226
AUSTEN RIGGS
between 264-900 mp (Enoki and Tyuma, 1964; Anderson and Antonini,
1968). However, the possibility of deviations from linearity should be
borne in mind. Many fish hemoglobins are quite sensitive to oxidation.
Many investigators of fish hemoglobins have experienced difficulties in
maintaining the pigment in the ferrous state after hemolyzing the cells,
but it is seldom clear whether this difficulty is owing to an intrinsic
tendency toward autoxidation of the hemoglobin or is owing to the presence of oxidizing substances formed upon hemolysis after the disruption
of the cellular reducing machinery. If significant methemoglobin forms
during the oxygenation measurements, spurious data will be produced
which cannot properly be corrected because methemoglobin increases
the oxygen affinity of the remaining ferrous hemoglobin, even if the
methemoglobin content is constant. One explanation for this effect
is that oxidation of the heme in one subunit excrts much the same effect
on the unoxidized subunits as does oxygenation. That is, in a tetramer
in which one heme is ferric, the remaining hemes have a higher affinity
for oxygen than in completely ferrous unoxygenated hemoglobin. These
general problems are discussed in detail elsewhere (Riggs, 1965). It
should be pointed out, however, that none of the techniques for removing
methemoglobin are without drawbacks, and so the best procedure is to
minimize oxidativc processes. Enzymic reduction of methemoglobin
(Benesch et al., 1964; Rossi-Fanelli et al., 1957) is probably the most
satisfactory technique, but it appears not always to be free of problems.
One of the lamprey hemoglobin components, for example, remained
brown even after enzymic reaction ( Antonini et al., 1964).
All studies of whole blood need to consider the intracellular environment of the hemoglobin. Fish erythrocytes arc relatively large, nucleated,
and carry on a substantial metabolism. Although little in detail is known
of the function of this metabolism in fish the presumption is that the
overall functions are similar to those of the red cells of other animals.
The major functions appear to be: maintenance of the heme iron in thc
ferrous state, control of a proper ionic environment for optimal function
of the hemoglobin, and maintenance of suitable concentrations of substances which serve as allosteric effcctors to modify the oxygen affinity
of the hemoglobin. But many unsolved problems exist. Why, for example,
should it be important for the dogfish, Squalus acanthias, to maintain an
intracellular Na+ Concentration of only 20 mAl compared with the 250
mlll extracellular concentration ( Bricker et aZ., 1968)? Dogfish hemoglobin might be particularly sensitive to Na' ions, but this possibility has
not been studied. In addition, changes in thc shape of the red cells
are important and often may be linked to the oxygen transport function. It has often been stated that one function of the red cell is to reduce
AUSTEN RIGGS
between 264-900 mp (Enoki and Tyuma, 1964; Anderson and Antonini,
1968). However, the possibility of deviations from linearity should be
borne in mind. Many fish hemoglobins are quite sensitive to oxidation.
Many investigators of fish hemoglobins have experienced difficulties in
maintaining the pigment in the ferrous state after hemolyzing the cells,
but it is seldom clear whether this difficulty is owing to an intrinsic
tendency toward autoxidation of the hemoglobin or is owing to the presence of oxidizing substances formed upon hemolysis after the disruption
of the cellular reducing machinery. If significant methemoglobin forms
during the oxygenation measurements, spurious data will be produced
which cannot properly be corrected because methemoglobin increases
the oxygen affinity of the remaining ferrous hemoglobin, even if the
methemoglobin content is constant. One explanation for this effect
is that oxidation of the heme in one subunit excrts much the same effect
on the unoxidized subunits as does oxygenation. That is, in a tetramer
in which one heme is ferric, the remaining hemes have a higher affinity
for oxygen than in completely ferrous unoxygenated hemoglobin. These
general problems are discussed in detail elsewhere (Riggs, 1965). It
should be pointed out, however, that none of the techniques for removing
methemoglobin are without drawbacks, and so the best procedure is to
minimize oxidativc processes. Enzymic reduction of methemoglobin
(Benesch et al., 1964; Rossi-Fanelli et al., 1957) is probably the most
satisfactory technique, but it appears not always to be free of problems.
One of the lamprey hemoglobin components, for example, remained
brown even after enzymic reaction ( Antonini et al., 1964).
All studies of whole blood need to consider the intracellular environment of the hemoglobin. Fish erythrocytes arc relatively large, nucleated,
and carry on a substantial metabolism. Although little in detail is known
of the function of this metabolism in fish the presumption is that the
overall functions are similar to those of the red cells of other animals.
The major functions appear to be: maintenance of the heme iron in thc
ferrous state, control of a proper ionic environment for optimal function
of the hemoglobin, and maintenance of suitable concentrations of substances which serve as allosteric effcctors to modify the oxygen affinity
of the hemoglobin. But many unsolved problems exist. Why, for example,
should it be important for the dogfish, Squalus acanthias, to maintain an
intracellular Na+ Concentration of only 20 mAl compared with the 250
mlll extracellular concentration ( Bricker et aZ., 1968)? Dogfish hemoglobin might be particularly sensitive to Na' ions, but this possibility has
not been studied. In addition, changes in thc shape of the red cells
are important and often may be linked to the oxygen transport function. It has often been stated that one function of the red cell is to reduce