Biology Reference
In-Depth Information
II. Rationale
A. Which Ca
2
þ
Bu
er Should You Use?
When selecting the appropriate Ca
2
þ
bu
V
V
er for your application, the main
consideration is to choose one with a dissociation constant (K
d
) close to the desired
free [Ca
2
þ
]. The ability of a bu
er to absorb or release ions and thus to hold the
solution at a given concentration of that ion is greatest at its K
d
. Just as you should
not choose PIPES (pK
a
¼
V
er a solution at pH 7.8, choosing a Ca
2
þ
bu
er
with a K
d
far from the desired [Ca
2
þ
] set point is a mistake. As a rule of thumb, the
bu
6.8) to bu
V
V
er's K
d
should not lie more than a factor of 10 from your desired [Ca
2
þ
].
In addition, the bu
V
nity for Ca
2
þ
than Mg
2
þ
since intracellular [Mg
2
þ
] is typically 10,000-fold higher than [Ca
2
þ
]
i
. Fortunately,
about a dozen suitable bu
V
er should exhibit a much greater a
Y
V
ers are available spanning the range from 10 nM to
). There are also a large number of fluorescent Ca
2
þ
indicators
(see Chapter 5) that can also serve as Ca
2
þ
bu
100
m
M(
Table
I
V
ers, giving one the opportunity to
er and measure free [Ca
2
þ
] with the same reagent. We will not focus on
both bu
V
Table I
Mixed stability constants for useful Ca
2
þ
bu
V
ers at 0.15 M ionic strength in order of Ca
2
þ
a
Y
nity
log K
0
Ca
K
d
K
0
Ca
(pH 7.4)/
K
0
Ca
(pH 7.0)
Ca
2
þ
bu
er
a
V
(pH 7.4)
K
0
Ca
/K
0
Mg
(pH 7.4)
References
CDTA
7.90
13 nM
2.7
120
Martell and Smith (1974, 1977),
Bers and MacLeod (1988)
EGTA
7.18
67 nM
6.2
72,202
Martell and Smith (1974, 1977),
Bers and MacLeod (1988)
Quin 2
6.84
144 nM 1.15
25,114
Tsien (1980)
BAPTA
6.71
192 nM 1.14
158,244
Tsien (1980)
Fura-2
6.61
242 nM 1.14
72,373
Grynkiewicz et al. (1985)
Dibromo-BAPTA
5.74
1.83
m
M 1.02
63,000
Tsien (1980)
4,4
0
-Difluoro-BAPTA
1.7
m
M
b
5.77
-
-
Pethig et al. (1989)
6.3
m
M
b
Nitr-5 photolyzed
5.2
-
-
Tsien and Zucker (1986)
5-Methyl-5
0
-nitro-BAPTA
22
m
M
b
4.66
-
-
Pethig et al. (1989)
40
m
M
b
5-Mononitro-BAPTA
4.4
-
-
Pethig et al. (1989)
NTA
3.87
134
m
M 2.5
8
Martell and Smith (1974, 1977),
Bers and MacLeod (1988)
ADA
3.71
191
m
M 1.24
32
Nakon (1979)
Citrate
3.32
471
m
M 1.03
1.3
Martell and Smith (1974, 1977),
Bers and MacLeod (1988)
5,5
0
-Dinitro-BAPTA
7 mM
b
2.15
-
-
Pethig et al. (1989)
a
Abbreviations: CDTA, cyclohexilinedinitrilo-N-N-N
0
-N
0
-tetraacetic acid; EGTA, Ethylene glycol bis (
b
-aminoethylester)
N-N-N
0
-N
0
-tetraacetic acid; BAPTA, 1,2-bis(o-aminophenoxy)ethane-N-N-N
0
-N
0
-tetraacetic acid; NTA, nitriloacetic acid;
ADA, acetamidominodiacetic acid.
b
Measured at pH 7 and 0.1 M ionic strength.
