A Practical Guide to the Preparation of Ca Buffers - Methods in Cell Biology - page 17

Some people include 1-2 m M TPEN, a heavy metal chelator in Ca 2 þ -bu

er
solutions. This can chelate submicromolar amounts of heavy metals, which may
or may not be chelated by the dominant Ca 2 þ bu

V

er. This may not be important in
routine applications, but may ensure that the Ca 2 þ -sensitive process under study
will not be altered by trace amounts of other metals. All solutions should be made
and stored in clean plastic ware (careful washing and extensive rinsing in deionized
water is required). Glass containers should be avoided. EGTA can leach Ca 2 þ out
of glass leading to gradual increase in free [Ca 2 þ ] in the solutions. We have often
been able to store Ca 2 þ calibration solutions for more than 6 months in polypro-
pylene bottles (provided that there is no organic substrate to foster bacterial
growth).
An accurate [Ca 2 þ ] standard is important for making Ca 2 þ bu

V

V ers. It is di Y cult

to make accurate [Ca 2 þ ] using CaCl 2 2H 2 O typically used to make physiological

solutions. This is because the hydration state varies making stoichiometric weigh-
ing imprecise. CaCO 3 can be more accurately weighed, but has the disadvantage
that you must then drive o

the CO 2 with prolonged heating and HCl, unless
HCO 3 is desired in the solutions (which is a weak Ca 2 þ bu

V

er itself). A convenient
alternative is to buy a CaCl 2 standard solution and we use a 100 mM CaCl 2
solution from Orion (BDH also sells an excellent 1 M CaCl 2 standard). To save
money, one can titrate a larger volume of CaCl 2 to the same free [Ca 2 þ ] as the
Orion standard using a Ca 2 þ -electrode.
It is also important to prepare accurate stock solutions of Ca 2 þ chelators. EGTA
from di

V

er somewhat in purity ( Bers, 1982; Miller
and Smith, 1984 ), but manufacturers provide purity estimates that help (we find
that purity typically ranges from 95 to 100% of the stated purity). BAPTA has also
been reported to contain 20% water by weight ( Harrison and Bers, 1987 ), but can
be dried at 150 C until the weight is constant to assure removal of water. If one
measures the total bu

V erent commercial sources di V

V

er concentration (as described in section above) this prob-
lem can be largely obviated. We typically measure the purity of each lot of EGTA
or BAPTA that we use, taking this approach. Then we often keep track on the
bottle itself, so that we can confirm the value upon subsequent tests with the same
batch. EGTA (in the free acid form) is also not very soluble because of the acid pH.
For neutral pH solutions, it is practical to dissolve EGTA with KOH in a 1:2
stoichiometry, since at neutral pH two of the four protons on EGTA are disso-
ciated (vs. all four for BAPTA).
When Ca 2 þ is added to EGTA solutions, 2 mol of H þ are released for each mole
of Ca 2 þ bound. Thus, the pH should always be adjusted as the Ca 2 þ is being added
or afterward. The strong pH dependence of the K 0 Ca of EGTA ( Fig. 1 ) emphasizes
the importance of this point. We typically measure [Ca 2 þ ] and pH simultaneously
just before the solutions are brought up to final volume (for approximate pH
adjustment) and after, for final pH adjustment (as close to the third decimal
place as possible) and [Ca 2 þ ] measurement. The solutions are also checked again
later to assure consistency. The rigorous attention to pH adjustment will obviously
be less crucial for the BAPTA bu

V ers.