Biology Reference
In-Depth Information
The absorbance of Ca
2
þ
-saturated and free DM-nitrophen is 4330 and
4020 M
1
cm
1
, respectively, and 0.18.
A serious complication of DM-nitrophen is that it shares the cation-binding
properties of its parent molecule EDTA. In particular, H
þ
and Mg
2
þ
compete for
Ca
2
þ
at the hexacoordinate-binding site. The a
nity of DM-nitrophen for Mg
2
þ
at pH 7.2 is 1.7
m
M, whereas the photoproducts bind Mg
2
þ
with a
Y
Y
nities of about
2 mM. Further, both the Ca
2
þ
- and Mg
2
þ
-a
nities of DM-nitrophen are highly
pH-dependent (
Grell et al., 1989
), doubling for each 0.3 units of pH increase. Thus,
in the presence of typical [Mg
2
þ
]
i
levels of 1-3 mM, DM-nitrophen that is not
already bound to Ca
2
þ
will be largely in the Mg
2
þ
-bound form. Further, excess
DM-nitrophen will suck Mg
2
þ
o
Y
ATP, which binds it substantially more weakly,
compromising the ability of ATP to serve as an energy source or as a substrate for
ATPases. Finally, photolysis of DM-nitrophen will lead to a jump in [Mg
2
þ
]
i
as
well as [Ca
2
þ
]
i
, and to a rise in pH. Unless controlled by native or exogenous pH
bu
V
ers, this pH change can alter the Ca
2
þ
and Mg
2
þ
a
nities of the remaining
DM-nitrophen. In the absence of Ca
2
þ
-loading, DM-nitrophen even may be used
as a caged Mg
2
þ
chelator (
Ellis-Davies, 2006
). Attributing physiological responses
to a [Ca
2
þ
]
i
jump, therefore, requires control experiments in which DM-nitrophen
is not charged with Ca
2
þ
. DM-nitrophen currently is sold by CalBiochem.
To circumvent the problems arising fromMg
2
þ
competing for the Ca
2
þ
-binding
site of DM-nitrophen, a second generation derivative of ethylene glycol bis(
b
-
aminoethylether)-N,N,N
0
,N
0
-tetraacetic acid (EGTA, which binds Mg
2
þ
only
very weakly) coupled to a light-sensitive ortho-nitrophenyl group was developed
(
Ellis-Davies and Kaplan, 1994
). This compound, nitrophenyl-EGTA or NP-
EGTA, is very rapidly cleaved (
V
Y
2
m
s) (
Ellis-Davies, 2003
)toH
þ
-absorbing
imidodiacetic acid photoproducts with e
t
¼
ective Ca
2
þ
-K
D
of 1 mM, 12,500-fold
V
Y
higher (lower a
nity) than that of the unphotolyzed cage (80 nM) at pH 7.2, with
pH-dependence similar to that of EGTA, EDTA, and DM-nitrophen. Unlike DM-
nitrophen, Mg
2
þ
binding to NP-EGTA is negligible (9 mM before and after
photolysis). Quantum e
ciency (0.23) is similar to that of DM-nitrophen, and
higher than for the nitr compounds, but less than that of azid-1. However,
photolysis e
Y
ciency is seriously limited by its low absorbance (975 M
1
cm
1
),
only 1/6—1/4 those of the nitr compounds and DM-nitrophen, and less than 3% of
azid-1's absorbance.
More recently, a dimethoxy-ortho-nitrophenyl derivative of EGTA (DMNPE-4)
was introduced (
Ellis-Davies and Barsotti, 2006
), with somewhat higher Ca
2
þ
a
Y
nity (48 nM), dropping with time constants of 10 and 17
m
s to 1 mM on
photolysis, low Mg
2
þ
-a
Y
ciency
(0.09) but over five times the absorbance (5140 M
1
cm
1
), thus twice the photoly-
sis e
Y
nity (7 mM), and under half the quantum e
Y
ciency of NP-EGTA. An additional very slow phase releasing 30% of caged
Ca
2
þ
with
Y
667 ms was observed.
Ellis-Davies' lab has also produced a new generation of EGTA-based chelators
using the novel photosensitive chromophore nitrodibenzofuran or NDBF-EGTA
(
Momotake et al., 2006
). This compound binds Ca
2
þ
with K
D
¼
t
100 nM at pH 7.2,
