Biology Reference
In-Depth Information
these fluorescent indicators here, but they can be substituted for the bu
ers de-
scribed (especially when the fundamental binding properties have been measured).
Ethylene glycol bis(
b
-aminoethylether)-N,N,N
0
,N
0
-tetraacetic acid (EGTA) is one
of the best-known Ca
2
þ
bu
V
V
V
er in the range of
10 nM-1
m
M [Ca
2
þ
] at the typical intracellular pH of 7.2. However, if your goal is
tomake bu
ers, and it can be a reliable bu
ers in the 1-10
m
Mrange, BAPTA (1,2-bis(o-aminophenoxy)ethane-N,
N,N
0
,N
0
-tetraacetic acid) or dibromo-BAPTA (Br
2
-BAPTA) would be better
choices.
V
B. EGTA: The Workhorse of Biological Ca
2
þ
Chelators
By far the most popular Ca
2
þ
bu
V
er has been EGTA. This molecule has been
used extensively because its apparent dissociation constant (K
d
) at pH 7 (0.4
m
M) is
close to intracellular Ca
2
þ
levels and it has a much higher a
nity for Ca
2
þ
than for
Y
Mg
2
þ
(
100,000 times higher around neutral pH). However, the preparation of
Ca
2
þ
bu
ers using EGTA is complicated by the strong pH dependence of its Ca
2
þ
V
). Thus, while the free [Ca
2
þ
] would be about 400 nM
when EGTA is half saturated with Ca
2
þ
at pH 7, the free [Ca
2
þ
] in this same
solution would decrease by nearly 10-fold to 60 nM by simply raising the pH to
7.4! Therefore, the pH of Ca
2
þ
bu
a
Y
nity (see
Fig. 1
and
Table
I
ers made with EGTA must be very carefully
controlled, and the calculation of the appropriate amounts of EGTA and Ca
2
þ
to
use must be made at the desired pH. The purity of the EGTA is also a variable that
can cause substantial errors, as large as 0.2 pCa units in the free [Ca
2
þ
](
Bers, 1982;
Miller and Smith, 1984
).
V
EGTA
9
0.001
8
0.01
BAPTA
7
0.1
Br
2
-BAPTA
6
1
5
10
4
6
7
8
pH
Fig. 1
The pH dependence of apparent a
Y
nities (K
0
Ca
) for EGTA, BAPTA, and Br
2
-BAPTA at 20
C
and 150 mM ionic strength.
