Biology Reference
In-Depth Information
pH, etc.) are kept constant, the same eigenvalues should hold for all concen-
tration conditions so that global fitting can resolve the individual constants.
Furthermore, the kinetic information can be combined to yield thermody-
namic parameters, that is, binding constants or free energy of binding.
In the rapid-mixing experiment, the protein sample is at equilibrium
prior to encounter with the ligand L. Thus, the initial concentrations of
Hb(6c) and Hb(5c) are dictated by
K
X
k
þ
X
/
k
X
. Two versions of the flash
photolysis experiment can be implemented. In the first, there is no added
exogenous ligand. X is flashed off, leaving Hb(5c) behind. The kinetics
of X rebinding is then obtained (
Vos et al., 2008
). In the second, HbL is
prepared and L is flashed off, again leaving Hb(5c) behind and allowing
for measurements of L rebinding. This has the advantage of simpler initial
conditions for the determination of
k
þ
L
. Nevertheless, the kinetic behaviour
of the haemoglobins considered in this chapter has not been easy to inter-
pret. This is in part because the mechanism can be considerably more com-
plicated than captured by the two-equation model and because the
concentration regimes necessary for complete description may be difficult
to access experimentally. Higher complexity is also expected of proteins that
are not monomeric, although so far, the studied algal and cyanobacterial glo-
bins have no quaternary structure interfering with the measurements.
The function of a globin in the cell depends on the flux of ligands, itself
dependent on all the rate constants pertaining to the system and the intra-
cellular concentrations. For realistic modelling, rate-determining constants
are required under the conditions that best represent cellular metabolic
states. The organisms considered here vary widely in their physiology and
the conditions they encounter. In fact, cyanobacteria form an extremely
complex phylum (
Bryant & Frigaard, 2006
). The two cyanobacteria
Syn-
echocystis
6803 and
Synechococcus
7002 differ in the enzymes participating
in nitrogen metabolism, which compromises extension of physiological data
from one globin to another. Even with well-determined
in vitro
rate con-
stants, we are far from the goal of understanding the role of globins in
the cell.
¼
5.2.2 Binding of endogenous and exogenous ligands
Table 6.6
lists the binding and equilibrium constants for
N. commune
GlbN,
CtrHb and
Synechocystis
6803 GlbN along with selected reference proteins.
The parameters were obtained with rapid-mixing and flash photolysis
methods. The first observation is that dioxygen affinity spans a wide range
of values (see also
Table 6.7
). As observed for other globins, this is due to
