Biology Reference
In-Depth Information
form, and interpretation depends on understanding the nature of the product. These
can be distinguished by careful measurement of the turbidity (OD) yield per polymer
concentration, and by the turbidity as a function of wavelength (see below).
Fluorescence enhancement upon polymerization provides a simple and sensitive
method for measuring polymerization that is less sensitive to different polymer forms
than is turbidity. A drawback is the need for equipment that is less widespread than
spectrophotometers: spectrofluorometers or fluorescence-based plate readers.
A number of compounds have been described whose fluorescence increase can be
used to monitor MT polymerization; DAPI has been most widely applied for this
purpose (
Bane, Ravindra, & Zaydman, 2007; Barron et al., 2003
). DAPI fluores-
cence increases on binding to tubulin, and the affinity of DAPI for tubulin increases
upon tubulin polymerization. Thus, the fluorescence emission of a given concentra-
tion of DAPI will increase upon tubulin addition and increase even more so when that
tubulin polymerizes. This relatively straightforward effect allows assay of polymer-
ization that is less affected by polymorphic assembly than is turbidity (
Heusele et al.,
1987
). A small cautionary note is that addition of DAPI may influence the polymer
form being measured (
Vater, B
¨
hm, & Unger, 1993
).
Although, in principle, a fluorescence assay might be expected to be more sen-
sitive (requiring less of the assayed reagents, i.e., tubulin here) than one based on
optical density, in this application that may not be so. This is because the amount
of tubulin required is driven by the critical concentration of tubulin polymerization
and the volume of the assay rather than by the sensitivity of the optical method.
14.1.2
Critical concentration
An important parameter to consider in the design of any
in vitro
tubulin polymeri-
zation experiment is the critical concentration (
C
C
). This is the total tubulin concen-
tration below which no polymerization occurs, or equivalently, the concentration of
dimeric (nonpolymerized) tubulin (
C
D
) in steady-state equilibrium with MT poly-
mer. For any experiment designed to measure polymerization, the initial (total) tu-
bulin concentration (
C
T
) must be higher than the critical concentration (
C
C
). The
critical concentration is not an intrinsic property of tubulin, but rather is a system
parameter that is dependent on the solution composition (buffer, pH, ionic strength,
divalent cations, etc.) and temperature. It is equivalent to the
K
d
(
1/
K
a
) for tubulin
at the MT ends and therefore gives thermodynamic information about the system.
The most thorough way to measure the critical concentration for a given set of con-
ditions is to prepare multiple samples with increasing
C
T
, allow them to polymerize to
steady state, measure some parameter that is proportional to polymerized tubulin, such
as OD or fluorescence, and plot that parameter versus
C
T
. When the line through the
data is extrapolated to zero polymer, the intersection on the
C
T
axis is the
C
C
. Since the
system is in steady-state equilibrium, an equivalent procedure is to allow polymeriza-
tion to occur at high
C
T
and then dilute to several lower
C
T
, allow those to come to
steady state, and plot the result. Performing both a series of increasing initial (unpo-
lymerized) concentrations and dilution-based decreasing concentrations (from a poly-
merized sample) should yield the same
C
T
and overlapping data sets. This is illustrated
¼
