Biology Reference
In-Depth Information
Table 14.1
Buffer effects on polymerization reactions
Critical concentration
C
C
(g/l)
A
350
/(mg/ml
polymer)¼A/C
P
¼
«
*
Buffer
Pipes/Mg (0.1 M/0.5 mM) pH 7.0
2.51
0.23
Pipes/Mg (0.1 M/0.5 mM)
þ
1M
1.41
0.22
Glycerol pH 7.0
Pipes/Mg (0.1 M/0.5 mM)
þ
1M
0.16
0.24
TMAO pH 7.0
1 M Glutamate pH 6.6
0.22
0.65
Pipes/Mg (0.1 M/0.5 mM) pH
7.0
0.07
0.50
þ
10
m
M paclitaxel
Critical concentrations and turbidity coefficients are presented for polymerization in several buffers.
Critical concentrations in the different buffer conditions were determined by pelleting assay. Buffers were
prepared beforehand except for the paclitaxel-containing buffer where the paclitaxel was added
immediately before the reaction. In addition, DMSOwas kept at 1% for all buffers tomaintain consistency
due to its necessity in the case of paclitaxel. Reactions of 50
l were carried out directly in centrifuge
tubes so as not to lose protein in transfer. Reactions were prepared by adding to the buffer 0.5
m
m
lof
0.1 MGTP to a final concentration of 1 mM, followed by paclitaxel in that one case, then 6
l of 25 mg/ml
purified tubulin (3 mg/ml final concentration), except in the case of paclitaxel-containing buffer where 1
m
m
l
tubulin was added (0.5 mg/ml final concentration). The solutions were placed in a 30
C water bath
for 30 min. The tubes were removed from the bath and centrifuged in the Airfuge for 5 min. The
supernatant was assayed with BioRad Protein Assay to determine soluble tubulin concentration (C
D
),
corresponding to C
C
.
Turbidity coefficients were measured using optical density measurements carried out in a
spectrophotometer and reactions prepared in 50
l cuvette. Reactions were prepared as similarly as
possible to the reactions performed for critical concentration measurements. The components of
the polymerization solution were added to the cuvette, buffer, GTP, paclitaxel (in the one case), and
tubulin. These were mixed quickly, but thoroughly by pipetting up and down, and then placed into the
spectrophotometer at 30
C. Optical density at 350 nm was monitored for 30 min at 2 s intervals (this
short interval is only necessary for conditions that give a short lag time, i.e., paclitaxel). The DOD is
determined by subtracting the initial OD value in the lag phase from the plateau OD in the steady-state
phase. The DOD divided by the calculated polymer concentration gives e*.
m
quantitated in
Table 14.1
. The high OD seen in the upper plot (OD vs. time) in glu-
tamate is not due to an increased mass of MT produced, but rather is due to production
of some fraction of sheet polymers, indicated by the
* value of 0.65. All that is re-
quired to check that the OD is due to production of MT is to measure the OD at steady
state, measure
C
P
as described earlier, and divide OD by
C
P
. It should be noted that
production of non-MT polymers does not mean an invalid assay. Indeed, the increased
OD yield by glutamate polymerization gives a stronger signal of polymerization, and
the polymers have the expected sensitivity to temperature and tubulin-binding drugs as
do MT (
Hamel, 2003
). It simply means that changes in OD can be influenced by poly-
mer
type
as well as by polymer
quantity
, and a simple control will check this.
This is an important control because often the addition of a test compound may
result in increased or decreased OD, relative to a control solution, and this will be
interpreted (and reported) as an increase or decrease in the quantity of MT produced.
Often it can be that the compound altered the
type
of polymer produced, rather than
the
quantity
. This simple procedure can avoid such an incorrect interpretation.
e
