Biology Reference
In-Depth Information
2
H
þ
½
K
1
K
2
a
2
¼
ðA1:11Þ
4
3
2
H
þ
H
þ
H
þ
H
þ
½
þ
½
K
1
þ
½
K
1
K
2
þ
½
K
1
K
2
K
3
þ K
1
K
2
K
3
K
4
The plots shown in
Fig. 7
were generated using the above expressions for
a
2
,
a
3
,
and
a
4
in conjunction with the four stepwise dissociation constants for EGTA.
In footnote 14, it was stated that K
d
represents the ''absolute'' or intrinsic
dissociation constant characterizing the fully deprotonated form of the chelator
(e.g., A
4
in the case above). At any pH where not all of the chelator is in the fully
deprotonated form, K
d
must be corrected for the weakening e
V
ect of acidic pH; the
corrected, or ''conditional,'' dissociation constant is K
0
d
. As one would expect, the
correction factor is the fraction of chelator that exists in the fully deprotonated
form at the desired pH (e.g.,
a
4
in the case of a tetrabasic acid like EGTA). Thus,
for a tetrabasic chelator,
¼
K
d
a
4
0
d
K
ðA1:12Þ
The plots shown in
Fig. 8
were generated using
Eq. (A1.12)
.
Appendix 2. Deriving an expression for the amount of
fluorescence emitted by a solution of fluorescent indicator
Light absorption by a solution containing a light-absorbing molecule, such as a
colorimetric or fluorescent indicator, is described by the Beer-Lambert Law:
A ¼log
I
I
0
¼ elc
ðA2:1Þ
where A is the absorbance (or ''optical density'') of the solution, I
0
is the intensity of
a light beam impinging on the solution, I is the intensity after the beam has passed
through the solution (I
0
I
¼
I
abs
is the amount of light absorbed),
e
is the molar
extinction coe
cient (also known as the molar absorptivity), l is the thickness of the
solution through which the light beampasses, and c is the concentration of the light-
absorbing molecule. The equation can be rearranged to the exponential form:
Y
I ¼ I
0
e
2
:
303
elc
ðA2:2Þ
By convention,
e
has units of M
1
cm
1
(i.e., l mol
1
cm
1
), l is measured in cm,
and c is measured in units of molarity (M, or mol l
1
). For typical imaging
experiments where fluorescent indicators are loaded into cells, numerical limits
may be defined for the three parameters of interest:
000M
1
cm
1
e <
50
;
10
4
cm
l<
50
10
6
M
c<
100
