Biology Reference
In-Depth Information
It may well be asked, why not just use BAPTA rather than EGTA? The main
reason is expense, BAPTA is about 30 times more expensive. The other reason is
that EGTA is the ''Devil we know'' and indeed we do know much about its
chemistry (e.g., metal binding constants, D H values). For applications with small
volumes of solution though, it may be quite reasonable to replace EGTA with
BAPTA.
The ionic strength contribution of the pH bu
V
er should also be included in the
ionic strength calculation (I e ¼
0.5 S C i |z i |). This requires calculation of the fraction
of bu
V
er in ionized form (i.e., not protonated).
2. Potential Complications
Not all of the desired constants have been determined for the metals and
chelators of interest. This places some limitations on how accurately one can
predict the free [Ca 2 þ ] of a given complex solution or determine how much total
Ca 2 þ is required to achieve a desired free [Ca 2 þ ]. The same is true for other species
of interest (e.g., Mg 2 þ ,Mg 2 þ -ATP). Some Ca 2 þ bu
ers also can interact with Ca 2 þ
V
nity Ca 2 þ bu
in multiple stoichiometries (e.g., the low a
er, NTA (nitrilotriacetic
acid) can form Ca 2 þ -NTA 2 complexes). There can also be systematic errors in pH
measurements ( Illingworth, 1981 ) or purity of reagents. Purity can be estimated as
described above.
The pH problem is actually quite common, especially with combination pH
electrodes. To put it simply, the reference junction of some electrodes (particularly
with ceramic junctions) can develop junction potentials which are sensitive to ionic
strength. This problem can be exacerbated when the ionic strength of the experi-
mental solutions di
Y
V
ers greatly from the pH standards (typically low ionic strength
phosphate pH standard bu
V
V
ers). A systematic error in solution pH of about 0.2 pH
units is not at all uncommon. As is clear from Fig. 1 , this could translate into a 0.4
error in log K 0 Ca and produce a two- to threefold di
erence in free [Ca 2 þ ] even
V
where EGTA is at its best in terms of bu
er capacity.
While measuring the free [Ca 2 þ ] with an electrode can be extremely valuable, it is
not foolproof either. Ca 2 þ electrodes are not perfectly selective for Ca 2 þ (see
Chapter 3). For example, the selectivity of these electrodes for Ca 2 þ over Mg 2 þ
is about 30,000-100,000 ( Schefer et al., 1986 ). This roughly corresponds to the
di
V
erence in intracellular concentrations. Thus, a 100 nM Ca 2 þ solution with
1mMMg 2 þ would look to the electrode like a 110-130 nM Ca 2 þ solution. For
the Ca 2 þ electrodes described in Chapter 3 (using the ETH 129 chelator), the
interference by Na or K is less. For 140 mM Na or K in a 100 nM Ca 2 þ solution,
the apparent [Ca 2 þ ] would be only about 101 nM.
Some Ca 2 þ bu
V
ers can also interfere with Ca 2 þ electrodes. Citrate, DPA (dipi-
colinic acid), and ADA (acetamidoiminodiacetic acid), three low a
V
nity Ca 2 þ
Y
ers were found to interfere with Ca 2 þ electrode measurements, while NTA
did not ( Bers et al., 1991 ). Interestingly, citrate, DPA, and ADA (which modified
electrode behavior) also modified Ca 2 þ channel characteristics, but NTA did not.
bu
V
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