6. PROPERTIES OF FISH HEMOGLOBINS
223
whether these components occur in the same cell or whether they are
confined to different cells. Some components interact with one another
in such a way that the oxygen equilibrium differs from that expected
from a noninteracting mixture. Such interaction could only occur in
zjioo if the hemoglobins occurred in the same cells. These questions
should now bc answerable because techniques have been developed both
for thcx measurement of the electrophoresis of hemoglobin from individual erythrocytes (Matioli and Niewisch, 1965) and the determination of
the oxygen equilibrium of single erythrocytes ( Huckauf et al., 1969).
From a physiological viewpoint, the most important parameters in
the description of the oxygen equilibrium are: shape of the curve (plot
of y, the degree of oxygenation, vs. p , the oxygen pressure)-whether
sigmoid, hyperbolic, or “undulating”; pH and CO, dependence ( Bohr
effect) ; the “oxygen affinity,” usually expressed in terms of the pressure
requircd for 50% oxygenation; temperature dependence; allosteric effectors, substances (ions and organic molecules which can alter the
equilibrium). Ontogenetic variation of such substances may play a
crucial role in determining the adaptation of the function of hemoglobin.
These five parametcm determine the amount of oxygen transported by
a given quantity of hemoglobin.
A. Critique of Measurements
Only two procedures are in common use for the determination of
the equilibrium between hemoglobin and oxygen: gasometric and spectrophotometric. The gasometric technique involves direct determination of
the oxygen content of blood or a hemoglobin solution at a measured
oxygen pressure. Thus, it provides unambiguous data of immediate
practical use in physiological studies of gas transport. This procedure,
as applied to blood, gives no information about the state of the hemoglobin, and is a “black box” technique insofar as the red cell contents
are concerned. Furthermore, the gasometric procedure with whole cells
does not take into consideration the possible effects of changes in cell
shapc on oxygen transport (see discussion of shape changes; Riggs,
1965). Thc spectrophotomctric technique is difficult to apply with precision to whole cells and has been most widely used with hcrnoglobin
solutions. Although easier and faster than the gasometric procedure,
the spcctrophotometric tcchnique has several potential pitfalls. The
basic spectrophotometric assumption is that the change in absorbance
at any wavelength is linearly related to the fraction of the total number
of fcrroheme groups oxygenated. This appears to be a valid assumption
223
whether these components occur in the same cell or whether they are
confined to different cells. Some components interact with one another
in such a way that the oxygen equilibrium differs from that expected
from a noninteracting mixture. Such interaction could only occur in
zjioo if the hemoglobins occurred in the same cells. These questions
should now bc answerable because techniques have been developed both
for thcx measurement of the electrophoresis of hemoglobin from individual erythrocytes (Matioli and Niewisch, 1965) and the determination of
the oxygen equilibrium of single erythrocytes ( Huckauf et al., 1969).
From a physiological viewpoint, the most important parameters in
the description of the oxygen equilibrium are: shape of the curve (plot
of y, the degree of oxygenation, vs. p , the oxygen pressure)-whether
sigmoid, hyperbolic, or “undulating”; pH and CO, dependence ( Bohr
effect) ; the “oxygen affinity,” usually expressed in terms of the pressure
requircd for 50% oxygenation; temperature dependence; allosteric effectors, substances (ions and organic molecules which can alter the
equilibrium). Ontogenetic variation of such substances may play a
crucial role in determining the adaptation of the function of hemoglobin.
These five parametcm determine the amount of oxygen transported by
a given quantity of hemoglobin.
A. Critique of Measurements
Only two procedures are in common use for the determination of
the equilibrium between hemoglobin and oxygen: gasometric and spectrophotometric. The gasometric technique involves direct determination of
the oxygen content of blood or a hemoglobin solution at a measured
oxygen pressure. Thus, it provides unambiguous data of immediate
practical use in physiological studies of gas transport. This procedure,
as applied to blood, gives no information about the state of the hemoglobin, and is a “black box” technique insofar as the red cell contents
are concerned. Furthermore, the gasometric procedure with whole cells
does not take into consideration the possible effects of changes in cell
shapc on oxygen transport (see discussion of shape changes; Riggs,
1965). Thc spectrophotomctric technique is difficult to apply with precision to whole cells and has been most widely used with hcrnoglobin
solutions. Although easier and faster than the gasometric procedure,
the spcctrophotometric tcchnique has several potential pitfalls. The
basic spectrophotometric assumption is that the change in absorbance
at any wavelength is linearly related to the fraction of the total number
of fcrroheme groups oxygenated. This appears to be a valid assumption