6. PROPERTIES OF FISH HEMOGLOBINS
215
group will change the net charge on the molecule and thus result
in multiplicity of components if the reaction is incomplete. So, for
example, the hemoglobin of Lampetra fluuiatilis consists of two major
components which appear to have the same amino acid sequence and
differ only at the NH, terminus: 20% of the molecules have freely reactive NH,-terminal proline, while the rest are blocked to reaction with
NH,-terminal reagents ( Braunitzer, 1966). Such groups (often acetyl)
appear, in hemoglobins, at least, to be added after chain synthesis and
depend on the presence of an active enzymic acetylation system
( Marchis-Mouren and Lipmann, 1965) I The presence of this variation,
therclfore, apparently has nothing directly to do with the genetics of
hemoglobin structure itself, although blocking of this group might be
important in modifying the role of hemoglobin in CO, transport (see
Section 111, C on the mechanism of the Bohr effect). An acetyl NH,
terminus would, of course, be genetically determined if the presence or
absence of the enzyme system were so determined.
1). Allo,steric Efectors. Barcroft showed more than 50 years ago
that dialysis of hemolyzates modified the oxygen affinity of the hemoglobin, and he noted that phosphates had particularly large effects on
the oxygen equilibrium (see Barcroft, 1928). Had hemoglobin been
considered an enzyme, an attempt surely would have been made to
determine what was removed by dialysis and to reconstitute by adding
back what had been removed. But only recently has it become established that diphosphoglycerate (DPG) is normally present in human
hemolyzates in the approximate ratio of one mole of DPG per hemoglobin molecule, and, most important, the DPG combines with the
hemoglobin and produces an electrophoretically distinct component,
which has a much lower oxygen affinity (Chanutin and Curnish, 1964,
1967; Bcnesch and Benesch, 1967, 1969). The binding of DPG is
primarily to the deoxygenated pigment. Diphosphoglycerate appears,
therefore, to act as an “allosteric effector” in controlling the oxygen
transport function of hemoglobin. Other organic phosphates, especially
ATP, are also present in red cells and have similar effects, but they
are quantitatively much less important. Inositol hexaphosphate appears
to serve ii function in bird and turtle red cells similar to DPG in mammalian cells. Diphosphoglycerate is not removed by dialysis against
distilled water and only slowly by dialysis against 0.1 M NaCl. Therefore, these common procedures of “purification” are likely to result
in a mixture of two electrophoretically distinct components which will
have different oxygenation properties.
The quantity of DPG in the red cell depends on the metabolism;
different environmental conditions may result in substantial changes
in the quantity present. The point to be made here is that considerable
215
group will change the net charge on the molecule and thus result
in multiplicity of components if the reaction is incomplete. So, for
example, the hemoglobin of Lampetra fluuiatilis consists of two major
components which appear to have the same amino acid sequence and
differ only at the NH, terminus: 20% of the molecules have freely reactive NH,-terminal proline, while the rest are blocked to reaction with
NH,-terminal reagents ( Braunitzer, 1966). Such groups (often acetyl)
appear, in hemoglobins, at least, to be added after chain synthesis and
depend on the presence of an active enzymic acetylation system
( Marchis-Mouren and Lipmann, 1965) I The presence of this variation,
therclfore, apparently has nothing directly to do with the genetics of
hemoglobin structure itself, although blocking of this group might be
important in modifying the role of hemoglobin in CO, transport (see
Section 111, C on the mechanism of the Bohr effect). An acetyl NH,
terminus would, of course, be genetically determined if the presence or
absence of the enzyme system were so determined.
1). Allo,steric Efectors. Barcroft showed more than 50 years ago
that dialysis of hemolyzates modified the oxygen affinity of the hemoglobin, and he noted that phosphates had particularly large effects on
the oxygen equilibrium (see Barcroft, 1928). Had hemoglobin been
considered an enzyme, an attempt surely would have been made to
determine what was removed by dialysis and to reconstitute by adding
back what had been removed. But only recently has it become established that diphosphoglycerate (DPG) is normally present in human
hemolyzates in the approximate ratio of one mole of DPG per hemoglobin molecule, and, most important, the DPG combines with the
hemoglobin and produces an electrophoretically distinct component,
which has a much lower oxygen affinity (Chanutin and Curnish, 1964,
1967; Bcnesch and Benesch, 1967, 1969). The binding of DPG is
primarily to the deoxygenated pigment. Diphosphoglycerate appears,
therefore, to act as an “allosteric effector” in controlling the oxygen
transport function of hemoglobin. Other organic phosphates, especially
ATP, are also present in red cells and have similar effects, but they
are quantitatively much less important. Inositol hexaphosphate appears
to serve ii function in bird and turtle red cells similar to DPG in mammalian cells. Diphosphoglycerate is not removed by dialysis against
distilled water and only slowly by dialysis against 0.1 M NaCl. Therefore, these common procedures of “purification” are likely to result
in a mixture of two electrophoretically distinct components which will
have different oxygenation properties.
The quantity of DPG in the red cell depends on the metabolism;
different environmental conditions may result in substantial changes
in the quantity present. The point to be made here is that considerable