New strategy for quantifying biological zinc by a modified zinpyr fluorescence sensor

Article abstract of DOI:10.1021/ja807156b

Substitution of one pyridine by pyrazine in each DPA appendage of ZP1 leads to a new zinc sensor, ZPP1, with a modified background fluorescence and zinc affinity. ZPP1 exhibits a two-step zinc response during fluorescence titrations, which leads to a new method for zinc quantification. The ability of ZPP1 to image and quantify zinc was demonstrated in pancreatic Min6 cells. Copyright

Full text of DOI:10.1021/ja807156b

Published on Web 10/31/2008
New Strategy for Quantifying Biological Zinc by a Modified Zinpyr
Fluorescence Sensor
Xiao-an Zhang, Dugan Hayes, Sarah J. Smith, Simone Friedle, and Stephen J. Lippard*
Department of Chemistry, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139
Received September 9, 2008; E-mail: lippard@mit.edu
3
,9
Mobile zinc ions both facilitate essential biological functions and
of sensors, protonation at the quenching units increases the
fluorescence intensity of ZPP1 in a pH titration experiment (Figure
1,2
mediate the pathophysiology of disease. An array of fluorescence
sensors for mobile zinc(II) have been devised to help unravel these
a
S2), resulting in an apparent pK value of 6.52, which is below
physiological pH and significantly lower than that of ZP1. Accord-
ingly, the background fluorescence of ZPP1 at physiological pH is
3
-6
processes.
No single sensor is universally suited for all zinc
imaging studies, however. New sensors with modified chemical
and photophysical properties are therefore in high demand, largely
due to the variable concentrations, cellular locales, and organelle
requirements for biological zinc. Sensors with different zinc
affinities, lower background fluorescence, higher turn-on, and the
ability to accurately quantify zinc levels are desired, and a single
sensor comprising several of the properties would be ideal. Here
we report a new fluorescence sensor for mobile zinc, ZPP1,
modified from our previously reported Zinpyr (ZP) family of zinc
7
almost seven times lower than that reported for ZP1 (Φapo ) 0.38),
with an observed quantum yield of 0.052. Hence, upon the addition
of excess zinc, ZPP1 exhibits a significantly higher fluorescence
increase (ΦZn ) 0.70; ∼13-fold turn-on) compared to ZP1. These
results are similar to those obtained for ZP3, an earlier ZP1
a
derivative with a pK value suppressed by introduction of electron-
1
1
withdrawing groups on the fluorophore. ZPP1, however, shows
an even higher turn-on value than ZP3. ZPP1 also displays a good
selectivity for zinc, similar to that of ZP1 and ZP3 (Figure S3). No
7,8
probes. ZPP1 exhibits lower background fluorescence and higher
fluorescence turn-on when complexed with zinc, with lower
apparent zinc affinity compared to its parent ZP1 (Chart 1). Most
interestingly, ZPP1 exhibits a distinct two-step fluorescence turn-
on upon binding 1 vs 2 equiv of zinc, which can be utilized to
quantify zinc via a novel protocol.
+
+
2+
fluorescence response occurs upon addition of Na , K , Mg
,
2
+
and Ca to ZPP1, whereas divalent transition metals, such as
Mn , Fe2+, Co , Ni , and Cu , quench the fluorescence.
2
+
2+
2+
2+
Our first-generation zinc sensor ZP1, although very bright, has
relatively high background fluorescence and hence relatively low
zinc turn-on due to protonation of the quenching units at physi-
9
ological pH. The fluorescence of ZP1 increases by 3-fold upon
7
d
zinc binding with an apparent affinity (K ) in the sub-nM range.
We anticipated that we could generate a new zinc sensor, ZPP1,
with a lower pK and zinc affinity by substituting one pyridine arm
a
of ZP1 at each dipicolylamine (DPA) moiety with pyrazine, a
stronger electron-withdrawing group and weaker zinc-binding ligand
Figure 1. Fluorescence response of ZPP1 (5 µM) upon the addition of
2
+
(
Chart 1). ZPP1 was synthesized in a manner analogous to that
used for ZP1 by a Mannich reaction of 2′,7′-dichlorofluorescein
DCF) with 1-(pyrazin-2-yl)-N-(pyridin-2-ylmethyl)methanamine,
which was obtained from coupling 2-picolylamine with (chloro-
zinc (A) and fluorescence titration of Zn (5 µM) upon stepwise addition
of ZPP1 (B). Both experiments were performed in pH 7 PIPES buffer
solution (50 mM) containing KCl (100 mM).
(
10
A remarkable property of ZPP1 was discovered in an experiment
in which a constant amount of zinc chloride (5 µM) was titrated
with ZPP1. As shown in Figure 1B, there is a distinctive two-step
fluorescence response upon addition of increasing amounts of ZPP1.
The reason for this behavior is clear from Figure 1A, in which
zinc is added to ZPP1. Addition of up to 1 equiv of zinc results in
only a slight fluorescence increase; addition of more than 1 equiv
leads to a significant, almost linear increase in fluorescence intensity
that reaches its maximum near 2 equiv. Since ZPP1 contains two
apparent zinc-binding pockets, the following stepwise equilibria
describe the binding events.
methyl)pyrazine (Supporting Information, SI). We confirmed the
composition of ZPP1 by MS, NMR, UV-vis, and fluorescence
spectroscopy and the structure by X-ray crystallography (SI).
The apo form of ZPP1 (5 µM) emits at 532 nm (λmax) upon
excitation at 505 nm in pH 7 buffered solution (50 mM PIPES,
1
00 mM KCl). In its zinc-saturated state, the emission maximum
of ZPP1 shifts to 523 nm. As with other members of the ZP family
Chart 1. ZP1 and ZPP1 (Left) and X-ray Structure (Supporting
Information) of ZPP1 in Its Lactone Form (Right)
K1
[
ZPP1] + [Zn] y\ z [ZPP1-Zn1]
K2
[
ZPP1-Zn ] + [Zn] y\ z [ZPP1-Zn2]
1
Mass balance equations corresponding to the above equilibria
were solved by Newton-Raphson techniques using Excel soft-
1
5788 9 J. AM. CHEM. SOC. 2008, 130, 15788–15789
10.1021/ja807156b CCC: $40.75  2008 American Chemical Society

C O M M U N I C A T I O N S
1
2
ware to compute the concentrations of individual species, which
were subsequently used to fit the fluorescence titration data (SI).
The experimental results agree well with the calculated values, and
the fitting procedure returned two apparent association constants
was harvested and its zinc content was quantified by ZPP1 titration
as described above. The background fluorescence of free ZPP1 was
subtracted from the titration curve, and the resulting plot exhibited
a clear fluorescence maximum at 0.67 µM ZPP1, revealing the
concentration of released zinc to be 1.34 µM (Figure S6). Based
on the number of cells used in the experiment, we estimate that
∼3.6 fmol of zinc on average were released per cell under these
stimulation conditions. From the computed average cell volume of
4.5 pL (30 µm × 30 µm × 5 µm), we estimate the intracellular
concentration of released zinc to be ∼800 µM. This value is subject
to errors in the estimated cell volume and will vary between
intracellular compartments.
1
1
-1
8
-1
(
K
1app ) 1.52 × 10
M
, K2app ) 1.02 × 10 M ) (Figure S5).
Since most of the fluorescence increase occurs at the second zinc
binding event, K2app should be considered the apparent zinc affinity
for fluorescence response, which is more than 10 times lower than
7
that of ZP1.
We realized that the special zinc-response properties of ZPP1
offered a unique opportunity for zinc quantification. By fluorescence
titration of a mobile zinc sample with ZPP1, we could determine
the zinc concentration from the concentration of sensor giving the
maximum fluorescence response. This expectation was experimen-
tally confirmed, as mentioned above and shown in Figure 1B. As
predicted, titration of ZPP1 into a zinc solution first generated the
In conclusion, we have prepared a new zinc sensor, ZPP1,
modified from the ZP sensor family by substitution of one pyridine
by pyrazine at each DPA unit. ZPP1 exhibits low background
fluorescence and hence a higher zinc-responsive fluorescence turn-
highly fluorescent species ZPP1-Zn
2
, since excess zinc was avail-
a
on due to the lower pK value of the quenching units compared to
able, producing an almost linear increase in fluorescence intensity
at the beginning of the titration. When the concentration of ZPP1
that of ZP1. ZPP1 exhibits a novel two-step fluorescence response
toward zinc binding that can be applied to quantify zinc concentra-
tion levels. ZPP1 selectively stains mobile zinc in the secretory
granules of Min6 cells. Moreover, the amount of zinc released from
these granules upon stimulation was quantified by ZPP1 using the
newly discovered method. The advantages of incorporating pyrazine
into the metal-binding units in this sensor can be generally applied
to detect zinc, and possibly mercury and cadmium (Figure S3),
sensors as well. The detailed mechanism of stepwise zinc response
by ZPP1 is under investigation.
reached half the concentration of total zinc ([ZPP1]total
)
1
2+
/
2
[Zn
]total), the addition of more ZPP1 shifted the equilibrium
1
to form ZPP1-Zn which is only weakly fluorescent. The fluores-
cence therefore decreased, even though more ZPP1 was added. At
the sharp maximum point in the titration curve, the amount of ZPP1
added was equal to half of the total amount of zinc in solution,
which allows for an accurate determination of mobile zinc
concentration (Figure 1B). To the best of our knowledge, this type
of quantification method has never been reported previously for
zinc, and possibly for any other analyte.
Acknowledgment. This work was supported by Grant GM65519
from the National Institute of General Medical Sciences. X.-a.Z.
thanks the Swiss National Science Foundation and Roche Research
Foundation for postdoctoral fellowship support. D.H. was supported
by a Novartis UROP fellowship. Min6 cells were kindly provided
by Dr. Andrey Kuznetsov from Prof. Louis Philipson’s laboratory
at the University of Chicago. We thank Brian Wong for helpful
comments on the manuscript and valuable discussion.
The properties of ZPP1 render it suitable for biological applica-
tions. To demonstrate the ability of ZPP1 as a fluorescent probe to
image and quantify endogenous zinc, we chose Min6, an insulin-
oma cell line that contains a relatively high concentration of mobile
13
zinc packaged in insulin granules. Min6 cells were incubated with
0 µM ZPP1 overnight, although 2 h will suffice, and imaged using
2
an inverted epifluorescence microscope. A vivid granule-like
staining pattern localized mostly at the periphery of the cells was
observed, with bright spots consistent with zinc in the secretory
granules. The bright fluorescence was quenched by addition of an
excess of the membrane-permeant zinc chelator, N,N,N′,N′-tetra(2-
picolyl)ethylenediamine (TPEN), consistent with fluorescence stain-
ing due to mobile zinc (Figure 2).
Supporting Information Available: Synthetic procedures, crystal-
lographic details in CIF format, experimental details, and data fitting
method. This material is available free of charge via the Internet at
http://pubs.acs.org.
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JA807156B
J. AM. CHEM. SOC. 9 VOL. 130, NO. 47, 2008 15789