Biology Reference
In-Depth Information
There are many papers explaining how to calculate the proper amounts of
EGTA and Ca
2
þ
that must be combined to obtain a given free [Ca
2
þ
] (some are
listed below). Due to the steep pH dependence and slight Mg
2
þ
sensitivity, both pH
and Mg
2
þ
must be considered in the calculation and it is best accomplished by
computer. We provide a program for such calculations and describe it below.
Systematic errors in EGTA purity and pH can be a real practical problem (
Bers,
1982
), even with the best calculations for solution preparation. Thus, we also
recommendmeasuring the free [Ca
2
þ
] whenever possible (see below and Chapter 3).
C. BAPTA Family of Ca
2
þ
Bu
V
ers
Roger Tsien developed an analogue of EGTA in which the methylene links
between oxygen and nitrogen atoms were replaced with benzene rings to yield a
compound called BAPTA (
Tsien, 1980
;
Fig. 2
). This compound exhibits a much
lower pH sensitivity and much higher rates of calcium association and dissocia-
tion. These characteristics are mainly due to the fact that BAPTA is almost
completely deprotonated at neutral pH. Moreover, modifications of BAPTA
have been made to provide Ca
2
þ
bu
ers with a range of K
d
values covering the
biologically significant range of 0.1
m
M-10 mM (see
Table
V
;
Pethig et al., 1989
).
However, one disadvantage compared with EGTA is that the BAPTA family of
bu
I
ers exhibits a greater ionic strength dependence (see
Figs. 3-5
). In particular,
increasing ionic strength from 100 to 300 mM decreases the apparent a
V
nity
constant, K
0
Ca
for BAPTA or Br
2
-BAPTA by almost threefold, whereas the
Y
COO
-
-
OOC
COO
-
-
OOC
N
N
O
O
EGTA
COO
-
-
OOC
COO
-
-
OOC
N
N
O
O
X
X
X = H; BAPTA
X=Br; Br
2
-BAPTA
Fig. 2
Structural formulas for the Ca
2
þ
chelators EGTA (top) and BAPTA and Br
2
-BAPTA
(bottom).
