Biology Reference
In-Depth Information
Ca
2+
−
O
2
C
CO
−
CO
−
−
O
2
C
Ca
2+
CO
−
−
O
2
C
−
O
2
C
CO
−
N
N
N
N
h
n
O
O
O
O
O
H
CH
3
CH
3
HO
NO
2
NO
+
H
2
O
nitr-5
(high Ca
2+
affinity)
Photolyzed nitr-5
(low Ca
2+
affinity)
O
O
O
O
Fig. 2
Reaction scheme for the photorelease of nitr-5.
with significantly higher Ca
2
þ
a
nities (54 nM, decreasing to 3
m
M after photol-
ysis at 120 mM ionic strength). To increase the change in Ca
2
þ
-binding a
Y
nity on
photolysis, nitr-8 was created with a 2-nitrobenzyl group on each aromatic ring of
BAPTA. Photolysis of each group reduces a
Y
nity only about 40-fold, as for nitr-
5 and nitr-7, but photolysis of both nitrobenzyl groups reduces a
Y
Y
nity nearly
3000-fold, to 1.37 mM, with a quantum e
ciency of 0.026. Finally, nitr-9 is a
dicarboxylate 2-nitrobenzhydrol with a low Ca
2
þ
a
Y
Y
nity that is una
V
ected by
photolysis; this compound can be used to control for nonspecific e
V
ects of the
photoproducts.
Initially, nitr-5 was the substance most often applied in biological experiments,
largely because it was the first photolabile chelator to have most of the qualities of
the ideal substance. The limited a
nity for Ca
2
þ
of this substance in the unpho-
tolyzed form requires that it be lightly loaded with Ca
2
þ
when introduced into
cells; otherwise, the resting [Ca
2
þ
]
i
will be too high. However, the compound in a
lightly loaded state contains little Ca
2
þ
to be released on photolysis. Nitr-7 alle-
viates this problem with an a
Y
nity closer to that of normal resting [Ca
2
þ
]
i
, but its
Y
synthesis is more di
cult and its photochemical kinetics are significantly slower.
Both compounds permit less than two orders of magnitude increase in [Ca
2
þ
]
i
,
generally to only the low micromolar range, and then only with very bright flashes
or prolonged exposures to steady light to achieve complete photolysis. Nitr-8 per-
mits a much larger change in [Ca
2
þ
]
i
. Photolysis kinetics for this compound have
not yet been reported. Neither nitr-8 nor the control compound nitr-9 is presently
commercially available; nitr-5 and nitr-7 are supplied by CalBiochem (La Jolla,
California).
Y
