Biology Reference
In-Depth Information
photolyzable diazoketones. Absorption of one photon increases the Ca
2
þ
a
nity
from 89 to 2.2
m
M (with a 10% probability of producing a side-product with one
inactivated group). Absorption of two photons (with a probability assumed to
equal the square of the probability of one group absorbing one photon, and with a
measured quantum e
Y
Y
Y
ciency of 0.015) results in further increase of the a
nity to
55 nM, a total increase of 1600-fold. This large increase in a
Y
nity is, to some
extent, o
set by the small fraction of diazo-4 that can be doubly photolyzed
readily. Thus, a flash of light produces a variety of species: unphotolyzed, singly
photolyzed, doubly photolyzed, singly inactivated, doubly inactivated, and singly
photolyzed-singly inactivated, with a variety of transition probabilities among
species (
Fryer and Zucker, 1993
). Unphotolyzed diazo-4 is highly absorbent
(46,000 M
1
cm
1
at 371 nm for the free form; about 4600 M
1
cm
1
for the
Ca
2
þ
-bound form). The singly photolyzed species have absorbances of half these
values and doubly photolyzed diazo-4 has negligible absorbance at this wave-
length. Inactivation causes little change in absorbance.
A third member of this series, diazo-3, has a diazoketone attached to half the
cation-coordinating structure of BAPTA, and has negligible Ca
2
þ
a
V
nity.
On photolysis, diazo-3 produces the photochemical intermediates of diazo-2 plus
a proton, and may be used to control for these e
Y
ects of photolysis of the diazo
series. At one time, diazo-2 and diazo-3 (but not diazo-4) were commercially
available (Molecular Probes, Eugene, and Oregon), but these stocks appear to
have been exhausted.
V
B. Calculating E
V
ects of Photolysis
As for the nitr compounds, equilibration is faster than photolysis, so a flash of
light leads to a smooth step transition in the concentration of Ca
2
þ
chelator
species. If the percentage of photolysis caused by a light flash is known, the
proportions of photolyzed and inactivated diazo-2, or of the six species of diazo-
4, can be calculated. Usually, diazo is injected without any added Ca
2
þ
, so the
e
ers is to reduce the [Ca
2
þ
]
i
from its resting value. This
change can be calculated only if the total Ca
2
þ
bound to the native bu
V
ect of photoreleased bu
V
V
er in
cytoplasm as well as the characteristics of that bu
er are known. These character-
istics often can be inferred from available measurements on cytoplasmic Ca
2
þ
bu
V
er power and the normal resting [Ca
2
þ
]
i
level. The more usual application of
these substances is to reduce the e
V
ect of a physiologically imposed rise in [Ca
2
þ
]
i
.
In many cases, the magnitude of the source of this Ca
2
þ
is known, as in the case of
aCa
2
þ
influx measured as a Ca
2
þ
current under voltage clamp or the influx
through single channels estimated from single channel conductances. Also, the
magnitude of the total Ca
2
þ
increase in a response can be estimated frommeasured
increases in [Ca
2
þ
]; and estimates of cytoplasmic bu
V
V
ering. With this information,
the expected e
ect of newly formed diazo photoproducts on a physiological rise in
[Ca
2
þ
]
i
can be calculated by solving di
V
usion equations that are appropriate for the
distribution of Ca
2
þ
sources before and after changing the composition of the
V
