Biology Reference
In-Depth Information
III. DM-Nitrophen
A. Chemical Properties
Graham Ellis-Davies followed a di
erent strategy for releasing Ca
2
þ
—by
attaching a 2-nitrobenzyl group to one of the chelating amines of ethylenediami-
netetraacetic acid (EDTA) to form the photosensitive chelator dimethoxynitro-
phenyl-EDTA or DM-nitrophen (
Ellis-Davies and Kaplan, 1988; Kaplan and
Ellis-Davies, 1988
). Photolysis by UV light in the wavelength range 330-380 nm
cleaves the DM-nitrophen with a quantum e
V
ciency of 0.18 through multiple
intermediate pathways (
McCray et al., 1992
) to form iminodiacetic acid and a
H
þ
-absorbing 2-nitrosoacetophenone derivative, with 65% of the photoproducts
formed with a time constant of 15
m
s and the rest with
Y
1.9 ms (
Faas et al., 2005,
2007
). A simplified reaction is shown in
Fig. 3
. Although DM-nitrophen binds
Ca
2
þ
with an a
t
¼
nity of 7 nM at pH 7.2, Ca
2
þ
-bound chelator forms a photoprod-
uct-binding Ca
2
þ
with a 4-mM a
Y
nity, while the free form (and Mg
2
þ
-bound
forms, see below) photolyze to a 90
m
M-K
D
photoproduct at an ionic strength of
150 mM. These values are from the most recent of the continually evolving models
of DM-nitropen photolysis (
Ayer and Zucker, 1999; Bollmann and Sakmann,
2005; Faas et al., 2005, 2007; Kaplan and Ellis-Davies, 1988; Neher and Zucker,
1993
). Thus, complete photolysis of Ca
2
þ
-DM-nitrophen can elevate Ca
2
þ
over
50,000-fold, much more than photolysis of the nitr compounds or azid-1. This
significant advantage is counterbalanced to some extent by the facts that the
photoproducts bu
Y
er Ca
2
þ
so weakly that the final [Ca
2
þ
]
i
will be determined
largely by native cytoplasmic bu
V
ers, and that the Ca
2
þ
liberated by photolysis of
DM-nitrophen will be removed more readily by extrusion and uptake pumps.
V
CO
−
Ca
2+
CO
−
−
O
2
C
CO
−
CO
−
−
O
2
C
N
N
Ca
2+
N
O
NO
2
NO
CO
−
CO
−
h
n
+
N
H
OCH
3
OCH
3
OCH
3
OCH
3
DM-nitrophen
(high Ca
2+
affinity)
DM-nitrophen photolysis products
(low Ca
2+
affinity)
Fig. 3
Structure of and reaction scheme for DM-nitrophen, which releases calcium on exposure to
light.