueq(non-H), and hydrogen atoms on oxygens were found from
as a hygroscopic white solid. The product was employed the difference Fourier maps and refined isotropically with
in crystallization experiments as isolated, without further restrained O–H distances of 0.84 Å. All the tested crystals of
purification.
the magnesium complex were either twinned or polycrystals,
1
K3APTRA·6KOH: H NMR (400 MHz, D2O, δ): 6.87 (m, 2H), therefore a dataset on a twinned crystal was collected. The data
6.80 (dd, 3J = 7 Hz, 4J = 2 Hz, 1H), 6.74 (dd, 3J = 7 Hz, 4J = 2 Hz, analysis revealed
a non-merohedral twin; the two twin
1H), 4.44 (s, 2H), 3.81 (s, 4H). 13C{1H} NMR (125 MHz, D2O, δ): domains are related by a 2-fold axis, and the molar ratio
180.2, 178.0, 150.1, 140.2, 122.0, 121.9, 118.6, 113.5, 67.9, 57.3. between two domains is 58.6% : 41.4%. The crystals of the zinc
ESI-MS (m/z): [M + K]+ calcd for C12H13NO7, 322.0; found complexes were also twinned: a pseudo non-merohedral twin
322.0.
with a monoclinic angle of almost 90°. Careful examination of
Na3APTRA·6NaOH: 1H NMR (400 MHz, D2O, δ): 6.87 (m, the observed reflections that violate the systematic absences
3H), 6.77 (d, J = 7 Hz, 1H), 4.44 (s, 2H), 3.79 (s, 4H). 13C{1H} revealed the true symmetry in the space group P21/n (see the
3
NMR (125 MHz, D2O, δ): 180.0, 177.7, 150.7, 140.3, 123.2, details in the ESI†). Crystallographic data collection and refine-
122.0, 119.5, 113.4, 67.7, 57.8. ESI-MS (m/z): [M + Na]+ calcd ment parameters are summarized in Table S1.† The crystallo-
for C12H13NO7, 306.1; found 306.0.
graphic information files (CIFs) including the HKL and RES
data were deposited in the Cambridge Crystallographic Data
Centre with the numbers CCDC 1474449 (K+ complex),
Crystallization of [Ca(OH2)3]2{[(APTRA)Ca(OH2)]2(μ-H2O)2}Cl2
A solution of Na3APTRA·6NaOH (0.10 mmol) in water (250 μL) 1474450 (Mg2+ complex), 1474451 (Ca2+ complex), and
was treated with Me4NCl (2 equiv.) and brought to pH 7 with 1474452 (Zn2+ complex).
small additions of 1 M HCl. The solution was filtered and
Spectrophotometric determination of Mg2+ and Ca2+ binding
constants
treated with 1.0 M aqueous CaCl2 (250 μL, 0.25 mmol). Color-
less
crystals
(plates)
of
[Ca(OH2)3]2{[(APTRA)Ca
(OH2)]2(μ-H2O)2}Cl2·2(H2O) were obtained by liquid diffusion A solution of ∼50 μM APTRA in aqueous PIPES buffer (from
of dioxane into the aqueous solution of the complex at room stock solution prepared above) was treated with aliquots of
temperature.
MgCl2 or CaCl2 aqueous solutions (from 1.0 M MgCl2 or 1.0 M
CaCl2 and 0.1 M CaCl2 solutions) to cover the range of 0 mM
to 100 mM total Mg2+ concentration, or 0 μM to 1300 μM total
Crystallization of [Mg(OH2)6][(APTRA)Mg]2
A solution of K3APTRA·6KOH (0.10 mmol) in water (250 μL) Ca2+ concentration. The sample was allowed to re-equilibrate
was treated with Me4NCl (2 equiv.) and brought to pH 7 with in the cuvette holder at the desired temperature (15 °C, 25 °C,
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37 °C or 45 °C) for 5 min before collecting each spectrum. Foundation under Award Number CHE-01162222. The Bruker
Absorbance values at 287 nm were fitted using a 1 : 1 binding AXS SMART APEXII single crystal diffractometer was acquired
model as described in the ESI.† All the reported apparent through the support of the Molecular Design Institute in the
binding constants represent the average of a minimum of Department of Chemistry at New York University.
three independent titrations.
Spectrophotometric determination of the Zn2+ binding
Notes and references
constant
Analysis of zinc binding was conducted using a Zn2+/EGTA
1 B. Valeur and I. Leray, Coord. Chem. Rev., 2000, 205, 3.
2 In older reports, the acronym APTA is used instead.
3 L. A. Levy, E. Murphy, B. Raju and R. E. London, Biochemis-
try, 1988, 27, 4041.
buffered system, modified from the protocol described by
Fahrni and O’Halloran.34 A solution containing 50 μM APTRA,
1 mM ZnCl2 and 1 mM EGTA in aqueous PIPES buffer was
mixed at various ratios with a solution containing 50 μM
APTRA and 1 mM EGTA in the same aqueous buffer, and
allowed to equilibrate for 10 min at 25 °C before acquiring the
absorption spectrum. The absorbance at 287 nm as a function
of [Zn2+] was analyzed using a simple 1 : 1 binding model. The
free metal ion concentrations in each mixture was calculated
from the total Zn2+ concentration, [Zn2+]t, using an apparent
stability constant for Zn2+–EGTA of log K′ZnEGTA = 8.24 at pH
7.0. This apparent stability constant was calculated from the
absolute binding constant log KZnEGTA = 12.6 (25 °C, μ = 0.1)35
using Schwarzenbach’s α coefficient method, with pKa values
for EGTA corrected by 0.11 units to account for the fact that
tabulated pKa values are determined using concentrations and
not the activity of the hydrogen ion. Further details are pro-
vided in the ESI.†
4 R. E. London, Annu. Rev. Physiol., 1991, 53, 241.
5 For selected examples, see: (a) B. Raju, E. Murphy,
L. A. Levy, R. D. Hall and R. E. London, Am. J. Physiol.: Cell
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H. Q. N. Gunaratne and G. E. M. Maguire, J. Chem. Soc.,
Chem. Commun., 1994, 1213; (c) E. Cielen, A. Stobiecka,
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6 The Molecular Probes Handbook: A Guide to Fluorescent
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7 B. Metten, M. Smet, N. Boens and W. Dehaen, Synthesis,
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Isothermal titration calorimetry experiments were conducted
at 25 °C with a TA Instruments NanoITC LV. Data were ana-
lyzed using NanoAnalyze (v. 3.5.0). Solutions of the metal
(50 mM MgCl2 or 30 mM CaCl2 in 50 mM PIPES, 100 mM KCl
aqueous buffer at pH 7.0; or 3.0 mM ZnCl2 in deionized water)
were titrated into samples of APTRA (3.0 mM for Mg2+ and
Ca2+ titrations, 0.30 mM for Zn2+ titrations) in 50 mM PIPES,
100 mM KCl aqueous buffer at pH 7.0. Heat values were
recorded against a reference cell filled with 350 μL of the same
aqueous PIPES buffer. Blank titrations of the metal into buffer
were subtracted from the APTRA titration data to correct for
the effects of metal dilution. The heats of metal buffer inter-
actions were determined36 and used to correct the heat of
metal–APTRA binding. Additional titrations were conducted to
account for the heats of ligand dilution and buffer dilution;
these were found to be negligible with respect to heat values
arising from the metal–ligand interaction and were, therefore,
neglected. Buffer protonation effects were neglected.
9 A 15N-labeled APTRA chelator was recently reported for the
low-affinity detection of Ca2+ by hyperpolarized NMR tech-
niques. See: R. Hata, H. Nonaka, Y. Takakusagi,
K. Ichikawa and S. Sando, Chem. Commun., 2015, 51,
12290.
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signal per unit change of the metal concentration being
measured.
Acknowledgements
This work was supported in part by the National Science Foun- 14 R. A. Colvin, W. R. Holmes, C. P. Fontaine and W. Maret,
dation (CHE-1555116). The authors acknowledge Tal Moriah Metallomics, 2010, 2, 306.
for her assistance in acquiring spectrophotometric data. The 15 W. Maret, Metallomics, 2015, 7, 202.
Bruker Avance-400 and Avance-500 NMR spectrometers were 16 R. D. Grubbs, Biometals, 2002, 15, 251.
acquired through the support of the National Science 17 A. M. P. Romani, Arch. Biochem. Biophys., 2011, 512, 1.
Dalton Trans.
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