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Hemoglobin and oxygen: Different affinities in two species of rodents (Mus musculus and Pitymys duodecimcostatus)
0300-9629/86$3.00+ 0.00 Pergamon Journals Ltd
Camp. Biochem. Physiol. Vol. 84A, No. 3, pp. 409-411, 1986
Printed in Great Britain
HEMOGLOBIN AND OXYGEN: DIFFERENT AFFINITIES IN TWO SPECIES OF RODENTS (MUS MUSCULUS AND PITYMYS DUODECIMCOSTATUS) G. PEREZ-SUAREZ, F. ARBVALO and P. LOPEZ-LUNA Departamento de Biologia-Fisiologia Animal, Facultad de Ciencias, Universidad de Alcala de Henares, Madrid, Spain (Received 22 October 1985)
Abstract-l. Five different hemoglobins have been demonstrated by polyacrylamide-gel disk electrophoresis in the species Mus musculus. 2. Oxygen affinities of hemoglobin (P,,) from Mus musculus and Pitymys duodecimcostatus hemolysates were determined at pH 7.4 and 37°C. 3. Values obtained for A log P-/A DH in hemolvsates from both species point out a more pronounced Bohr effect in Pitymys duode~m&tdtus.
INTRODUCTION It is well known that the hemoglobin molecule from mammalian blood varies with the species, its relative oxygen affinity being the most characteristic variant. The position of the oxygen equilibrium curve is related to environmental limitations and to metabolic requirements of the species. The adaptative value of hemoglobin must be considered from the point of view of its ability to combine with oxygen in the lungs and its ability to release this oxygen to the functioning cells of the body. Multiple hemoglobins have been reported in numerous species (Foreman, 1960; Condo et al., 1981; Perez-Suarez et al., 1985) and the numbers and quantities of these hemoglobins, which seem to be genetically determined, may vary among specimens of a single species (Foreman, 1966; Rasmussen et al., 1968), depending on their geographic location. Wild house mice are particularly variable in this respect, and differences have been reported among strains of laboratory mice (Gluecksohn-Waelsch et al., 1957; Popp and St. Amand, 1960). There is no experimental evidence that multiple hemoglobins offer any physiological advantages over single ones, but it has been suggested that multiple hemoglobins may differ in their oxygen affinities and thereby permit the carrier to adapt to different environmental situations (Gluecksohn-Waelsch, 1960; Brunori et al., 1979) or that multiple hemoglobins might, according to the phase rule, permit higher solubilities within red cells than would be possible with single hemoglobins (Perutz et al., 1959). In this paper we describe the oxygen binding properties of solutions of hemoglobins from two species of small mammals, Pitymys duodecimcostatus, trapped alive in the Mediterranean area of Spain, and Mus musculus v. albino. MATERIALS AND
Alcala de Henares (Madrid, Spain). Taxonomic designation was according to Corbet (1978). Blood for hemoglobin was obtained by heart puncture using heparin as a&coagulant. Hemoglobin was prepared from washed red cells. lvsed as described bv Riggs (1981). Hemoglobin was analyzed by polyacryiamide-gel disk electrophoresis according to Davis (1964) and Omstein (1964). Hemoglobin samples were diluted in buffer (pH 8.3) containing 0.1 M B-mercaptoethanol and a small amount of dithionite. The mixture was bubbled with carbon monoxide and applied immediately to the gel. The gels were stained for 12 hr in 0.025% Coomassie Brilliant Blue R in 12.5% trichloroacetic acid and destained by diffusion. The gels were analyzed and the relative percentage of each protein was determined by using a densitometer (Model Gilford 250). Planimetry was done manually by resolving the scan tracings into individual peaks. Hemoglobin-concentration was determined by the spectroohotometric method of Drabkin and Austin (1935). Blood was centrifuged in microhematocrit capillaries at 11,000 rev/min for 10 min and the hematocrit determined. Red cells were counted in a Thoma chamber. A solution of hemoglobin was prepared in pH 7.4 0.1 M Tris-HCl buffer containing 1mM EDTA and 0.1 M NaCl (Powers et al., 1979). Fromthis solution 3 ml was taken and the oxygen equilibrium curves (OEC) were determined spectrophotometrically by means of the special tonometer and the procedure of Rossi-Fanelli and Antonini (1958). The oxygen-binding properties of the hemolysate from blood of Mu.r musculus v. albino and Pitymys duodecimcostatus were investigated at pH 7, 7.2 and 7.4 at 37°C. The curves were drawn through the average values obtained.
METHODS
Mice used in the present study came from the Department facilities (Mus musculus v. albino) while the voles were trapped alive in the neighbourhood of the University of 409
RESULTS The hematological values, hematocrit (Hc), hemoglobin concentration (Hb) and number of red blood cells (RBC) found in A4us musculus (N = 10; average weight = 19.67 f 2.3 g) were: 47.4 f 2.8%, 15.0 + 1.6 g/100 ml and 9.0 f 1.4 ( 106/mm3), respectively. The corresponding values for Pitymys duodecimcostatus were previously published by PerezSuarez et al. (1985). Electrophoretic analysis of the hemolysate from blood of Mus musculus showed five different hemo-
G. P~~JZZ-SU~REZ et ai.
410
\ \ ‘0
1
i \
.
t
I\
\
I ’ x0
I 7.2
l\ ’ -- 0,86 0.95
It
J
I
--+
I 7.4
PH
III Fig. 1. Densitometric hemoglobins
x5
IZLP
scan of typical tube gel showing five (I-V) from MUS musculus.
globin components which were designated I-V in accordance with their anionic mobility. Densitometric analysis of polyacrylamide-gel disks (Fig. 1) provided the relative proportions of the components (Table 1). Band III represented approximately 66%. This component was the major one and its mobility was intermediate between those of components II and IV. Electrophoresis of hemoglobins from vole blood (Pitymys duodecimcostatus) exhibits six distinct hemoglobin components (Pkrez-Su&rez et al., 1985). Oxygen equilibrium curves of blood from both small mammals shbwed that the oxygen partial pressures at which the pigment was 50% saturated (P,,) at pH 7.4 and 37°C varied for both species: 21 mmHg for Mus musculus and 19 mmHg for Pitymys duodecimcostatus.
pH-de~ndent changes in oxygen affinity between 7 and 7.4 were determined for hemoglobins from both species. The slopes of these lines were -0.86 for -0.95 for Pitymys duoMus musculus and decimcostatus (Fig. 2). DISCUSSION
Electrophoretic studies carried out in the hemolysate from Mus musculus have revealed five hemoglobin components (I-V). Much confusion has surrounded the interpretation of the electrophoretic patterns of mouse hemogiobins. Foreman (1960) Table 1. Relative proportions and electrophoretic mobility of Mus musculus hemoglobin components Component
Amount WI
Electrophoretic mobility
I II III IV V
3 20 66 9 2
0.25 0.30 0.35 0.40 0.45
Fig. 2. Alkaline Bohr effect of hemoglobin of MUS mz~sculus and Pitymys ~ao~ctmcostat~. P?, 0, partial pressure (mmJ&) at which 50% of h~o~obln is saturated with 4. Key: (0) M. musculus (aibino); (e) P. duadecimcostatus.
found in Mw musculus two hemoglobin bands on cellulose acetate, while the densitomet~c analysis showed three or four peaks. According to several authors (Smith et al., 1966; Bonaventura and Riggs, 1967; Russell and McFarland, 1974), the fact that, in the mouse, one or two of the components appeared in greater proportions prompted researchers to talk about the so-called “unique” mouse hemoglobin electrophoretic band and a second hemoglobin termed “diffuse”. In our case, diffuse hemoglobin could be represented by component II, in a relative proportion of 20% and with an electrophoretic mobility lower than that of component III (Table 1). The other three hemoglobin bands would correspond to minor components, already noticed by Rhinesmith et al. (1964) by chromatography. It appears that different strains of laboratory mice present great variability in the number of hemo~obin components (Popp and St. Amand, 1960). The affinity of hemoglobin for oxygen is of possible significance in two processes: (a) the uptake of oxygen in the lungs and (b) the unloading of oxygen in the tissues. The OEC for Pitymys ~5~cimcostat~ at pH 7.4 and 37°C is shifted to the left with respect to that of the mouse, which points to a greater affinity of vole hemoglobin for oxygen, because of its saturation at oxygen pressures at which mouse hemoglobin is only partially saturated. Schmidt-Nielsen and Larimer (1958) has shown that the equilibrium curves of blood from several mammals is related to body size. Those of lower size possess a hemoglobin with lower affinity for oxygen due to their higher metabolic rate, their tissues presenting greater requirements of oxygen. Besides the size, the animals’ habitat also has an influence on the curve situation, so that animals living under hypoxia, such as the vole is supposed to live under due to its prevailing underground life, will present their OEC shifted to the left. This fact would account for the observation that
0, affinities of rodent Hb of a similar size present different OECs because the mouse lives in normal oxygen-tension environments. This intkence of habitat of OECs has been reported for several animals (Hall, 1965; Powers et al., 1979). The values obtained for A log PJA pH (Fig. 2) in both animals point out that the Bohr effect is more pronounced in the vole, which could be interpreted as a facititating mechanism to produce a greater oxygen liberation in the tissues. The Microtines, in fact, present a higher metabolic rate than was to be expected regarding only their body weight (Morrison et nl., i959). In both species studied, a decreased oxygen affinity could be observed accompanied by an increased Bohr effect. Foreman (1954) reported this fact in other species. animals
REFERENCES
Bonaventura J. and Riggs A. (1967) Polymerization of hemoglobins of mouse and man: Structural basis. Science 158, 800-802. Brunori M., Bonaventura J., Focesi A., Galdames-Portus M. I. and Wilson M. T. (1979) Separation and characterization of the hemoglobin components of Prerygoplichthys pardalis, the Acaribodo. Comp. Biochem. Physiol. 62A, 1733178. Condo S. G., Giardina B., Barra D., Gill S. J. and Brunori M. (1981) Purification and functional properties of the hemo~iobin components from the rat (Wistar). Eur. J. Biochem. 116, 243-247. Corbet B. G. (1978) The Mammals of the Paleartic Repion. A Taxonomk Reiiew, pp. 1061 IO.-British Museum (Natural History), London; Cornell University Press, Ithaca. Davis B. J. (1964) Disk electrophoresis. II. Method and application to human serum proteins. Ann. N.Y. Acad. sci. 121, 404-427. Drabkin D. L. and Austin J. H. (1935) Spectrophotometric studies. V.A. Technique for the analysis of undiluted blood and concentrated hemoglobin solution. J. biol. Chem. 112, 105-115. Foreman C. W. (1954) A comparative study of the oxygen dissociation of mammalian hemoglobin. J. cell. camp. Physiol. 44, 421430. Foreman C. W. (1960) Electromigration properties of mam-
411
malian hemoglobins as taxonomic criteria. Am. Midl. Nat. 74, 177-186. Foreman C. W. (I 966) Inheritance of multipIe hemoglobins in Peromyscus. Genetics 54, 1007-1012. Gluecksohn-Waelsch S., Ranney H. M. and Sisken B. F. (1957) The hereditary transmission of hemoglobin differences in mice. J. clin. Invest. 36, 753-756. Gluecksohn-Waelsch S. (1960) The inheritance of hemoglobin types and other biochemical traits in mammals. J. cell. camp. Physiol. 56, 89-101. Hall F. G. (1965) Hemoglobin and oxygen: affinities in seven species of Sciuridae. Science 148, 1350-1351. Morrison P. R., Ryser F. A. and Dawe A. R. (1959) Studies on the physiology of the masked shrew Sorex cinerus. Physiol. Zoof. 32, 25&271. Ornstein L. (1964) Disk electrophoresis. I. Background theory. Ann. N.Y. Acad. Sci. 121, 321-349. Perez-Suarez G., Artvalo F. and Lopez-Luna P. (1985) Hemoglobin components and plasma proteins in Pitymyi duodecimcostatus. Comp. Biochem. Physiol. 80, 145-147. Perutz M. F., Steinrauf L. K., Stockell A. and Bangham A. D. (1959) ChemicaI and C~staIlo~aohic studv of the two fractions of adult horse hemoglobin. J. mo&. Biof. 1, 402404. Popp R. A. and St. Amand W. (1960) Studies on the mouse hemoglobin locus. J. Herediry 51, 141-144. Powers D. A., Fyhn H. J., Fyhn U. E. H., Martin .I. P., Garlick R. L. and Wood S. C. (1979) A comparative study of the oxygen equilibria of blood from 40 genera of Amazonian fishes. Camp. Biochem. Phvsiol. 62. 67-88. Rasmussen D. I., Jensen ‘J. N. and Koehn R. K. (1968) Hemoglobin nolvmorohism in the deer mouse Peromys& mani&tus. kochem. Genet. 2, 87. Rhinesmith H. S., Li H. H.. Miiliten B. L. and Strone L. C. (1964) Mouse hemoglobin I. Chromatographic agalysis. Analyt. Eiochem. 8, 407-414. Riggs A. (1981) Preparation of blood hemoglobins of vertebrates, Meth. Enzymol. 76, 5-29. Rossi-Fanelli A. and Antonini E. (1958) Studies on the oxygen and carbon monoxide equilibria of human myoglobin. Archs Biochem. Biophys. 77, 478-492. Russell E. S. and McFarland E. C. (1974) Genetics of mouse hemoglobin. Ann. N.Y. Acad. Sk 241, 25-37. Schmidt-Nielsen K. and Larimer J. L. (1958) Oxvzen dissociation curves of mammalian blood in relation lo body size. Am. J. Physioi. 195, 424-428. Smith D. B., Brunori M., Antonini E. and Wyman J. (1966) The oxygen Bohr effect in mouse hemoglobin. Archs Biochem. Biophys. 113, 725-729.
Camp. Biochem. Physiol. Vol. 84A, No. 3, pp. 409-411, 1986
Printed in Great Britain
HEMOGLOBIN AND OXYGEN: DIFFERENT AFFINITIES IN TWO SPECIES OF RODENTS (MUS MUSCULUS AND PITYMYS DUODECIMCOSTATUS) G. PEREZ-SUAREZ, F. ARBVALO and P. LOPEZ-LUNA Departamento de Biologia-Fisiologia Animal, Facultad de Ciencias, Universidad de Alcala de Henares, Madrid, Spain (Received 22 October 1985)
Abstract-l. Five different hemoglobins have been demonstrated by polyacrylamide-gel disk electrophoresis in the species Mus musculus. 2. Oxygen affinities of hemoglobin (P,,) from Mus musculus and Pitymys duodecimcostatus hemolysates were determined at pH 7.4 and 37°C. 3. Values obtained for A log P-/A DH in hemolvsates from both species point out a more pronounced Bohr effect in Pitymys duode~m&tdtus.
INTRODUCTION It is well known that the hemoglobin molecule from mammalian blood varies with the species, its relative oxygen affinity being the most characteristic variant. The position of the oxygen equilibrium curve is related to environmental limitations and to metabolic requirements of the species. The adaptative value of hemoglobin must be considered from the point of view of its ability to combine with oxygen in the lungs and its ability to release this oxygen to the functioning cells of the body. Multiple hemoglobins have been reported in numerous species (Foreman, 1960; Condo et al., 1981; Perez-Suarez et al., 1985) and the numbers and quantities of these hemoglobins, which seem to be genetically determined, may vary among specimens of a single species (Foreman, 1966; Rasmussen et al., 1968), depending on their geographic location. Wild house mice are particularly variable in this respect, and differences have been reported among strains of laboratory mice (Gluecksohn-Waelsch et al., 1957; Popp and St. Amand, 1960). There is no experimental evidence that multiple hemoglobins offer any physiological advantages over single ones, but it has been suggested that multiple hemoglobins may differ in their oxygen affinities and thereby permit the carrier to adapt to different environmental situations (Gluecksohn-Waelsch, 1960; Brunori et al., 1979) or that multiple hemoglobins might, according to the phase rule, permit higher solubilities within red cells than would be possible with single hemoglobins (Perutz et al., 1959). In this paper we describe the oxygen binding properties of solutions of hemoglobins from two species of small mammals, Pitymys duodecimcostatus, trapped alive in the Mediterranean area of Spain, and Mus musculus v. albino. MATERIALS AND
Alcala de Henares (Madrid, Spain). Taxonomic designation was according to Corbet (1978). Blood for hemoglobin was obtained by heart puncture using heparin as a&coagulant. Hemoglobin was prepared from washed red cells. lvsed as described bv Riggs (1981). Hemoglobin was analyzed by polyacryiamide-gel disk electrophoresis according to Davis (1964) and Omstein (1964). Hemoglobin samples were diluted in buffer (pH 8.3) containing 0.1 M B-mercaptoethanol and a small amount of dithionite. The mixture was bubbled with carbon monoxide and applied immediately to the gel. The gels were stained for 12 hr in 0.025% Coomassie Brilliant Blue R in 12.5% trichloroacetic acid and destained by diffusion. The gels were analyzed and the relative percentage of each protein was determined by using a densitometer (Model Gilford 250). Planimetry was done manually by resolving the scan tracings into individual peaks. Hemoglobin-concentration was determined by the spectroohotometric method of Drabkin and Austin (1935). Blood was centrifuged in microhematocrit capillaries at 11,000 rev/min for 10 min and the hematocrit determined. Red cells were counted in a Thoma chamber. A solution of hemoglobin was prepared in pH 7.4 0.1 M Tris-HCl buffer containing 1mM EDTA and 0.1 M NaCl (Powers et al., 1979). Fromthis solution 3 ml was taken and the oxygen equilibrium curves (OEC) were determined spectrophotometrically by means of the special tonometer and the procedure of Rossi-Fanelli and Antonini (1958). The oxygen-binding properties of the hemolysate from blood of Mu.r musculus v. albino and Pitymys duodecimcostatus were investigated at pH 7, 7.2 and 7.4 at 37°C. The curves were drawn through the average values obtained.
METHODS
Mice used in the present study came from the Department facilities (Mus musculus v. albino) while the voles were trapped alive in the neighbourhood of the University of 409
RESULTS The hematological values, hematocrit (Hc), hemoglobin concentration (Hb) and number of red blood cells (RBC) found in A4us musculus (N = 10; average weight = 19.67 f 2.3 g) were: 47.4 f 2.8%, 15.0 + 1.6 g/100 ml and 9.0 f 1.4 ( 106/mm3), respectively. The corresponding values for Pitymys duodecimcostatus were previously published by PerezSuarez et al. (1985). Electrophoretic analysis of the hemolysate from blood of Mus musculus showed five different hemo-
G. P~~JZZ-SU~REZ et ai.
410
\ \ ‘0
1
i \
.
t
I\
\
I ’ x0
I 7.2
l\ ’ -- 0,86 0.95
It
J
I
--+
I 7.4
PH
III Fig. 1. Densitometric hemoglobins
x5
IZLP
scan of typical tube gel showing five (I-V) from MUS musculus.
globin components which were designated I-V in accordance with their anionic mobility. Densitometric analysis of polyacrylamide-gel disks (Fig. 1) provided the relative proportions of the components (Table 1). Band III represented approximately 66%. This component was the major one and its mobility was intermediate between those of components II and IV. Electrophoresis of hemoglobins from vole blood (Pitymys duodecimcostatus) exhibits six distinct hemoglobin components (Pkrez-Su&rez et al., 1985). Oxygen equilibrium curves of blood from both small mammals shbwed that the oxygen partial pressures at which the pigment was 50% saturated (P,,) at pH 7.4 and 37°C varied for both species: 21 mmHg for Mus musculus and 19 mmHg for Pitymys duodecimcostatus.
pH-de~ndent changes in oxygen affinity between 7 and 7.4 were determined for hemoglobins from both species. The slopes of these lines were -0.86 for -0.95 for Pitymys duoMus musculus and decimcostatus (Fig. 2). DISCUSSION
Electrophoretic studies carried out in the hemolysate from Mus musculus have revealed five hemoglobin components (I-V). Much confusion has surrounded the interpretation of the electrophoretic patterns of mouse hemogiobins. Foreman (1960) Table 1. Relative proportions and electrophoretic mobility of Mus musculus hemoglobin components Component
Amount WI
Electrophoretic mobility
I II III IV V
3 20 66 9 2
0.25 0.30 0.35 0.40 0.45
Fig. 2. Alkaline Bohr effect of hemoglobin of MUS mz~sculus and Pitymys ~ao~ctmcostat~. P?, 0, partial pressure (mmJ&) at which 50% of h~o~obln is saturated with 4. Key: (0) M. musculus (aibino); (e) P. duadecimcostatus.
found in Mw musculus two hemoglobin bands on cellulose acetate, while the densitomet~c analysis showed three or four peaks. According to several authors (Smith et al., 1966; Bonaventura and Riggs, 1967; Russell and McFarland, 1974), the fact that, in the mouse, one or two of the components appeared in greater proportions prompted researchers to talk about the so-called “unique” mouse hemoglobin electrophoretic band and a second hemoglobin termed “diffuse”. In our case, diffuse hemoglobin could be represented by component II, in a relative proportion of 20% and with an electrophoretic mobility lower than that of component III (Table 1). The other three hemoglobin bands would correspond to minor components, already noticed by Rhinesmith et al. (1964) by chromatography. It appears that different strains of laboratory mice present great variability in the number of hemo~obin components (Popp and St. Amand, 1960). The affinity of hemoglobin for oxygen is of possible significance in two processes: (a) the uptake of oxygen in the lungs and (b) the unloading of oxygen in the tissues. The OEC for Pitymys ~5~cimcostat~ at pH 7.4 and 37°C is shifted to the left with respect to that of the mouse, which points to a greater affinity of vole hemoglobin for oxygen, because of its saturation at oxygen pressures at which mouse hemoglobin is only partially saturated. Schmidt-Nielsen and Larimer (1958) has shown that the equilibrium curves of blood from several mammals is related to body size. Those of lower size possess a hemoglobin with lower affinity for oxygen due to their higher metabolic rate, their tissues presenting greater requirements of oxygen. Besides the size, the animals’ habitat also has an influence on the curve situation, so that animals living under hypoxia, such as the vole is supposed to live under due to its prevailing underground life, will present their OEC shifted to the left. This fact would account for the observation that
0, affinities of rodent Hb of a similar size present different OECs because the mouse lives in normal oxygen-tension environments. This intkence of habitat of OECs has been reported for several animals (Hall, 1965; Powers et al., 1979). The values obtained for A log PJA pH (Fig. 2) in both animals point out that the Bohr effect is more pronounced in the vole, which could be interpreted as a facititating mechanism to produce a greater oxygen liberation in the tissues. The Microtines, in fact, present a higher metabolic rate than was to be expected regarding only their body weight (Morrison et nl., i959). In both species studied, a decreased oxygen affinity could be observed accompanied by an increased Bohr effect. Foreman (1954) reported this fact in other species. animals
REFERENCES
Bonaventura J. and Riggs A. (1967) Polymerization of hemoglobins of mouse and man: Structural basis. Science 158, 800-802. Brunori M., Bonaventura J., Focesi A., Galdames-Portus M. I. and Wilson M. T. (1979) Separation and characterization of the hemoglobin components of Prerygoplichthys pardalis, the Acaribodo. Comp. Biochem. Physiol. 62A, 1733178. Condo S. G., Giardina B., Barra D., Gill S. J. and Brunori M. (1981) Purification and functional properties of the hemo~iobin components from the rat (Wistar). Eur. J. Biochem. 116, 243-247. Corbet B. G. (1978) The Mammals of the Paleartic Repion. A Taxonomk Reiiew, pp. 1061 IO.-British Museum (Natural History), London; Cornell University Press, Ithaca. Davis B. J. (1964) Disk electrophoresis. II. Method and application to human serum proteins. Ann. N.Y. Acad. sci. 121, 404-427. Drabkin D. L. and Austin J. H. (1935) Spectrophotometric studies. V.A. Technique for the analysis of undiluted blood and concentrated hemoglobin solution. J. biol. Chem. 112, 105-115. Foreman C. W. (1954) A comparative study of the oxygen dissociation of mammalian hemoglobin. J. cell. camp. Physiol. 44, 421430. Foreman C. W. (1960) Electromigration properties of mam-
411
malian hemoglobins as taxonomic criteria. Am. Midl. Nat. 74, 177-186. Foreman C. W. (I 966) Inheritance of multipIe hemoglobins in Peromyscus. Genetics 54, 1007-1012. Gluecksohn-Waelsch S., Ranney H. M. and Sisken B. F. (1957) The hereditary transmission of hemoglobin differences in mice. J. clin. Invest. 36, 753-756. Gluecksohn-Waelsch S. (1960) The inheritance of hemoglobin types and other biochemical traits in mammals. J. cell. camp. Physiol. 56, 89-101. Hall F. G. (1965) Hemoglobin and oxygen: affinities in seven species of Sciuridae. Science 148, 1350-1351. Morrison P. R., Ryser F. A. and Dawe A. R. (1959) Studies on the physiology of the masked shrew Sorex cinerus. Physiol. Zoof. 32, 25&271. Ornstein L. (1964) Disk electrophoresis. I. Background theory. Ann. N.Y. Acad. Sci. 121, 321-349. Perez-Suarez G., Artvalo F. and Lopez-Luna P. (1985) Hemoglobin components and plasma proteins in Pitymyi duodecimcostatus. Comp. Biochem. Physiol. 80, 145-147. Perutz M. F., Steinrauf L. K., Stockell A. and Bangham A. D. (1959) ChemicaI and C~staIlo~aohic studv of the two fractions of adult horse hemoglobin. J. mo&. Biof. 1, 402404. Popp R. A. and St. Amand W. (1960) Studies on the mouse hemoglobin locus. J. Herediry 51, 141-144. Powers D. A., Fyhn H. J., Fyhn U. E. H., Martin .I. P., Garlick R. L. and Wood S. C. (1979) A comparative study of the oxygen equilibria of blood from 40 genera of Amazonian fishes. Camp. Biochem. Phvsiol. 62. 67-88. Rasmussen D. I., Jensen ‘J. N. and Koehn R. K. (1968) Hemoglobin nolvmorohism in the deer mouse Peromys& mani&tus. kochem. Genet. 2, 87. Rhinesmith H. S., Li H. H.. Miiliten B. L. and Strone L. C. (1964) Mouse hemoglobin I. Chromatographic agalysis. Analyt. Eiochem. 8, 407-414. Riggs A. (1981) Preparation of blood hemoglobins of vertebrates, Meth. Enzymol. 76, 5-29. Rossi-Fanelli A. and Antonini E. (1958) Studies on the oxygen and carbon monoxide equilibria of human myoglobin. Archs Biochem. Biophys. 77, 478-492. Russell E. S. and McFarland E. C. (1974) Genetics of mouse hemoglobin. Ann. N.Y. Acad. Sk 241, 25-37. Schmidt-Nielsen K. and Larimer J. L. (1958) Oxvzen dissociation curves of mammalian blood in relation lo body size. Am. J. Physioi. 195, 424-428. Smith D. B., Brunori M., Antonini E. and Wyman J. (1966) The oxygen Bohr effect in mouse hemoglobin. Archs Biochem. Biophys. 113, 725-729.