Biomedical Engineering Reference
In-Depth Information
With equations of this type the titration pattern as in Figure 10.5(b) were
obtained. Here, the semi-equivalence point lies half way. The calculation
indicates that dissociation of the last proton (from charge 211 to 212) costs
3.7 kcal M
21
more than the first (from charge 26to27). While the details of
the experimental titration data were not captured by this model, the essential
characteristics certainly are. A more elaborate model was not pursued, because
that would also have to include pH-dependent conformational changes as are
expected to occur by the changing electrostatics. Such conformational changes
could possibly explain the total lack of titration of the blue signal between pH
7.5 and 9.5 in Figure 10.3. We have also observed backwards titration for one
of the phosphate groups of IP5 (not shown here, but see ref.
11
), which can
certainly not be accounted for by electrostatic theory alone.
In summary, the electrostatic interaction between the phosphate groups of
IP6 give rise to a proton association/dissociation thermodynamics extending
over five pH units. NMR showed that the electrostatic interactions affect the
entire molecule. The presented thermodynamic model can account for the
anomalous semi-equivalence points in the proton binding.
10.3 The Binding of Inositol Hexakis Phosphate to
Hemoglobin: Fast-Exchange Kinetics for
Nanomolar Affinity
Inositol hexakis phosphate, IP6, (see Figure 10.1) is known to bind to the
positively charged N-termini of the hemoglobin (Hb) b-chains, with a
stoichiometry of one IP6 per Hb tetramer.
12
This binding site is referred to
as the 'cavity'. At neutral pH, IP6 binds to deoxyhemoglobin with a K
D
5 62
nM,
13
while the binding to oxyhemoglobin occurs with a K
D
5 100 mM.
15
IP6
thus binds much tighter to deoxyHb than to oxyHb, hence lowering the Hb
oxygen affinity by allostery.
9
The allostery has a structural explanation: the
IP6-binding cavity is more accessible in deoxyHb than in oxyHb.
12
The
binding of IP6 to hemoglobin is accompanied by proton uptake, a property
that was utilised to measure the IP6 binding affinity.
13,14
The proton uptake is
caused by formation of salt bridges between the IP6 phosphates and the a-
amino group of valine 1, the side-chains of histidines 2 and 143, and of lysine
82.
12
The a-amino group and the histidines titrate around neutral pH; below
pH 6 these groups are fully protonated. In those conditions, they interact even
more strongly with IP6 because no thermodynamic work to protonate the
groups upon interaction is expended. The exact value of the IP6-Hb affinityas
a function of pH was calculated from a quantitative treatment of this effect.
15
One finds that the affinity of IP6 for deoxyHb at pH 6 is extremely tight, witha
K
D
of 1 nM or less (see Table 10.1). The low pH values are of relevance only
because the NMR studies of the IP6-deoxyHb interaction described in the
following were carried out at or below this pH.
16
