A R T I C L E S
Komatsu et al.
Zinquin. The high quantum yield in aqueous solution provides
high sensitivity in the cellular environment, and the excitation
wavelength in the visible range minimizes cell damage during
irradiation. These sensors have led to great advances in Zn2+
biology, and further development of sensor molecules would
be very valuable.21
is desirable to use a sensor molecule whose apparent dissociation
constant (Kd) is near the target concentration of Zn2+. Almost
all Zn2+ sensor molecules recently developed have Kd values
in the nanomolar region (ZnAF-2, 2.7 nM), because cytosolic
free Zn2+ concentration is controlled at ∼1 nM or lower.7
Neither Zn2+ deficiency nor Zn2+ excess affects cell viability
and function,23 and these sensors can easily be used in
cytoplasm, etc. However, at higher concentrations of Zn2+, the
fluorescence intensity of these sensors would be saturated. Zn2+
is known to be sequestered in synaptic vesicles of many
excitatory forebrain neurons, and the concentration of Zn2+
inside the vesicle was reported to be in the micro- to millimolar
range.8 This vesicular Zn2+ is also released into synaptic space,
where it is estimated to achieve peak levels of 10-30 µM.24 In
such circumstances, sensor molecules with high affinity would
have no ability to detect changes of Zn2+ concentration. So,
for precise analysis of the biological roles of Zn2+, we require
a range of sensor molecules with Kd values not only in the nM
range, but also higher.
We set out several criteria for developing novel fluorescent
Zn2+ sensor molecules. First, for intensity-based measurement,
the sensor should have no signal in the absence of Zn2+, and
the signal should increase in the presence of Zn2+ in an all-or-
none fashion. Second, the sensor should be selective for Zn2+
,
without interference by biologically important metal ions, such
as Ca2+ or Mg2+. Third, complexation and decomplexation with
Zn2+ should be rapid, to provide a fast response. Fourth, for
intracellular study, the sensor should be derivatizable into a cell-
permeable form that can penetrate through the cell membrane
and be hydrolyzed intracellularly to afford the sensor, which is
then trapped in the cell. Fifth, their affinity for Zn2+ should be
appropriate for the Zn2+ concentration range of interest.
We previously showed that ZnAFs, which satisfy most of
the requirements mentioned above, can serve as useful tools
for biological applications.19,22 However, the affinity for Zn2+
remains an issue. To follow changes of Zn2+ concentration, it
Among Zn2+ sensor molecules so far reported, only Newport
Green (Kd ∼ 1 µM) has been used as a low-affinity Zn2+ sensor
to detect synaptically released Zn2+, for which purpose the Zn2+
concentration was calculated on the basis of the Kd value.24
There is little other evidence to support the idea that the release
of Zn2+ reaches micromolar levels, so confirmation remains
desirable.
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Our purpose in this study is to develop a range of low-affinity
sensor molecules whose Kd values are higher than nanomolar
order without any loss of favorable characteristics, such as low
basal fluorescence and selectivity for Zn2+
.
In designing low-affinity sensor molecules, we chose ZnAF-2
as a basal structure. ZnAF-2 has a fluorescein fluorophore
conjugated to N,N-bis(2-pyridylmethyl)ethylenediamine as a
Zn2+ chelator. The design of the chelator was based on the
structure of TPEN (N,N,N′,N′-tetrakis(2-pyridylmethyl)ethyl-
enediamine). These groups offer the following advantages.
Derivatives of fluorescein that are amino-substituted at the
benzoic acid moiety emit little fluorescence due to the photo-
induced electron transfer (PeT) quenching pathway, resulting
in low basal fluorescence and high sensitivity.20a TPEN is known
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For
.
intracellular application, a cell-permeant form can be obtained
readily, because the TPEN moiety itself cannot be protonated
at physiological pH, so the sensor remains intact. Moreover,
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