Biology Reference
In-Depth Information
The latest addition to the nitr-like class of compounds based on BAPTA is
Azid-1 (
Adams et al., 1997
). This compound was derived from the high-a
nity
fluorescent indicator derivative of BAPTA, fura-2, by addition of an azido substit-
uent to fura's benzofuran-3 position. Unlike fura-2, neither this compound nor its
photoproducts are fluorescent; and unlike the other nitr compounds and the
dimethoxynitrophenyl class of Ca
2
þ
chelators (see below), it relies on the photo-
sensitivity of an aromatic azide rather than a nitrobenzyl group. UV absorption
peaking at 372 nm (342 nm for the Ca
2
þ
-bound form) probably leads to formation
of a nitrene which steals hydrogen from water to produce an amidine, which with
another hydrogen converts to a nitrenium that rapidly combines with water to
form an amidinium that reacts with OH
to produce the final low-a
Y
nity electron-
withdrawing benzofurane-3-one photoproduct plus ammonia. Thus, photolysis
absorbs one net proton and produces one molecule of ammonia for each molecule
of azid-1 photolyzed, which can lead to an elevation of pH
i
in weakly bu
Y
ered cells.
This disadvantage is counterbalanced by substantial advantages. Photolysis of
both Ca
2
þ
-bound and Ca
2
þ
-free forms of zaid-1 is phenomenally e
V
Y
cient
1), and azid-1 is very UV-dark, absorbing at 33,000 M
1
cm
1
when
Ca
2
þ
-bound (or 27,000 M
1
cm
1
when free); these factors combine to make it
250-300 times more sensitive to light than nitr-5! Moreover, its Ca
2
þ
-a
(Q.E.
nity drops
from 230 nM to 120
m
M, on photolysis, a change that is 12 times the change in nitr-
5a
Y
nity on photolysis. Like the nitr compounds, it hardly binds Mg
2
þ
at all
(K
D
¼
Y
8 mM), and its Ca
2
þ
-binding (
10
9
M
1
s
1
) and photolysis rates (
2
m
s)
are equally rapid. In most respects, azid-1 comes closest to the ideal-caged Ca
2
þ
compound. Unfortunately, its synthesis is quite di
t
<
cult, and it has never been
commercially available; at present, apparently none exists at all.
Y
B. Calculating [Ca
2þ
]
i
Changes in Cells
If nitr-5 or azid-1 is photolyzed partially by a flash of light, the reduction in Ca
2
þ
a
Y
nity of a portion of the chelator occurs within
0.3 ms. During this period of
photolysis, low-a
Y
nity bu
V
er is being formed and high-a
Y
nity bu
V
er is vanishing
while the total amount of Ca
2
þ
remains unchanged. As the bu
V
er concentrations
change, Ca
2
þ
ions reequilibrate among the new bu
V
er concentrations by shifting
from the newly formed low-a
Y
nity photoproduct to the remaining unphotolyzed
high-a
usion
limit (as calculated from
Adams et al., 1988
; see also
Ashley et al., 1991b
), this
equilibration occurs much faster than photolysis, and Ca
2
þ
remains in quasi-
equilibrium throughout the photolysis period. The [Ca
2
þ
]
i
in a cell rises smoothly
in a step-like fashion over a period of 0.3 ms from the low level determined by the
initial concentrations of total Ca
2
þ
, and unphotolyzed chelator to a higher level
determined by the final concentrations of all the chelator species after partial
photolysis. At least in the case of nitr-5, [Ca
2
þ
]
i
remains under the control of the
low- and high-a
Y
nity caging chelator. Since the on-rate of binding is close to the di
V
nity species, so the elevated Ca
2
þ
is removed only gradually by
extrusion and uptake into organelles. Thus, nitr-5 and azid-1 are well suited to
Y
