Biology Reference
In-Depth Information
centrifugation of the same sample. This will also reveal how much of the tubulin is
polymerization competent. Once steady state is reached in the spectrophotometer,
remove an aliquot from each sample (to determine total tubulin concentration,
C
T
) and a second aliquot for centrifugation. Pellet the polymer from this aliquot
by centrifugation at
g
for
15 min in a microcentrifuge). Remove an aliquot of the supernatant and determine
protein concentration. This is the concentration of the soluble dimer,
C
D
. The con-
centration of the polymer (
C
P
) can now be calculated, since
C
T
¼
200,000
g
for 5 min in an Airfuge (or at
20,000
C
D
. Plot the
data as shown in
Fig. 14.1
. The measurement of
C
D
and
C
P
, plotted as a function of
C
T
, should show
C
D
increasing up to
C
C
and then remaining constant above that. If
the slope of
C
D
as a function of
C
T
is greater than 0 above
C
C
, this is an indication that
some fraction of the tubulin dimers are not competent to polymerize (dead tubulin). If
the slope is 0, that is, if
C
D
remains nearly constant at
C
T
>
C
P
þ
C
C
, then this may be taken
as evidence that essentially all of the tubulin is polymerization competent.
A simpler way to determine
C
C
is to centrifuge a polymerized sample at a
single
value of
C
T
, yielding
C
D
from the supernatant, which is equal to the
C
C
as long as
there is no significant incompetent tubulin. This condition can be shown by a (pre-
vious) experiment with the same tubulin preparation, using the procedure described
earlier, or in quick form, by repeated centrifugation on a second sample prepared at a
higher
C
T
. If the supernatants yield the same
C
D
, then this may be taken to be the
C
C
.
14.1.3
Polymerization curves and the “turbidity coefficient”
An important but often overlooked component of a polymerization assay monitored
by OD is the shape of the polymerization curve. Tubulin assembly is characterized by
a lag time, a period of net growth, and a steady state (plateau). An increase in the
amount of polymerized tubulin is accompanied by an increase in the maximum slope
as well as the plateau of the curve.
Figure 14.2
shows assembly curves in a few crit-
ical concentration experiments, quantitated in
Table 14.1
. Note how the maximum
slope increases as the amount of assembled tubulin (plateau) increases. The three
parameters of the curve to examine are the lag period (the time before the OD begins
to increase), the maximum rate of OD increase, and the OD reached at steady state.
These parameters are often highly correlated, but need not be. Note that the lag time
may be relatively long or barely observable. The lag time generally reflects the num-
ber of nucleation events, which affects the
number
of MT (an increased number of
nucleation events produces an increased number of shorter MT), but may not affect
the total mass of polymer produced. Thus, in comparing two solutions, a shorter lag
phase in one could indicate an increased number of nucleation events producing an
increased number of shorter MT, even if the total mass of MT at plateau is the same
between the two solutions.
The relationship between steady-state OD and polymer mass is a very useful pa-
rameter. The turbidity coefficient, which we will refer to as
e
* (by analogy to
e
, the
extinction coefficient for absorbance), is the turbidity (OD) of the polymer per con-
centration, thus OD/
C
P
. The value of
* is different for different polymers. A solution
containing pure MT at a concentration of 1 mg/ml (
C
P
) will have an OD of not more
e
