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CHAPTER 28
Hemoglobin
His a 122, His
fi
143, and Lys /1144. The amount each of
these groups contributes depends on pH and other condi-
tions prevailing at the time of measurement.
A primary allosteric effector of hemoglobin in hu-
man erythrocytes, in addition to H+ and CO
2
, is the or-
ganic phosphate 2,3-DPG. Deoxyhemoglobin binds 1 mol
of 2,3-DPG per mole of hemoglobin, whereas oxyhe-
moglobin does not. The reason for this differential binding
is the location of the binding site (Figure 28-6). The nega-
tively charged 2,3-DPG molecule fits between the
fi
sub-
units, where it binds to positively charged residues (see
below). In oxyhemoglobin, the
fi
subunits are close to-
gether, the cleft between them being too small for entry
of the 2,3-DPG molecule. Binding of 2,3-DPG inhibits
this movement of the
fi
chains and helps to stabilize the
deoxy form. This binding is an equilibrium phenomenon,
and the amount of 2,3-DPG bound depends on the con-
centrations of 2,3-DPG, O
2
, CO
2
, H+, and hemoglobin.
Chloride promotes H+ binding to deoxyhemoglobin and
acts as an allosteric effector, so its concentration must also
be considered. All of these factors interact to determine the
amount of oxygen bound to hemoglobin at any particular
Po2-
Function, Metabolism, and Regulation of
Organic Phosphates in Erythrocytes
2,3-DPG, ATP, inositol hexaphosphate (IHP), and other
organic phosphates bind to hemoglobin and decrease its
affinity for oxygen. IHP, the principal organic phosphate
in avian erythrocytes, is the most negatively charged of
these compounds and binds the most tightly; however, it
is not found in human red cells.
2,3-DPG
levels
in
the
erythrocytes
help
regulate
hemoglobin oxygenation (Figure 28-7). Unloading of oxy-
gen at the Po
2
in tissue capillaries is increased by 2,3-DPG,
and small changes in its concentration can have significant
effects on oxygen release. At the pH prevailing in the ery-
throcyte, the net charge on the 2,3-DPG molecule is —5.
The binding site between the two
fi
chains of hemoglobin
contains eight positively charged amino acid residue side
chains contributed by the Vail, His2, Lys82, and His 143
of each chain (Figure 28-8). These residues are highly
conserved in mammalian hemoglobins, indicating their
importance for normal hemoglobin function. In placental
mammals, a fetus receives its oxygen by diffusion from
the maternal circulation, across the placenta, into the fetal
circulation. To ensure that the oxygen flow is adequate,
the pressure gradient from mother to fetus is increased
by increasing the affinity of fetal hemoglobin for oxygen.
This decreases the partial pressure of oxygen in the fetal
circulation, thereby increasing the rate of transplacental
diffusion.
F I G U R E 2 8 - 7
Effect of 2,3-bisphosphoglycerate (2,3-DPG) on the oxygen saturation
curve of hemoglobin. Note that 2,3-DPG decreases the affinity of
hemoglobin for oxygen.
Different species increase transplacental diffusion in
different ways. In humans and other primates, adult
hemoglobin contains two
a
and two
fi
chains, while fe-
tal hemoglobin has two
a
and two
y
chains. Although the
amino acid sequences of
fi
and /chains are similar, they
differ in position 143, which is part of the 2,3-DPG binding
site. In the
y
chain, His 143 is replaced by Ser, thus reduc-
ing the charge in the 2,3-DPG binding site from
+8
to +
6
.
Thus, 2,3-DPG binds less tightly to fetal hemoglobin, the
oxygen affinity of which is thereby increased relative to
that of HbA at the same concentration of 2,3-DPG. Thus,
erythrocytes in fetal primates, despite having 2,3-DPG
concentrations equal to those in adult erythrocytes, have
higher oxygen affinity than maternal red blood cells, al-
lowing for maternal to fetal oxygen transport.
In other mammals, including horse, dog, pig, and guinea
pig, fetal hemoglobin is not structurally different from
adult hemoglobin, and transplacental diffusion is facil-
itated by a reduced concentration of 2,3-DPG in fetal
erythrocytes. In ruminants, hemoglobin does not bind
2,3-DPG because the
fi
chains are too far apart. How-
ever, fetal hemoglobin in ruminants has a higher affinity
for oxygen than does adult hemoglobin because of other
structural differences. These three different solutions to
the problem of the need for transfer of oxygen to the fetus
are an example of
convergent evolution.
In humans, 2,3-DPG is the most abundant phos-
phate compound in the red cell. Its concentration is
5 mmol/L, approximately the same as that of hemoglobin
tetramer.
The
concentration
of
ATP
is
also
high,
1.3 mmol/L. Although ATP has roughly the same affin-
ity for hemoglobin as does 2,3-DPG, it has little ef-
fect on oxygen affinity because it is mostly present as
ATP-Mg2+, which binds weakly to hemoglobin. 2,3-DPG
is formed by rearrangement of 1,3-bisphosphoglycerate,
