Biology Reference
In-Depth Information
total [Ca
2
þ
], and photolysis rate (
Zucker, 1993
) then may be used to calculate
photolysis rate per flash or per second of steady light exposure.
Most photosensitive compounds also undergo substantial absorbance changes
after photolysis. These changes can be monitored during repeated exposure to the
light source without a Ca
2
þ
indicator; the number of flashes or the duration of light
exposure required to reach a given percentage photolysis then can be determined.
Realizing that photolysis proceeds exponentially to completion (
Zucker, 1993
),
these data can be used to determine the photolysis rate directly. Ideally, both
methods should be used to check for consistent results. A final method for
determining photolysis rate is using high pressure liquid chromatography
(HPLC) to separate and quantify parent chelators and photoproducts in the
reaction solution after partial photolysis (
Walker, 1991
).
VIII. Purity and Toxicity
When experiments do not work as planned, the first suspected source of error is
the integrity of the photolabile chelator. Di
V
erent procedures have proved most
useful for testing the di
erent classes of compounds. The nitr and diazo com-
pounds undergo large absorbance changes on binding calcium and photolysis.
A 100
m
M solution (nominally) of the chelator is mixed with 50
m
MCa
2
þ
in
100 mM chelexed HEPES solution (pH 7.2), and 0.3 ml is scanned in a 1-mm
pathlength spectrometer. Then 1
m
l1MK
2
EGTA is added to bring the [Ca
2
þ
]to0,
and the sample is scanned again. Finally, 1
m
l 5 MCaCl
2
is added to provide excess
Ca
2
þ
, and a third scan is recorded. The first scan should be midway between the
other two. If the first scan is closer to the excess Ca
2
þ
scan, it is indicative of a lower
than expected concentration of the chelator, probably because of an impurity.
Alternatively, Ca
2
þ
may have been present with the chelator, which may be
checked by running a scan on the chelator with no added Ca
2
þ
and comparing
the result with a scan with added EGTA; they should be identical. Ca
2
þ
free and
Ca
2
þ
-saturated chelator solutions also are scanned before and after exposure to
UV light su
V
cient to cause complete photolysis; the spectra are compared with
published figures (
Adams et al., 1988, 1989; Kaplan and Ellis-Davies, 1988
)to
determine whether the sample was partially photolyzed at the outset. The Ca
2
þ
a
Y
nities of unphotolyzed and photolyzed chelators can be checked by measuring
the [Ca
2
þ
] of 50%-loaded chelators with a Ca
2
þ
-selective electrode.
The absorbance of DM-nitrophen and some related chelators is almost Ca
2
þ
independent, so these procedures are not e
Y
ective. A solution of DM-nitrophen
nominally of 2 mM concentration is titrated with concentrated CaCl
2
until the
[Ca
2
þ
] measured with an ion-selective electrode suddenly increases; this change
indicates the actual concentration of the chelator and gives an estimate of purity.
The a
V
nity of the photolysis products can be measured as for the other chelators;
spectra before and after photolysis indicate whether the sample was already
partially photolyzed.
Y
