Biology Reference
In-Depth Information
is 4 nmol. If the cells are 20
m
m in diameter, then the 1 million cells have a total
intracellular volume of 4.2
m
L. Further, if cells are loaded to a final intracellular
indicator concentration of 150
m
M (a generous estimate), then the total amount of
AM uptake by cells is 0.63 nmol, which is still much less than the 4 nmol available
in the loading medium.
IV. Manipulation of [Ca
2
þ
]
In studying Ca
2
þ
-dependent cellular processes, raising or lowering intracellular
or extracellular [Ca
2
þ
] is frequently desirable. Conventional techniques for achiev-
ing these ends require the use of Ca
2
þ
bu
V
ers or ionophores and will be discussed in
this section.
A. Using EGTA and BAPTA as Extracellular Ca
2
þ
Bu
ers
Because it is highly selective for binding Ca
2
þ
over Mg
2
þ
,
12
EGTA is the most
commonly used Ca
2
þ
bu
V
V
er. However, because two of the ligand atoms in EGTA
are tertiary alkylamino nitrogens, the two highest pK
a
s of EGTA are 8.90 and
9.52,
13
implying that at physiological pH EGTA will exist primarily as protonated
species—a fact that is illustrated more quantitatively in
Fig. 7
. For example,
Fig. 7
shows that, at pH 7.2,
98% of EGTA in solution exists as H
2
EGTA
2
,
2% as
HEGTA
3
, and only a negligible fraction is in the EGTA
4
form. Therefore, the
Ca
2
þ
-binding reaction near physiological pH is fairly represented as
2H
þ
That two H
þ
ions are liberated in the binding reaction means that the binding of
Ca
2
þ
by EGTA should have very steep pH dependence, as a plot of pK
0
d
(Ca)
14
versus pH indeed shows (
Fig. 8
). For a concrete example, a drop in pH from 7.2 to
7.1 changes the K
0
d
(Ca) of EGTA by a factor of
H
2
EGTA
2
þ
Ca
2
þ
>
CaEGTA
2
þ
1.6, that is, small errors in pH
can lead to significant uncertainties in the dissociation constant. In contrast,
12
For EGTA,
D
pK
d
¼
pK
d
(Ca
2
þ
)
pK
d
(Mg
2
þ
)
¼
5.58; therefore, EGTAbinds Ca
2
þ
more tightly than
Mg
2
þ
by a factor of 380,000 (i.e., 10
5.58
). For comparison, in the case of EDTA,
D
pK
d
¼
1.78, which
represents only a 60-fold di
V
erence in EDTA's a
Y
nity for Ca
2
þ
and Mg
2
þ
. BAPTA [1,2-bis(o-aminophe-
noxy)ethane-N,N,N
0
,N
0
-tetraacetic acid] has a selectivity similar to that of EGTA:
D
pK
d
¼
5.20.
13
At 25
C and 0.10 M ionic strength. Data pertaining to EGTA that are used in this section are from
Martell and Smith (1974)
.
14
In the metal chelator literature, K
d
is used for the ''absolute'' (or intrinsic) dissociation constant
and represents the dissociation constant characterizing the fully deprotonated form of the chelator. K
0
d
represents K
d
that has been corrected for the weakening e
V
ect of acidic pH (thus K
0
d
is the working
dissociation constant at a specific pH). This convention (K
d
vs. K
0
d
) is not followed consistently in the
applications literature. Details of how pH correction is applied to convert K
d
into K
0
d
are described in
Appendix 1
.
