Biology Reference
In-Depth Information
With compounds that bind irreversibly, it is not possible to extract the compound
from the pellets due to the covalent bond (reversible MSAs are easily recovered from
the pellets). The stoichiometry of ZMP or any other irreversible ligand can be mon-
itored by its presence in the supernatant, which will increase as extra ligand is added
once saturation of the binding site has occurred (which is the same as for a reversible
binder). Only the supernatant samples need to be analyzed in this case. If 1:1 stoi-
chiometry is occurring, the test compound will not appear in the supernatant samples
until its concentration has increased to greater than the concentration of the total
number of binding sites in the reaction (concentration in which the binding sites
are saturated) (
Fig. 19.3
B).
19.2.5
Binding kinetics
If the test ligand is an irreversible MSA, there are no thermodynamic equilibrium
parameters to characterize. The kinetic parameters of the binding can, however,
be studied by two different methods using either Flutax-2 or HPLC.
19.2.5.1
Flutax-2 method
The kinetics of the reaction of compounds with cross-linked MTs is measured by
determining the inhibition of Flutax-2 binding.
1.
Incubate 5
M test compound (or equivalent
volume of DMSO) at 25
C in GAB buffer over a series of time points (30 min-
overnight). The time points can be staggered to all finish incubating at the same
time.
2.
After the desired incubation, add 10
m
M taxoid binding sites with 6
m
M Flutax-2, incubate for 5 min, and
centrifuge the samples (20 min, 37
C).
3.
Remove the supernatant and resuspend the pellet in 10 mM NaPi buffer with 1%
SDS, pH 7.0 buffer.
4.
Dilute 50
m
m
L of supernatant sample in 200
m
L of 10 mM NaPi buffer with 1%
SDS, pH 7.0, and 50
m
L of the pellet in 50
m
L GAB 0.1 mM GTP and 150
m
Lof
10 mM NaPi buffer with 1% SDS, pH 7.0.
5.
Prepare a standard curve (0-10
M Flutax-2) in 1:4 GAB 0.1 mM GTP:10 mM
NaPi buffer with 1% SDS, pH 7.0.
6.
Measure concentration of Flutax-2 in the pellet and supernatant
spectrofluorometrically (
m
l
ems
520 nm) in a black 96-well plate.
Calculate the free (supernatant) and bound (pellet) Flutax-2 concentrations from
the standard curve.
l
exe
495 nm and
Fast covalent binders such as ZMP will immediately bind to the MT in an irreversible
manner, and no Flutax-2 will be associated with the pellet after 30 min. Compounds
that do not bind irreversibly will have 100% of the Flutax-2 associated with the pel-
let, given that it is in excess concentration. Slow covalent binders such as dactylolide
bind covalently over time, will show a decrease in Flutax associated with the pellet
over time (
Fig. 19.4
).
