Unni E.H. Fyhn

Hemoglobin heterogeneity in Amazonian fishes

Abstract 1. The hemoglobin patterns of hemolysates from the lungfish,Lepidosiren paradoxa, four species of freshwater rays (Potamotrygon), and actinopterygian teleosts from 77 genera have been characterized by polyacrylamide disc gel electrophoresis. 2. These tropical fishes have hemolysates which are as complex as those reported from temperate zone fishes. 3. There are no obvious correlations between hemoglobin multiplicity and the fish behaviour or habitat preference. 4.Potamotrygon has blurred hemoglobin patterns,Lepidosiren shows a single hemoglobin, and the other teleosts show a mean of 4.0 hemoglobin bands per phenotype. 5. Ostariophysan fishes have a lower mean number of bands per phenotype than acanthopterygian fishes. 6. Patterns of a single hemoglobin band occurred in 8% of the phenotypes. 7. Two or more hemoglobin phenotypes were found in about 10 species. 8. SDS gel electrophoresis showed the fish hemoglobin chains to have molecular weights comparable to those of human Hb A.

A comparative study of the oxygen equilibria of blood from 40 genera of Amazonian fishes

Abstract 1. We have examined the oxygen-affinity of the fresh, whole blood from representatives of 40 genera of Amazonian fishes. 2. When all the air-breathing fishes and all the water-breathing fishes were considered, no differentiation in oxygen affinity could be distinguished, although in two cases of closely related species, i.e. Osteoglossum , a water breather, vs Arapaima , an air breather, and Hoplias , a water breather, vs Hoplerythrinus , an air breather; the air breathers have bloods with lower oxygen affinity than do the water breathers. 3. Significant differences in the oxygen affinity were found when fishes were considered by habitat: the oxygen affinities were in general correlated with available environmental oxygen and relative oxygen demand of the fishes.

Occurrence of root effect hemoglobinsin Amazonian fishes

Abstract o 1. The Root effect was measured in hemolysates from representatives of 56 genera of Amazonianfishes. 2. Hemolysates from several species of air-breathing fishes were found to have Root effects, contraryto published hypotheses. 3. Hemolysates from Potamotrygon, a freshwater ray, exhibit a Root effect under our experimental conditions. 4. The pattern of Root effect distribution correlates positively with the distribution of choroid retiamirabile and swimbladders, but not with the distribution of swimbladder retia mirabile; it is proposed that the former is the more primitive structure which is associated with the origin of Root effect hemoglobins 5. Some of the fish hemoglobins differ spectrally from one another. The positions of the absorptionmaxima of the deoxyhemoglobins range from 553 nm (Lepidosiren paradoxa and Potamotrygon sp.) to 560 nm (Plagioscion) 6. Occurrence of Root effects is not correlated with the complexity of the hemoglobin electrophoreticpattern, although several species are found to have multiple hemoglobin systems in which the Root effect is restricted to certain components.

Functional studies on the separated hemoglobin components of an air-breathing catfish,Hoplosternum littorale (Hancock)

1. The two hemoglobins, Hb I and II, of the obligate air-breathing catfish,Hoplosternum littorale have been isolated. 2. The unfractionated stripped hemoglobin has a high oxygen affinity, a normal alkaline Bohr effect, and a Root effect. 3. Both the Bohr and Root effects are enhanced by 1 mM ATP. 4. Stripped Hb I has a relatively high oxygen affinity, a reversed Bohr effect between pH 6.0 and 8.0 (Δlog P50†DpH> 0), but no Root effect. Addition of 1 mM ATP to Hb I causes a marked reduction in the oxygen affinity, a change to a normal alkaline Bohr effect (Δlog P50ΔpH< 0), but no Root effect. 5. Stripped Hb II has a lower oxygen affinity at low pH and a higher oxygen affinity at high pH than does Hb I. Hb II shows a large alkaline Bohr effect which is only slightly increased by 1 mM ATP and a Root effect at low pH which is enhanced by 1 mM ATP. 6. The observed rates of O2 dissociation and of CO combination with Hbs I and II show differences which parallel those observed in the oxygen equilibrium measurements.

A fetal-maternal shift in the oxygen equilibrium of hemoglobin from the viviparous caecilian,Typhlonectes compressicauda

1. The equilibria and kinetics of oxygen binding by blood and hemoglobin from adult and fetal caecilians,Typhlonectes compressicauda, have been measured. 2. The oxygen affinity of fetal blood is higher than that of adult blood. 3. Electrophoresis of adult and fetal hemoglobins suggests that they may be identical: a major and minor component occurs in each. 4. Adult and fetal hemoglobins have identical oxygen equilibria. Stripped hemoglobins have a high oxygen affinity and no Bohr effect between pH 6.5 and 10.0. An “acid”, reversed Bohr effect is present below pH 6.5. The addition of 1 mM ATP reduces the oxygen affinity markedly and produces a moderate, normal Bohr effect. 5. The major nucleoside triphosphate in fetal and adult erythrocytes is adenosine triphosphate: about 10% of the nucleoside triphosphates is guanosine triphosphate. Adult erythrocytes contain 3 times as much ATP as do the fetal erythrocytes. 6. The fetal to maternal shift in the oxygen equilibrium is mediated entirely by the difference in ATP content of the maternal and fetal red blood cells.

The effect of temperature on the oxygen equilibria of fish hemoglobins in relation to environmental thermal variability

Abstract 1. We have measured the temperature dependence of the oxygen equilibria of blood and hemoglobin solutions from four neotropical and three temperate zone fishes. 2. Significant differences exist in the heats of oxygenation of the bloods but not the hemoglobin solutions from different species. 3. The differences in thermal sensitivity of the oxygenation of the bloods appear to depend on differences in intracellular pH, organic phosphates and other ligand-linked variables and not the intrinsic enthalpies of the oxygenation of the hemoglobins themselves.

The isolation and characterization of the hemoglobin components ofMylossoma sp., an amazonian teleost

Abstract 1. The two hemoglobins of a characid fish, Mylossoma sp. have been isolated. The more anodally migrating electrophoretic component constitutes 89% of the total hemolysate. 2. The two native hemoglobins have apparent molecular weights of 57,000 by gel chromatography. The apparent molecular weights of the denatured subunits are 14,100 by sodium dodecyl sulfate gel electrophoresis. No polymerization occurs after oxidation with potassium ferricyanide. 3. Oxygen binding studies indicate that the more anodal hemoglobin component possesses a Root effect. At pH 5.9 in the presence of 1 mM ATP the hemoglobin is only 45% saturated when equilibrated with air at 1 atm. 4. The more anodal component possesses a normal Bohr effect which is enhanced in the presence of 1 mM ATP. The cooperativity, as determined by n of the Hill equation varies with pH; at and below pH 6.7 in the presence of 1 mM ATP, n . The presence of 1 mM ATP causes a reduction in n below pH 8.2. 5. The less anodal component evinces very different behavior having reverse Bohr effectΔlog p 1/2 /ΔpH= 0.14, between pH 7.0 and 8.0 which changes to a normal Bohr effect,Δlog p 1/2 /ΔpH= −0.13 upon addition of 1 mM ATP. This hemoglobin shows cooperativity, at all pH values studied. It does not show a Root effect. 6. Rapid kinetic studies of CO binding and O 2 dissociation of the isolated hemoglobin components show that both processes are pH dependent for each of the components. These results are consistent with the analysis of the oxygen equilibrium data. 7. Mylossoma hemoglobins resemble those of Hoplosternum , trout, salmon, sucker, eel and loach in the degree of their functional differentiation and may represent evolutionary specializations designed to serve diverse physiological functions.

Equilibria and kinetic of oxygen and carbon monoxide binding to the haemoglobin of the South American lungfish,Lepidosiren paradoxa

Abstract 1. The haemoglobin of the South American lungfishLepidosiren paradoxa has a single component. 2. The equilibria of this respiratory protein with oxygen have been investigated both in the blood and with the purified haemoglobin. There is a substantial, normal, alkaline Bohr effect and marked sensitivity to organic phosphates in the haemoglobin solutions. 3. Studies on the pH dependence of the kinetics of oxygen dissociation can be interpreted in terms of a normal Bohr effect. 4. The kinetics of combination of carbon monoxide have an unusual pH dependence. 5. These findings are discussed in terms of the two-state model of Monodet al. (1965)

Structural and functional studies of hemoglobins isolated from Amazon stingrays of the genusPotamotrygon

Abstract 1. The hemolysates isolated from Amazonian stingrays,Potamotrygon sp. are heterogeneous. Their apparent molecular weights, determined by gel filtration, vary from 54,000 to 58,000. Gel filtration experiments of ferro- and ferrihemoglobin show that all of the hemolysates are tetrameters which do not polymerize upon oxidation. 2. A Bohr effect is evident in oxygen equilibrium experiments of whole blood and of hemolysates. Thep1/2 of stripped hemolysates decreases 33-fold between pH 5.95 and 9.0. Neither NaCl nor urea in concentrations up to 4 M significantly affect the oxygen affinity of the stripped hemolysate. 3. The hemoglobin shows cooperative behavior. Then values, determined by the Hill equation, fall within the range 1.0–1.6 between pH 6.0 and 9.0. 4. The O2 dissociation and CO combination kinetics were measured by stopped flow spectrophotometry and flash photolysis, respectively. The oxygen dissociation rate decreases and carbon monoxide combination rate increases with increasing pH, both changing over four-fold between pH 6.3 and 8.8. The oxygen dissociation rate is also phosphate sensitive, increasing approx 22% in the presence of 1 mM ATP at pH 6.3. 5. Urea denaturation experiments indicate that Potamotrygonid hemoglobin is more stable at high urea concentrations than those of other elasmobranchs and teleosts, being only 50% denatured at urea concentrations greater than 9 M. 6. The oxygen affinity of the blood is higher than those previously reported for many species of marine rays and skates (p1/2 = 12mm HG at 30°C in the absence of CO2).

The hemoglobin ofpseudodoras, a South American catfish: Isolation, characterization and ligand binding studies

Abstract 1. The hemoglobin of the Amazonian catfish Pseudodoras sp. was isolated and characterized; it comprises a single component. 2. The hemoglobins subunit composition is similar to that of other teleost hemoglobins. The apparent native molecular weight as determined by gel filtration is 66,000. The apparent subunit molecular weight is 14,300 by sodium dodecyl sulfate electrophoresis. The hemoglobin does not polymerize after oxidation by potassium ferricyanide. 3. The hemoglobin lacks a Root effect. A small Bohr effect is evident in the phosphate-free hemoglobin:Δlog p 1/2 /ΔpH is no more than about −0.1 to −0.2 and increases toΔlog p 1/2 /ΔpH = −0.4 in the presence of 1 mM ATP. The cooperativity, as determined by n of the Hill equation, is low, varying from 0.8 to 1.7 between pH 6.1 and 8.6. 4. Thep 1/2 values of stripped hemoglobin solutions are extremely low, less than 0.5 mm Hg at all pH values examined between pH 6.1 and 9.0. The high oxygen affinity is reflected primarily in the CO combination rate which resembles that found in myoglobins and isolated subunits of human hemoglobin. 5. Both the CO combination rate and the O 2 dissociation rate determined by stopped-flow spectrophotometry are pH and phosphate sensitive. Between pH 6.2 and 8.1 the CO on rate increases about 5-fold in the phosphate-free hemoglobin. Addition of 1 mM ATP causes a depression in the rate at all pH values examined. The O 2 off rate decreases 7-fold going from pH 6.0 to 8.2 in stripped hemoglobin solutions. Addition of 1 mM ATP induces a 10-fold decrease over the same range. At pH values below 6.0 a depression in the O 2 off rate occurs in the stripped hemoglobin, indicative of an acid Bohr effect.