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Physiological significance of root effect hemoglobins in trout
Respiration Physiology (1982) 49, 1-10 Elsevier Biomedical Press
1
PHYSIOLOGICAL SIGNIFICANCE OF ROOT EFFECT HEMOGLOBINS IN TROUT
R O L F L. I N G E R M A N N Obstetrics and Gynecology Research, Oregon Health Sciences UniversitJ)~ Portland, OR 97201 U.S.A.
Abstract. Root effect hemoglobins are found in trout and salmon. These functionally unique hemoglobins are believed to be intricately involved in oxygen secretion to the swimbladders of many fishes. This has also been proposed as their primary physiological role in trout; however, such an oxygen secretory function is unlikely in these fish. Trout swimbladders characteristically contain very high concentrations of nitrogen and the anatomical structures associated with swimbladder gas secretion are absent from trout. Also, trout appear to fill their swimbladders by physical deposition of gas, with the swallowing of surface air, rather than by chemical secretion, thus obviating a role of Root effect hemoglobins at the swimbladder. A chemical secretion of gas is likely involved in oxygen secretion to the eye. The eyes of trout, as those of many fishes, contain very high concentrations of oxygen which exceed those found in the blood or ambient water. Data are consistent with a physiological role of trout Root effect hemoglobins in oxygen secretion to the eye; they are not consistent with a role in any gaseous secretion to the swimbladder. Eye Gasbladder Hemoglobin
Oxygen secretion Root effect Swimbladder
Root effect hemoglobins R o o t effect h e m o g l o b i n s are f o u n d in m a n y b o n y fishes i n c l u d i n g t r o u t a n d s a l m o n ( H a s h i m o t o et al., 1960; Black et al., 1966a,b; G i o v e n c o et al., 1970). These h e m o g l o b i n s are characterized by a n extreme p H d e p e n d e n c e o f ligan d affinity. A t p H values a b o v e a p p r o x i m a t e l y 7, these h e m o g l o b i n s are similar in ligand b i n d i n g to the m a m m a l i a n h e m o g l o b i n s . A t low p H , however, these R o o t effect h e m o g l o b i n s r e m a i n u n s a t u r a t e d in the presence o f relatively high oxygen tensions (fig. 1). This is t h o u g h t to involve two types o f p r o t o n - l i n k e d changes in the h e m o g l o b i n molecule ( N o b l e et al., 1970; Saffran a n d G i b s o n , 1981). P r o t o n s Accepted for publication 15 March 1982 0034-5687/82/0000-0000/$02.75 © Elsevier Biomedical Press
2
R.L. INGERMANN I00" /
75
pH 7.4
I Root Effect t L
50 ¸
V pH 6.4
25
2o
go
~o
so
,oo
,2o
PO 2
Fig. l. Hemoglobin oxygen saturation plotted against partial pressure of oxygen (in mm Hg) shows the Root effect of some fish hemoglobins.
apparently affect the confirmational equilibrium between the low affinity hemoglobin structure, usually associated with the deoxygenated state, and the high affinity form, usually associated with the oxygenated state. Also, high proton concentrations appear to decrease the ligand affinity of both confirmational states. Organic phosphates that have been found in high concentrations in most fish erythrocytes, including those of trout (Leray, 1979; Lane et al., 1981), accentuate the low-pH effect by stabilizing the low affinity hemoglobin structure as well (Tan et al., 1973; Gillen and Riggs, 1977). At low pH, especially in the presence of high intra-erythrocytic organic phosphate concentrations, Root effect hemoglobins are only partially saturated with oxygen. Consequently, oxygenated Root effect hemoglobins can unload oxygen, against high oxygen tensions, when exposed to low pH. This property of Root effect hemoglobins suggests a functional role in oxygen secretion to the swimbladder, and possibly to the eye, of many teleosts. The swimbladder and/or eyes of many teleosts contain oxygen tensions higher than those of blood or ambient water and therefore require a secretory mechanism (Scholander and van Dam, 1954; Wittenberg and Wittenberg, 1962, 1974).
3
FUNCTION OF TROUT ROOT EFFECT HEMOGLOBINS
Gas secretion to the swimbladder
Many fishes utilize gas-filled swimbladders to reduce body density and control buoyancy. This generally reduces or eliminates the energy required to maintain the animal's vertical position in the water column. Many research efforts have focused on the mechanism by which a teleost fills its swimbladder. An important theory of the secretory mechanism has emerged (see reviews by Steen, 1970; Blaxter and Tytler, 1978) and its basis is illustrated in fig. 2. This mechanism is thought to involve the secretion of primarily lactic acid by the gas gland cells. Lactic acid is secreted into the blood vessels of a countercurrent multiplier structure, the rete mirabile, located at or near the swimbladder. Countercurrent multiplication of the lactic acid results in high acid concentrations which promote gas secretion into the swimbladder by a dual mechanism. First, high acid concentrations are likely to cause the net dissociation of oxygen from Root effect hemoglobins as described. Second, since high solute concentrations reduce gas solubility, high lactic acid concentrations are likely to salt-out gases from solution. These gases, primarily oxygen, are also concentrated by the rete mirabile thereby developing sufficiently high pressures to fill the swimbladder. The trout is a physostome. Physostomous, as opposed to physoclistous, teleosts have a muscular pneumatic duct connecting the swimbladder with the gut (fig. 3). However, although the swimbladder communicates with the environment and con-
\
I "uc°.
Glucose
HbO2"~-~
> Hb f
I f//i /
i
I L~'itOl':!
L7-
SWIMBLADDER
02 N2 CO2
~- o 2 N2
RETEMIRABILE
; CO2 GLAND X CELLS
Fig. 2. Countercurrent multiplication of acid and gases by the rete mirabile and the unique properties of Root effect hemoglobins are likely involved in the chemical secretion of gases to the swimbladder (modified from F/inge, 1973).
4
R.L. I N G E R M A N N
ii f
Pneumatic duct
;Swimbladder
Fig. 3. The swimbladder of the physoclistous bass (A) does not communicate with the external environment while that of the physostomous trout (B) does via a muscular pneumatic duct (modified from Lagler et al., 1977).
ceivably could be filled by swallowing air, Sundnes (1963) and F~inge (1966) have postulated that gas secretion in the physostomes differs only quantitatively rather than qualitatively from that of the physoclists. It is therefore reasonable to propose that physostomous trout utilize their Root effect hemoglobins to inflate their swimbladders. Numerous reports have, in fact, supported this proposition (Binotti et al., 1971; Brunori, 1975; Brunori et al., 1973, 1978; Giardina et al., 1973, 1976). If trout do utilize their Root effect hemoglobins in swimbladder physiology in the manner described, several observations would be expected. One would expect the swimbladder to contain more than 21 ~o oxygen, the fraction of oxygen in air. One would also expect to find a rete mirabile and an ability to fill the swimbladder when the animal is denied access to surface air. However, experimental findings do not confirm these expectations. Evidence against oxygen secretion to the trout swimbladder
Unlike the swimbladders of many teleosts which contain primarily oxygen, those of trout contain primarily nitrogen. The swimbladder of the lake trout, Salvelinus namaycush, contains 90-99~ nitrogen (Saunders, 1953; Tait, 1956). The bladders of the arctic char, Salmo alpinus, and the sea char, Salmo salvelinus, contain 85-99~o and 99~ nitrogen, respectively (Sundnes, 1963). Jacobs (1934) found that the swimbladder of the rainbow trout, Salmo irideus, contained gas consisting of more than
FUNCTION OF TROUT ROOT EFFECT HEMOGLOBINS
5
90~o nitrogen. I have found the rainbow trout, Salmo gairdneri, to have a swimbladder content consisting of only 8~o oxygen (range 6-9~o, n = 6, Lex Oz CON-TL analyzer). Furthermore, Wittenberg (1958) found the newly deposited swimbladder gas of the rainbow trout, Salmo gairdneri, and the brown trout, Salmo trutta, to be essentially pure nitrogen. Wittenberg also found that the goldfish, Carassius auratus, as the trout, possesses high steady-state swimbladder concentrations of nitrogen, yet newly secreted gas in this fish is largely oxygen. He postulated that secretion of oxygen, and concomitantly nitrogen, followed by oxygen reabsorption occurred in the goldfish swimbladder. There is apparently no such oxygen secretion in the trout. A rete mirabile, along with appreciable concentrations of oxygen in swimbladder gas, have been found in the coregonid, Coregonus lavaretus, a close relative of trout (Sundnes et al., 1958; Fahl6n, 1959). Trout, however, do not possess retia mirabilia at their swimbladders (Jones and Marshall, 1953; Kriegsmann, 1975). The Root effect involves a marked change in ligand binding with change in pH. However, nitrogen does not function as a hemoglobin ligand. Root effect hemoglobins and countercurrent multipliers are not likely to be involved in nitrogen deposition and, hence, swimbladder physiology in trout. No known molecular mechanism exists for the selective secretion of nitrogen. It appears reasonable, therefore, to postulate that the physostomous trout fill their swimbladders by swallowing surface air, thus obviating the role of Root effect hemoglobins in that function. The importance of air swallowing by trout is indicated by the following findings. Ledebur (1928) reported that young brown trout originally fill their swimbladders by gulping surface air. In confirmation, Tait (1960) found that several trout species were unable to fill their swimbladders when denied access to surface air. Brown trout, for example, normally fill theirs by approximately 17 weeks after hatching; those prevented from reaching the water surface were gas-free even after 84 weeks. Jacobs (1934) noted that several members of the genus Salmo could fill experimentally emptied swimbladders by gulping surface air and would not refill them if prevented from reaching the surface. These swimbladders were still gas-free after 21-25 days. Wittenberg (1958) reported, however, that rainbow and brown trout that had been denied access to surface air could fill their emptied swimbladders, with approximately 97~o nitrogen, by 32-100~o of the original volume in 13 days. This was in sharp contrast to the filling rates of 100~o in 6 to 24 hours for most of the non-salmonid fishes examined. Furthermore, Fahl6n (1971) found, under similar conditions, that deflated rainbow trout had refilled their swimbladders by less than a third after 20 days. After 40 days, the swimbladders were still incompletely filled. Perhaps the swallowing of gas bubbles beneath the air surface could account for the slow and inconsistent filling rates noted by Wittenberg and Fahl6n. Such swallowing appears to occur in at least some salmonids (Saunders, 1965). Jacobs (1934) and Kriegsmann (1975) observed that normal trout became negatively buoyant if denied access to surface air for several days. In spite of these findings, Kriegsmann assumed that the brown trout, S. trutta lacustris (but not
6
R.L. INGERMANN
S. trutta fario) could secrete gas into its swimbladder. He applied a vacuum to a sealed vessel containing a negatively buoyant fish and monitored the pressure reduction required to make the fish neutrally buoyant. After repeated trials he found that most of the fish became less dense over a period of 100 days. Kriegsmann interpreted these results as indicating gas secretion to the swimbladder. The fish, however, were less healthy at the end than at the beginning of the experiment and it is possible that the fish became less dense with time due to catabolism of dense tissue. Nonetheless, trout become markedly negatively buoyant if denied access to surface air for several days. Surface gulping is likely the primary mode of swimbladder filling in trout. This is further supported by the findings of Wittenberg (1958) that the argon to nitrogen ratio of trout swimbladder gas is very close to that of air. In order to have such similar ratios, it appears more likely that these gases are deposited physically, as by gulping, than by being secreted chemically since argon and nitrogen have different solubilities in fluids and tissues (Abernethy, 1972). Filling of the swimbladder by gulping would result in fish being neutrally buoyant at or near the upper water surface. Due to the relationship of gas pressure and volume given by Boyle's law, such fish would become negatively buoyant as they descended; they would not remain neutrally buoyant. Although this concept may be used to argue against gulping, several studies have shown various trout to be negatively buoyant, even near the surface (Sundnes, 1959; Saunders, 1965). Furthermore, a swimbladder need not reduce body density to that of water to be of benefit to the animal; partially filled swimbladders are better than none at all. It appears likely, therefore, that no chemical mechanism is required to account for the deposition of gas to the trout swimbladder; the swallowing of air is the primary, if not sole, mode of swimbladder filling. The herring, Clupea harengus, is another fish believed to rely solely on gulping to fill its swimbladder (Brawn, 1962). Fahl6n (1971) reported that the internal surface of the rainbow trout swimbladder is lined with an epithelium, the cells of which contain mitochondria, thus indicating the presence of oxidative phosphorylation and oxygen utilization. Gas reabsorption by the blood and/or metabolic activity of this epithelium, with low gaseous permeability of the swimbladder wall, may account for an oxygen composition of less than 21~o.
Function of trout Root effect hemoglobins
Root effect hemoglobins are found in trout but, in spite of suggestions to the contrary, they are not involved in swimbladder physiology in these fish. Recent reports by Farmer et al. (1979) and Ingermann and Terwilliger (1980, 1982) have shown that numerous fishes lack a swimbladder (and therefore lack the need to secrete gas to a swimbladder) yet possess hemoglobins exhibiting the Root effect. Consequently, Root effect hemoglobins and swimbladder function are not inextri-
FUNCTION OF TROUT ROOT EFFECT HEMOGLOBINS
7
cably associated. Hence, what may be the physiological role of Root effect hemoglobins in trout? The inner surfaces of the retinas of fishes, in contrast to those of mammals, are poorly vascularized (Copeland, 1974), yet Wittenberg and Wittenberg (1962, 1974) found that the oxygen tensions within the eyes of some teleosts greatly exceeded those in the blood or water. These high oxygen concentrations require the operation of a secretory mechanism. Such a mechanism may be very similar to that depicted in fig. 2 for the swimbladder. Although the teleost retina is poorly vascularized, a vascular structure, the choroid rete, is found in the eyes of many fishes. The chorid rete is located behind the retina and is structurally analogous with the rete mirabile of the swimbladder, i.e., it appears to be a vascular countercurrent multiplier (Wittenberg and Wittenberg, 1962, 1974; Copeland, 1974). The Wittenbergs (1962, 1974) found that a good correlation exists between the presence and size of the choroid rete and elevated oxygen tensions within the eye. They postulated that the choroid rete is involved in oxygen secretion to the fish eye. The gas gland cells at the swimbladder convert glucose to lactic acid even in the presence of high oxygen tensions (Ball et al., 1955; D'Aoust, 1970); the rete mirabile can therefore build high lactic acid and oxygen concentrations simultaneously. There is evidence that the retina may serve the same function as the gas gland cells, i.e., lactic acid secretion, in oxygen secretion to the eye. Hoffert and Fromm (1970) and Bayens et al. (1971) have shown that trout retinas incubated in vitro convert 60~o of the glucose metabolized to lactic acid under aerobic conditions. The retina may therefore provide the choroid rete with the lactic acid presumably necessary for the concentration of oxygen. There is also evidence that links Root effect hemoglobins with oxygen secretion to the fish eye. In numerous fishes without a swimbladder, the presence of Root effect hemoglobins correlated very well with the presence of a choroid rete (Farmer et al., 1979; Ingermann and Terwilliger, 1980, 1982). It is therefore reasonable to propose that oxygen secretion to the fish eye is functionally analogous to oxygen secretion to the swimbladder in fishes whose swimbladders possess a rete mirabile. Specifically for trout, they do possess choroid retia (Wittenberg and Haedrich, 1974; Fairbanks et al., 1969; Fonner et al., 1973). They also have elevated ocular oxygen tensions. Fairbanks et al. (1969) showed that oxygen tensions in the rainbow trout reached 800 mm Hg and averaged 400 mm Hg. This average is approximately 20 times higher than average Pao2 values. High ocular oxygen concentrations exist in the lake trout as well (Fonner et al., 1973). Furthermore, trout retinas produce lactic acid by aerobic glycolysis. High trout ocular oxygen tensions may therefore be generated with Root effect hemoglobins, retina-derived lactic acid, and countercurrent multiplication of that lactic acid and oxygen by the choroid rete. It is more likely that the Root effect hemoglobins of trout function in eye, rather than swimbladder, physiology. Farmer et al. (1979) suggested that of the choroid rete and the rete mirabile, the choroid rete may be the more primitive structure and that the choroid rete therefore
8
R.L. INGERMANN
may be associated with the origin of the Root effect. As the trout are generally regarded as primitive teleosts, the data examined in this paper appear to support their suggestion.
Summary The gulping of surface air with subsequent removal of oxygen appears to be the sole mechanism of gas deposition to the trout swimbladder. There is no evidence indicating an appreciable deposition of oxygen to the swimbladder, the gas being primarily nitrogen. There is no evidence o f a rete mirabile at the trout swimbladder. Larval trout cannot fill their swimbladders, normal adults lose buoyancy, and adult animals cannot refill experimentally emptied swimbladders (or at least fill them very slowly) if denied access to surface air. Since there is no known molecular mechanism for the selective secretion of nitrogen, the gulping of surface air, and possibly submerged bubbles, appears to be the source of swimbladder gas in the trout. It is unlikely, therefore, that swimbladder filling mechanisms among the diverse teleosts differ only quantitatively; the data support the existence of qualitative differences in mechanism. It is unlikely that trout Root effect hemoglobins are involved in swimbladder physiology. However, the eyes of trout contain very high concentrations of oxygen and, hence, require a secretory mechanism. The correlation of Root effect hemoglobins with the presence of a well-developed choroid rete, and consequently, with high ocular oxygen tensions in trout as well as other fishes, along with their unique ligand binding properties, suggests an important role for Root effect hemoglobins in the oxygen secretory mechanism of the trout eye.
Acknowledgements I wish to thank Dr. J. Job Faber for his advice and encouragement. I am grateful to Mr. Jerry Bauer and J. Eugene Welch for assistance in obtaining and analyzing trout swimbladder gas samples and to Ms. Anneliese Ingermann for help with translations. This work was supported by grant H.D. 07084 from the United States Public Health Service.
References Abernethy, J.D. (1972). The mechanism of secretion of inert gases into the fish swimbladder. Aust. J. Exp. Biol. Med. Sci. 50:365 374. Baeyens, D.A., J. R. Hoffert and P.O. Fromm (1971). Aerobic glycolysisand its role in maintenance of high 02 tensions in the teleost retina. Proc. Soc. Exp. Biol. Med. 137: 740-744. Ball, E.G., C.F. Strittmatter and O. Cooper (1955). Metabolic studies on the gas gland of the swim bladder. Biol. Bull. 108: 1-17.
FUNCTION OF TROUT ROOT EFFECT HEMOGLOBINS
9
Binotti, I., S. Giovenco, B. Giardina, E. Antonini, M. Brunori and J. Wyman (1971). Studies on the functional properties of fish hemoglobins. II. The oxygen equilibrium of the isolated hemoglobin components from trout blood. Arch. Biochem. Biophys. 142: 274-280. Black, E.C., D. Kirkpatrick and H.H. Tucker (1966a). Oxygen dissociation curves of the blood of brook trout (Salvelinus fontinalis) acclimated to summer and winter temperatures. J. Fish. Res. Board Can. 23: 1-13. Black, E.C., H.H. Tucker and D. Kirkpatrick (1966b). Oxygen dissociation curves of the blood of Atlantic salmon (Salrno salar) acclimated to summer and winter temperatures. J. Fish. Res. Board Can. 23: 1187-1195. Blaxter, J. H. S. and P. Tytler (1978). Physiology and function of the swimbladder. Adv. Comp. Physiol. Biochem. 7: 311-367. Brawn, J.M. (1962). Physical properties and hydrostatic function of the swim bladder of herring (Clupea harengus L.). J. Fish. Res. Board Can. 19: 6354556. Brunori, M., B. Giardina, J. Bonaventura, D. Barra and E. Antonini (1973). Properties of fish hemoglobins: the hemoglobin system of trout (Salmo irideus). In: Comparative Physiology, Locomotion, Respiration, Transport and Blood, edited by L. Bolis, K. Schmidt-Nielsen and S. H.P. Maddrell. Amsterdam, North Holland Publ. Co., pp. 477-492. Brunori, M. (1975). Molecular adaptation to physiological requirements: the hemoglobin system of trout. Curr. Top. Cell. Reg. 9: 1-39. Brunori, M., M. Coletta, B. Giardina and J. Wyman (1978). A macromolecular transducer as illustrated by trout hemoglobin IV. Proc. Natl. Acad. Sci. U.S.A. 75: 4310-4312. Copeland, D.E. (1974). The anatomy and fine structure of the eye of teleost. I. The choroid body in Fundulus grandis. Exp. Eye Res. 18: 547-561. D'Aoust, B.G. (1970). The role of lactic acid in gas secretion in the teleost swimbladder. Comp. Biochem. Physiol. 32:637 668. Fahl6n, G. (1959). Rete mirabile in the gas bladder of Coregonus lavaretus. Nature (London) 184: 1001-1002. Fahl6n, G. (1971). The functional morphology of the gas bladder of the genus Salmo. Acta Anat. 78: 161 184. Fairbanks, M. B., J. R. Hoffert and P.O. Fromm (1969). The dependence of the oxygen-concentrating mechanism of the teleost eye (Salmo gairdneri) on the enzyme carbonic anhydrase. J. Gen. Physiol. 54 : 203-211. F~inge, R. (1966). Physiology of the swimbladder. Physiol. Rev. 46: 299-322. F~inge, R. (1973). The physiology of the swimbladder. In: Comparative Physiology, Locomotion, Respiration, Transport and Blood, edited by L. Bolis, K. Schmidt-Nielsen and S. H. P. Maddrell. Amsterdam, North Holland Publ. Co., pp. 135-159. Farmer, M., H.J. Fyhn, U. E. H. Fyhn and R. W. Noble (1979). Occurrence of Root effect hemoglobins in Amazonian fishes. Comp. Biochern. Physiol. 62A: 115-124. Fonner, D.B., J.R. Hoffert and P.O. Fromrn (1973). The importance of the counter current oxygen multiplier mechanism in maintaining retinal function in the teleost. Comp. Biochem. Physiol. 46A: 559-567. Giardina, B., E. Antonini and M. Brunori (1973). Hemoglobin in fishes: structural and functional properties of trout hemoglobins. Neth. J. Sea Res. 7 : 339-344. Giardina, B., M. Brunori, G. Hui Bon Hoa and P. Douzo (1976). Trout hemoglobin: oxygen binding at sub-zero temperatures. FEBS Lett. 72: 159-162. Gillen, R.G. and A. Riggs (1977). The enhancement of the alkaline Bohr effect of some fish hemoglobins with adenosine triphosphate. Arch. Biochem. Biophys. 183:678 685. Giovenco, S., I. Binotti, M. Brunori and E. Antonini (1970). Studies on the functional properties of fish haemoglobins. I. The 02 equilibrium of trout haemoglobin. Int. J. Biochem. 1 : 57451. Hashimoto, K., Y. Yamaguchi and F. Matsuura (1960). Comparative studies on two hemoglobins of salmon. IV. Oxygen dissociation curve. Bull. Jap. Soc. Sci. Fish. 26: 827-834.
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Hoffert, J.R. and P.O. Fromm (1970). Quantitative aspects of glucose catabolism by rainbow and lake trout ocular tissues including alterations resulting from various pathological conditions. Exp. Eye Res. 10: 263-272. Ingermann, R.L. and R. C. Terwilliger (1980). Root effect hemoglobins in fishes lacking a functional swimbladder. Am. Zool. 20: 767. Ingermann, R.L. and R.C. Terwilliger (1982). Presence and possible function of Root effect hemoglobins in fishes lacking a functional swimbladder. J. Exp. Zool. 220: 171-177. Jacobs, W. (1934), Untersucbungen zur Physiologie der Schwimmblase der Fische. III. Luftschlucken und Gassekretion bei Physostomen. Z. Vergl. Physiol. 20: 674-698. Jones, F. R. H. and N.B. Marshall (1953). The structure and function of the teleostean swimbladder. Biol. Rev. 28: 16-83. Kriegsmann, F. (1975). Zur Schwimmblasenfunktion der Seeforelle (Salmo trutta lacustris L.). Schweiz. Z. Hydrol. 37: 235-243. Lagler, K. F., J. E. Bardach, R. R. Miller and D. R. M. Passino (1977). Ichthyology, 2nd ed. New York, John Wiley and Sons. Lane, H. C., A.E. Rolfe and J. R. Nelson (1981). Changes in the nucleotide triphosphate/haemoglobin and nucleotide triphosphate/red cell ratios of rainbow trout, Salmo gairdneri Richardson, subjected to prolonged starvation and bleeding. J. Fish Biol. 18: 661-668. Ledebur, J.F. von (1928). Beitrage zur Physiologie der Schwimmblase der Fische. Z. Vergl. Physiol. 8 : 445-460. Leray, C. (1979). Patterns ofpurine nucleotides in fish erythrocytes. Comp. Biochem. Physiol. 64B : 77-82. Noble, R. W., L. J. Parkhurst and Q. H. Gibson (1970). The effect o f p H on the reactions of oxygen and carbon monoxide with hemoglobin of the carp, Cyprinus carpio. J. Biol. Chem. 245: 6628-6633. Saffran, W.A. and Q. H. Gibson (1981). Asynchronous ligand binding and proton release in a Root effect hemoglobin. J. Biol. Chem. 256: 4551-4556. Saunders, R.L. (1953). The swimbladder gas content of some freshwater fish with particular reference to the physostomes. Can. J. Zool. 31 : 547-560. Saunders, R. L. (1965). Adjustment of buoyancy in young Atlantic salmon and brook trout by changes in swimbladder volume. J. Fish. Res. Board Can. 22:335 352. Scholander, P. F. and L. van Dam (1954). Secretion of gases against high pressures in the swimbladder of deep sea fishes. I. Oxygen dissociation in blood. Biol. Bull. 107: 247-259. Steen, J.B. (1970). The swimbladder as a hydrostatic organ. In: Fish Physiology. Vol. IV, edited by W.S. Hoar and D.J. Randall. New York and London, Academic Press, pp. 413-443. Sundnes, G., T. Enns and P. F. Scholander (1958). Gas secretion in fishes lacking rete mirabile. J. Exp. Biol. 35:671 677. Sundnes, G. (1959). Gas secretion in coregonids. Nature (London) 183: 986-987. Sundnes, G. (1963). Studies on the high nitrogen content in the physostome swimbladder. Fiskerid. Skr. (Havundersok.) 13 : 1 8. Tait, J. S. (1956). Nitrogen and argon in salmonid swimbladders. Can. J. Zool. 34: 58-62. Tait, J. S. (1960). The first filling of the swimbladder in salmonids. Can. J. Zool. 38: 179-189. Tan, A. L., R.W. Noble and Q. H. Gibson (1973). Conditions restricting allosteric transitions in carp hemoglobin. J. Biol. Chem. 218: 2880-2888. Wittenberg, J.B. (1958). The secretion of inert gas into the swimbladder of fish. J. Gen. Physiol. 41 : 783 804. Wittenberg, J. B. and B.A. Wittenberg (1962). Active secretion of oxygen into the eye of fish. Nature (London) 194: 106-107. Wittenberg, J. B. and R.L. Haedrich (1974). The choroid rete mirabile of the fish eye. II. Distribution and relation to the pseudobranch. Biol. Bull. 146: 137-156. Wittenberg, J.B. and B.A. Wittenberg (1974). The choroid rete mirabile of the fish eye. I. Oxygen secretion and structure: comparison with the swimbladder rete mirabile. Biol. Bull. 146:116-136.
1
PHYSIOLOGICAL SIGNIFICANCE OF ROOT EFFECT HEMOGLOBINS IN TROUT
R O L F L. I N G E R M A N N Obstetrics and Gynecology Research, Oregon Health Sciences UniversitJ)~ Portland, OR 97201 U.S.A.
Abstract. Root effect hemoglobins are found in trout and salmon. These functionally unique hemoglobins are believed to be intricately involved in oxygen secretion to the swimbladders of many fishes. This has also been proposed as their primary physiological role in trout; however, such an oxygen secretory function is unlikely in these fish. Trout swimbladders characteristically contain very high concentrations of nitrogen and the anatomical structures associated with swimbladder gas secretion are absent from trout. Also, trout appear to fill their swimbladders by physical deposition of gas, with the swallowing of surface air, rather than by chemical secretion, thus obviating a role of Root effect hemoglobins at the swimbladder. A chemical secretion of gas is likely involved in oxygen secretion to the eye. The eyes of trout, as those of many fishes, contain very high concentrations of oxygen which exceed those found in the blood or ambient water. Data are consistent with a physiological role of trout Root effect hemoglobins in oxygen secretion to the eye; they are not consistent with a role in any gaseous secretion to the swimbladder. Eye Gasbladder Hemoglobin
Oxygen secretion Root effect Swimbladder
Root effect hemoglobins R o o t effect h e m o g l o b i n s are f o u n d in m a n y b o n y fishes i n c l u d i n g t r o u t a n d s a l m o n ( H a s h i m o t o et al., 1960; Black et al., 1966a,b; G i o v e n c o et al., 1970). These h e m o g l o b i n s are characterized by a n extreme p H d e p e n d e n c e o f ligan d affinity. A t p H values a b o v e a p p r o x i m a t e l y 7, these h e m o g l o b i n s are similar in ligand b i n d i n g to the m a m m a l i a n h e m o g l o b i n s . A t low p H , however, these R o o t effect h e m o g l o b i n s r e m a i n u n s a t u r a t e d in the presence o f relatively high oxygen tensions (fig. 1). This is t h o u g h t to involve two types o f p r o t o n - l i n k e d changes in the h e m o g l o b i n molecule ( N o b l e et al., 1970; Saffran a n d G i b s o n , 1981). P r o t o n s Accepted for publication 15 March 1982 0034-5687/82/0000-0000/$02.75 © Elsevier Biomedical Press
2
R.L. INGERMANN I00" /
75
pH 7.4
I Root Effect t L
50 ¸
V pH 6.4
25
2o
go
~o
so
,oo
,2o
PO 2
Fig. l. Hemoglobin oxygen saturation plotted against partial pressure of oxygen (in mm Hg) shows the Root effect of some fish hemoglobins.
apparently affect the confirmational equilibrium between the low affinity hemoglobin structure, usually associated with the deoxygenated state, and the high affinity form, usually associated with the oxygenated state. Also, high proton concentrations appear to decrease the ligand affinity of both confirmational states. Organic phosphates that have been found in high concentrations in most fish erythrocytes, including those of trout (Leray, 1979; Lane et al., 1981), accentuate the low-pH effect by stabilizing the low affinity hemoglobin structure as well (Tan et al., 1973; Gillen and Riggs, 1977). At low pH, especially in the presence of high intra-erythrocytic organic phosphate concentrations, Root effect hemoglobins are only partially saturated with oxygen. Consequently, oxygenated Root effect hemoglobins can unload oxygen, against high oxygen tensions, when exposed to low pH. This property of Root effect hemoglobins suggests a functional role in oxygen secretion to the swimbladder, and possibly to the eye, of many teleosts. The swimbladder and/or eyes of many teleosts contain oxygen tensions higher than those of blood or ambient water and therefore require a secretory mechanism (Scholander and van Dam, 1954; Wittenberg and Wittenberg, 1962, 1974).
3
FUNCTION OF TROUT ROOT EFFECT HEMOGLOBINS
Gas secretion to the swimbladder
Many fishes utilize gas-filled swimbladders to reduce body density and control buoyancy. This generally reduces or eliminates the energy required to maintain the animal's vertical position in the water column. Many research efforts have focused on the mechanism by which a teleost fills its swimbladder. An important theory of the secretory mechanism has emerged (see reviews by Steen, 1970; Blaxter and Tytler, 1978) and its basis is illustrated in fig. 2. This mechanism is thought to involve the secretion of primarily lactic acid by the gas gland cells. Lactic acid is secreted into the blood vessels of a countercurrent multiplier structure, the rete mirabile, located at or near the swimbladder. Countercurrent multiplication of the lactic acid results in high acid concentrations which promote gas secretion into the swimbladder by a dual mechanism. First, high acid concentrations are likely to cause the net dissociation of oxygen from Root effect hemoglobins as described. Second, since high solute concentrations reduce gas solubility, high lactic acid concentrations are likely to salt-out gases from solution. These gases, primarily oxygen, are also concentrated by the rete mirabile thereby developing sufficiently high pressures to fill the swimbladder. The trout is a physostome. Physostomous, as opposed to physoclistous, teleosts have a muscular pneumatic duct connecting the swimbladder with the gut (fig. 3). However, although the swimbladder communicates with the environment and con-
\
I "uc°.
Glucose
HbO2"~-~
> Hb f
I f//i /
i
I L~'itOl':!
L7-
SWIMBLADDER
02 N2 CO2
~- o 2 N2
RETEMIRABILE
; CO2 GLAND X CELLS
Fig. 2. Countercurrent multiplication of acid and gases by the rete mirabile and the unique properties of Root effect hemoglobins are likely involved in the chemical secretion of gases to the swimbladder (modified from F/inge, 1973).
4
R.L. I N G E R M A N N
ii f
Pneumatic duct
;Swimbladder
Fig. 3. The swimbladder of the physoclistous bass (A) does not communicate with the external environment while that of the physostomous trout (B) does via a muscular pneumatic duct (modified from Lagler et al., 1977).
ceivably could be filled by swallowing air, Sundnes (1963) and F~inge (1966) have postulated that gas secretion in the physostomes differs only quantitatively rather than qualitatively from that of the physoclists. It is therefore reasonable to propose that physostomous trout utilize their Root effect hemoglobins to inflate their swimbladders. Numerous reports have, in fact, supported this proposition (Binotti et al., 1971; Brunori, 1975; Brunori et al., 1973, 1978; Giardina et al., 1973, 1976). If trout do utilize their Root effect hemoglobins in swimbladder physiology in the manner described, several observations would be expected. One would expect the swimbladder to contain more than 21 ~o oxygen, the fraction of oxygen in air. One would also expect to find a rete mirabile and an ability to fill the swimbladder when the animal is denied access to surface air. However, experimental findings do not confirm these expectations. Evidence against oxygen secretion to the trout swimbladder
Unlike the swimbladders of many teleosts which contain primarily oxygen, those of trout contain primarily nitrogen. The swimbladder of the lake trout, Salvelinus namaycush, contains 90-99~ nitrogen (Saunders, 1953; Tait, 1956). The bladders of the arctic char, Salmo alpinus, and the sea char, Salmo salvelinus, contain 85-99~o and 99~ nitrogen, respectively (Sundnes, 1963). Jacobs (1934) found that the swimbladder of the rainbow trout, Salmo irideus, contained gas consisting of more than
FUNCTION OF TROUT ROOT EFFECT HEMOGLOBINS
5
90~o nitrogen. I have found the rainbow trout, Salmo gairdneri, to have a swimbladder content consisting of only 8~o oxygen (range 6-9~o, n = 6, Lex Oz CON-TL analyzer). Furthermore, Wittenberg (1958) found the newly deposited swimbladder gas of the rainbow trout, Salmo gairdneri, and the brown trout, Salmo trutta, to be essentially pure nitrogen. Wittenberg also found that the goldfish, Carassius auratus, as the trout, possesses high steady-state swimbladder concentrations of nitrogen, yet newly secreted gas in this fish is largely oxygen. He postulated that secretion of oxygen, and concomitantly nitrogen, followed by oxygen reabsorption occurred in the goldfish swimbladder. There is apparently no such oxygen secretion in the trout. A rete mirabile, along with appreciable concentrations of oxygen in swimbladder gas, have been found in the coregonid, Coregonus lavaretus, a close relative of trout (Sundnes et al., 1958; Fahl6n, 1959). Trout, however, do not possess retia mirabilia at their swimbladders (Jones and Marshall, 1953; Kriegsmann, 1975). The Root effect involves a marked change in ligand binding with change in pH. However, nitrogen does not function as a hemoglobin ligand. Root effect hemoglobins and countercurrent multipliers are not likely to be involved in nitrogen deposition and, hence, swimbladder physiology in trout. No known molecular mechanism exists for the selective secretion of nitrogen. It appears reasonable, therefore, to postulate that the physostomous trout fill their swimbladders by swallowing surface air, thus obviating the role of Root effect hemoglobins in that function. The importance of air swallowing by trout is indicated by the following findings. Ledebur (1928) reported that young brown trout originally fill their swimbladders by gulping surface air. In confirmation, Tait (1960) found that several trout species were unable to fill their swimbladders when denied access to surface air. Brown trout, for example, normally fill theirs by approximately 17 weeks after hatching; those prevented from reaching the water surface were gas-free even after 84 weeks. Jacobs (1934) noted that several members of the genus Salmo could fill experimentally emptied swimbladders by gulping surface air and would not refill them if prevented from reaching the surface. These swimbladders were still gas-free after 21-25 days. Wittenberg (1958) reported, however, that rainbow and brown trout that had been denied access to surface air could fill their emptied swimbladders, with approximately 97~o nitrogen, by 32-100~o of the original volume in 13 days. This was in sharp contrast to the filling rates of 100~o in 6 to 24 hours for most of the non-salmonid fishes examined. Furthermore, Fahl6n (1971) found, under similar conditions, that deflated rainbow trout had refilled their swimbladders by less than a third after 20 days. After 40 days, the swimbladders were still incompletely filled. Perhaps the swallowing of gas bubbles beneath the air surface could account for the slow and inconsistent filling rates noted by Wittenberg and Fahl6n. Such swallowing appears to occur in at least some salmonids (Saunders, 1965). Jacobs (1934) and Kriegsmann (1975) observed that normal trout became negatively buoyant if denied access to surface air for several days. In spite of these findings, Kriegsmann assumed that the brown trout, S. trutta lacustris (but not
6
R.L. INGERMANN
S. trutta fario) could secrete gas into its swimbladder. He applied a vacuum to a sealed vessel containing a negatively buoyant fish and monitored the pressure reduction required to make the fish neutrally buoyant. After repeated trials he found that most of the fish became less dense over a period of 100 days. Kriegsmann interpreted these results as indicating gas secretion to the swimbladder. The fish, however, were less healthy at the end than at the beginning of the experiment and it is possible that the fish became less dense with time due to catabolism of dense tissue. Nonetheless, trout become markedly negatively buoyant if denied access to surface air for several days. Surface gulping is likely the primary mode of swimbladder filling in trout. This is further supported by the findings of Wittenberg (1958) that the argon to nitrogen ratio of trout swimbladder gas is very close to that of air. In order to have such similar ratios, it appears more likely that these gases are deposited physically, as by gulping, than by being secreted chemically since argon and nitrogen have different solubilities in fluids and tissues (Abernethy, 1972). Filling of the swimbladder by gulping would result in fish being neutrally buoyant at or near the upper water surface. Due to the relationship of gas pressure and volume given by Boyle's law, such fish would become negatively buoyant as they descended; they would not remain neutrally buoyant. Although this concept may be used to argue against gulping, several studies have shown various trout to be negatively buoyant, even near the surface (Sundnes, 1959; Saunders, 1965). Furthermore, a swimbladder need not reduce body density to that of water to be of benefit to the animal; partially filled swimbladders are better than none at all. It appears likely, therefore, that no chemical mechanism is required to account for the deposition of gas to the trout swimbladder; the swallowing of air is the primary, if not sole, mode of swimbladder filling. The herring, Clupea harengus, is another fish believed to rely solely on gulping to fill its swimbladder (Brawn, 1962). Fahl6n (1971) reported that the internal surface of the rainbow trout swimbladder is lined with an epithelium, the cells of which contain mitochondria, thus indicating the presence of oxidative phosphorylation and oxygen utilization. Gas reabsorption by the blood and/or metabolic activity of this epithelium, with low gaseous permeability of the swimbladder wall, may account for an oxygen composition of less than 21~o.
Function of trout Root effect hemoglobins
Root effect hemoglobins are found in trout but, in spite of suggestions to the contrary, they are not involved in swimbladder physiology in these fish. Recent reports by Farmer et al. (1979) and Ingermann and Terwilliger (1980, 1982) have shown that numerous fishes lack a swimbladder (and therefore lack the need to secrete gas to a swimbladder) yet possess hemoglobins exhibiting the Root effect. Consequently, Root effect hemoglobins and swimbladder function are not inextri-
FUNCTION OF TROUT ROOT EFFECT HEMOGLOBINS
7
cably associated. Hence, what may be the physiological role of Root effect hemoglobins in trout? The inner surfaces of the retinas of fishes, in contrast to those of mammals, are poorly vascularized (Copeland, 1974), yet Wittenberg and Wittenberg (1962, 1974) found that the oxygen tensions within the eyes of some teleosts greatly exceeded those in the blood or water. These high oxygen concentrations require the operation of a secretory mechanism. Such a mechanism may be very similar to that depicted in fig. 2 for the swimbladder. Although the teleost retina is poorly vascularized, a vascular structure, the choroid rete, is found in the eyes of many fishes. The chorid rete is located behind the retina and is structurally analogous with the rete mirabile of the swimbladder, i.e., it appears to be a vascular countercurrent multiplier (Wittenberg and Wittenberg, 1962, 1974; Copeland, 1974). The Wittenbergs (1962, 1974) found that a good correlation exists between the presence and size of the choroid rete and elevated oxygen tensions within the eye. They postulated that the choroid rete is involved in oxygen secretion to the fish eye. The gas gland cells at the swimbladder convert glucose to lactic acid even in the presence of high oxygen tensions (Ball et al., 1955; D'Aoust, 1970); the rete mirabile can therefore build high lactic acid and oxygen concentrations simultaneously. There is evidence that the retina may serve the same function as the gas gland cells, i.e., lactic acid secretion, in oxygen secretion to the eye. Hoffert and Fromm (1970) and Bayens et al. (1971) have shown that trout retinas incubated in vitro convert 60~o of the glucose metabolized to lactic acid under aerobic conditions. The retina may therefore provide the choroid rete with the lactic acid presumably necessary for the concentration of oxygen. There is also evidence that links Root effect hemoglobins with oxygen secretion to the fish eye. In numerous fishes without a swimbladder, the presence of Root effect hemoglobins correlated very well with the presence of a choroid rete (Farmer et al., 1979; Ingermann and Terwilliger, 1980, 1982). It is therefore reasonable to propose that oxygen secretion to the fish eye is functionally analogous to oxygen secretion to the swimbladder in fishes whose swimbladders possess a rete mirabile. Specifically for trout, they do possess choroid retia (Wittenberg and Haedrich, 1974; Fairbanks et al., 1969; Fonner et al., 1973). They also have elevated ocular oxygen tensions. Fairbanks et al. (1969) showed that oxygen tensions in the rainbow trout reached 800 mm Hg and averaged 400 mm Hg. This average is approximately 20 times higher than average Pao2 values. High ocular oxygen concentrations exist in the lake trout as well (Fonner et al., 1973). Furthermore, trout retinas produce lactic acid by aerobic glycolysis. High trout ocular oxygen tensions may therefore be generated with Root effect hemoglobins, retina-derived lactic acid, and countercurrent multiplication of that lactic acid and oxygen by the choroid rete. It is more likely that the Root effect hemoglobins of trout function in eye, rather than swimbladder, physiology. Farmer et al. (1979) suggested that of the choroid rete and the rete mirabile, the choroid rete may be the more primitive structure and that the choroid rete therefore
8
R.L. INGERMANN
may be associated with the origin of the Root effect. As the trout are generally regarded as primitive teleosts, the data examined in this paper appear to support their suggestion.
Summary The gulping of surface air with subsequent removal of oxygen appears to be the sole mechanism of gas deposition to the trout swimbladder. There is no evidence indicating an appreciable deposition of oxygen to the swimbladder, the gas being primarily nitrogen. There is no evidence o f a rete mirabile at the trout swimbladder. Larval trout cannot fill their swimbladders, normal adults lose buoyancy, and adult animals cannot refill experimentally emptied swimbladders (or at least fill them very slowly) if denied access to surface air. Since there is no known molecular mechanism for the selective secretion of nitrogen, the gulping of surface air, and possibly submerged bubbles, appears to be the source of swimbladder gas in the trout. It is unlikely, therefore, that swimbladder filling mechanisms among the diverse teleosts differ only quantitatively; the data support the existence of qualitative differences in mechanism. It is unlikely that trout Root effect hemoglobins are involved in swimbladder physiology. However, the eyes of trout contain very high concentrations of oxygen and, hence, require a secretory mechanism. The correlation of Root effect hemoglobins with the presence of a well-developed choroid rete, and consequently, with high ocular oxygen tensions in trout as well as other fishes, along with their unique ligand binding properties, suggests an important role for Root effect hemoglobins in the oxygen secretory mechanism of the trout eye.
Acknowledgements I wish to thank Dr. J. Job Faber for his advice and encouragement. I am grateful to Mr. Jerry Bauer and J. Eugene Welch for assistance in obtaining and analyzing trout swimbladder gas samples and to Ms. Anneliese Ingermann for help with translations. This work was supported by grant H.D. 07084 from the United States Public Health Service.
References Abernethy, J.D. (1972). The mechanism of secretion of inert gases into the fish swimbladder. Aust. J. Exp. Biol. Med. Sci. 50:365 374. Baeyens, D.A., J. R. Hoffert and P.O. Fromm (1971). Aerobic glycolysisand its role in maintenance of high 02 tensions in the teleost retina. Proc. Soc. Exp. Biol. Med. 137: 740-744. Ball, E.G., C.F. Strittmatter and O. Cooper (1955). Metabolic studies on the gas gland of the swim bladder. Biol. Bull. 108: 1-17.
FUNCTION OF TROUT ROOT EFFECT HEMOGLOBINS
9
Binotti, I., S. Giovenco, B. Giardina, E. Antonini, M. Brunori and J. Wyman (1971). Studies on the functional properties of fish hemoglobins. II. The oxygen equilibrium of the isolated hemoglobin components from trout blood. Arch. Biochem. Biophys. 142: 274-280. Black, E.C., D. Kirkpatrick and H.H. Tucker (1966a). Oxygen dissociation curves of the blood of brook trout (Salvelinus fontinalis) acclimated to summer and winter temperatures. J. Fish. Res. Board Can. 23: 1-13. Black, E.C., H.H. Tucker and D. Kirkpatrick (1966b). Oxygen dissociation curves of the blood of Atlantic salmon (Salrno salar) acclimated to summer and winter temperatures. J. Fish. Res. Board Can. 23: 1187-1195. Blaxter, J. H. S. and P. Tytler (1978). Physiology and function of the swimbladder. Adv. Comp. Physiol. Biochem. 7: 311-367. Brawn, J.M. (1962). Physical properties and hydrostatic function of the swim bladder of herring (Clupea harengus L.). J. Fish. Res. Board Can. 19: 6354556. Brunori, M., B. Giardina, J. Bonaventura, D. Barra and E. Antonini (1973). Properties of fish hemoglobins: the hemoglobin system of trout (Salmo irideus). In: Comparative Physiology, Locomotion, Respiration, Transport and Blood, edited by L. Bolis, K. Schmidt-Nielsen and S. H.P. Maddrell. Amsterdam, North Holland Publ. Co., pp. 477-492. Brunori, M. (1975). Molecular adaptation to physiological requirements: the hemoglobin system of trout. Curr. Top. Cell. Reg. 9: 1-39. Brunori, M., M. Coletta, B. Giardina and J. Wyman (1978). A macromolecular transducer as illustrated by trout hemoglobin IV. Proc. Natl. Acad. Sci. U.S.A. 75: 4310-4312. Copeland, D.E. (1974). The anatomy and fine structure of the eye of teleost. I. The choroid body in Fundulus grandis. Exp. Eye Res. 18: 547-561. D'Aoust, B.G. (1970). The role of lactic acid in gas secretion in the teleost swimbladder. Comp. Biochem. Physiol. 32:637 668. Fahl6n, G. (1959). Rete mirabile in the gas bladder of Coregonus lavaretus. Nature (London) 184: 1001-1002. Fahl6n, G. (1971). The functional morphology of the gas bladder of the genus Salmo. Acta Anat. 78: 161 184. Fairbanks, M. B., J. R. Hoffert and P.O. Fromm (1969). The dependence of the oxygen-concentrating mechanism of the teleost eye (Salmo gairdneri) on the enzyme carbonic anhydrase. J. Gen. Physiol. 54 : 203-211. F~inge, R. (1966). Physiology of the swimbladder. Physiol. Rev. 46: 299-322. F~inge, R. (1973). The physiology of the swimbladder. In: Comparative Physiology, Locomotion, Respiration, Transport and Blood, edited by L. Bolis, K. Schmidt-Nielsen and S. H. P. Maddrell. Amsterdam, North Holland Publ. Co., pp. 135-159. Farmer, M., H.J. Fyhn, U. E. H. Fyhn and R. W. Noble (1979). Occurrence of Root effect hemoglobins in Amazonian fishes. Comp. Biochern. Physiol. 62A: 115-124. Fonner, D.B., J.R. Hoffert and P.O. Fromrn (1973). The importance of the counter current oxygen multiplier mechanism in maintaining retinal function in the teleost. Comp. Biochem. Physiol. 46A: 559-567. Giardina, B., E. Antonini and M. Brunori (1973). Hemoglobin in fishes: structural and functional properties of trout hemoglobins. Neth. J. Sea Res. 7 : 339-344. Giardina, B., M. Brunori, G. Hui Bon Hoa and P. Douzo (1976). Trout hemoglobin: oxygen binding at sub-zero temperatures. FEBS Lett. 72: 159-162. Gillen, R.G. and A. Riggs (1977). The enhancement of the alkaline Bohr effect of some fish hemoglobins with adenosine triphosphate. Arch. Biochem. Biophys. 183:678 685. Giovenco, S., I. Binotti, M. Brunori and E. Antonini (1970). Studies on the functional properties of fish haemoglobins. I. The 02 equilibrium of trout haemoglobin. Int. J. Biochem. 1 : 57451. Hashimoto, K., Y. Yamaguchi and F. Matsuura (1960). Comparative studies on two hemoglobins of salmon. IV. Oxygen dissociation curve. Bull. Jap. Soc. Sci. Fish. 26: 827-834.
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