Biomedical Engineering Reference
In-Depth Information
Table 10.1
The affinity of IP6 for deoxyhemoglobin (0.1 M KCl, 25 uC).
Adapted from ref.
15
.
pH
K
D
deoxyHb/M
,6.25 6 10
210
5.6
6.25 6 10
210
6.0
2.50 6 10
29
6.5
2.00 6 10
28
7.0
6.25 6 10
28
7.3
1.25 6 10
26
8.0
1.00 6 10
25
9.0
These IP6-Hb binding studies
16
using
31
P NMR are shown in Figure 10.6.
The conditions were pH 5.6, where K
D
#1 nM. The data are rather
unconventional for ligand-binding studies: we monitored the NMR signals of
the ligand, instead of those of the receptor. At conditions N ,1, there is excess
Hb over IP6, and one observes the spectrum of bound IP6. It is seen to vary
little between N 5 0.33 and N 5 0.75 [Figure 6(b)], in agreement with the very
tight binding. When N .1, there is excess IP6 over Hb. For instance, at N 5 3,
there is y2 mM free IP6, and y1 mM IP6 bound to Hb. If this was a slow-
exchange situation, the spectrum would show resonances for both bound and
free IP6. Since this is not the case, and because there is little or no exchange line
broadening, the NMR data establish a very fast exchange condition between
IP6 free and IP6 bound. Considering a typical chemical shift change of 0.5
ppm between IP6 free and bound, or 20 Hz (
31
P was at 41 MHz on this 1970s
Varian XL-100), we conclude that k
off
.. 2p 6 20 ..120 rad s
21
.
This fast exchange is very surprising, since a calculation of the maximum
possible off-rate for two-site exchange for a 1 nM binding event, using the
maximum value for a diffusion controlled on-rate
2
of 3 6 10
10
M
21
s
21
, yields
k
off
530 s
21
. Even at this maximum rate, when compared to a shift difference
of 120 rad s
21
, the binding would be in the slow-intermediate exchange regime,
and the NMR spectrum should consist of two resonances per phosphate and
be severely broadened.
Clearly, the NMR data is incompatible with a simple two-site exchange
model for the binding of IP6 to hemoglobin. The simplest extension is a three-
site exchange of the type
k
1f
k
2f
IzC
/
?
IC
/
?
IC
ð
10
:
19
Þ
k
1b
k
2b
where I represents IP6, C the cavity binding site of IP6 on hemoglobin, IC the
complex, and where IC* is some on-pathway intermediate. For the IP6
molecule this is a three-site exchange situation
