Biology Reference
In-Depth Information
pH, it is often essential to use Ca 2 þ bu
ers to control free [Ca 2 þ ] at the desired
biologically relevant concentrations. Fortunately, there are numerous available
Ca 2 þ bu
V
nities that make this practical. However, there are
numerous caveats with respect to making these solutions appropriately with
known Ca 2 þ bu
V
ers with di
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erent a
Y
ers. These include pH dependence, selectivity for Ca 2 þ (e.g., vs.
Mg 2 þ ), ionic strength and temperature dependence, and complex multiple equili-
bria that occur in physiologically relevant solutions. Here we discuss some basic
principles of Ca 2 þ bu
V
ering with respect to some of these caveats and provide
practical tools (including freely downloadable computer programs) to help in the
making and calibration of Ca 2 þ -bu
V
V
ered solutions for a wide array of biological
applications.
I. Introduction
Cell biologists quickly learn how important it is to control the ionic composition
of the solutions used when studying cellular biochemistry, physiology, and phar-
macology. Bu
ering the pH of the solutions we use is so routine that one can
hardly imagine making a biological solution without the careful selection of the
appropriate pH bu
V
er and measurement of pH in the resulting solution. Indeed,
there are an array of popular zwitterionic amino acid pH bu
V
ers introduced by
Good et al. (1966) that are in widespread use (e.g., HEPES) and which complement
the natural physiological pH bu
V
V
ers for these purposes. In contrast, there has been
ering and measuring [Ca 2 þ ] because extracellular [Ca 2 þ ] levels
are typically in the millimolar range and such concentrations are easily measured
and prepared. However, intracellular [Ca 2 þ ] ([Ca 2 þ ] i ) is quite another matter
because these levels are more typically in the 100 nM-10 m M range which is not
as easily prepared or measured. For example, your source of distilled water could
easily have trace Ca 2 þ contamination in the range of 1-10 m M. This range of
contaminant Ca 2 þ can also come from chemicals and biochemicals commonly
used to make solutions. Additionally, there is often a considerable amount of
endogenous Ca 2 þ in biological tissue or cell samples which is not easily removed
or controlled. Therefore, when we are interested in studying intracellular reactions,
Ca 2 þ bu
less attention to bu
V
ering is extremely important.
In this chapter, we will present a practical guide to the preparation of Ca 2 þ
bu
V
er solutions. Our goal is to emphasize the methods and important variables to
consider while making the procedure as simple as possible. We will also introduce
computer programs which may be of practical use to many workers in this field.
One is a spreadsheet useful in making and validating simple Ca 2 þ calibration
solutions. The others are more powerful and extensive programs for the calcula-
tion of [Ca 2 þ ] (and other metals and chelators) in complex solutions with multiple
equilibria. These programs have been developed and described with maximum ease
of use in mind.
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