Robert L. Garlick

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.

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. n n n2. The unfractionated stripped hemoglobin has a high oxygen affinity, a normal alkaline Bohr effect, and a Root effect. n n n3. Both the Bohr and Root effects are enhanced by 1 mM ATP. n n n4. 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. n n n5. 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. n n n6. 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. n n n2. The oxygen affinity of fetal blood is higher than that of adult blood. n n n3. Electrophoresis of adult and fetal hemoglobins suggests that they may be identical: a major and minor component occurs in each. n n n4. 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. n n n5. 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. n n n6. 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)

Purification and structure of the polypeptide chains of earthworm hemoglobin

Abstract Carboxyhemoglobin from the earthworm, Lumbricus terrestris, separates on isoelectric focusing into a major component A, and a minor component B, which comprises 4–9% of the total. The molecular weights of all the polypeptide chains from either component have been estimated to be near 15,000–16,000 by chromatography on Sephacryl S-200 in 6 m guanidine-HCl after oxidation with performic acid. Species of higher molecular weight were not detected under these conditions. The chains remain partially aggregated, however, in 8 m urea. Electrophoresis in 8 m urea at pH 3.5 on disc gels results in the separation of four protein bands. Analysis of chromatography of either globin A or B on carboxymethylcellulose in 8 m urea indicates that three of these bands are unique polypeptides chains. The fourth, most anodic, band appears to be a product of aggregation and not a unique polypeptide chain. The amino acid composition has been determined for the three chains isolated from each component. The NH2-terminal residues for the three isolated chains of globin A have been determined to be aspartic acid, alanine, and lysine. The unfractionated globin and that from components A and B have the same NH2 termini.

The hemoglobins of Phoronopsis viridis, of the primitive invertebrate phylum phoronida: Characterization and subunit structure☆

Abstract The primitive invertebrate, Phoronopsis viridis , of the phylum Phoronida, has intra-cellular hemoglobins composed of four unique polypeptide chains, two of which associate to form hetero- and homodimers and two which do not associate at all. The CO-derivatives of the associating chains are completely dimeric; removal of the ligand does not result in further aggregation as it does in several other invertebrate hemoglobins. Oxidation of the associating hemoglobins is accompanied by dissociation to monomers, but the cyanide derivative of the methemoglobin is dimeric. The four polypeptide chains all have molecular weights of about 16,000 as determined by iron content and gel electrophoresis with sodium dodecyl sulfate. The two associating chains form three components with isoelectric points at pH 5.6, 5.9, and 6.9 whereas those for the two monomeric chains are at pH 6.2 and 7.9. The chains have been characterized by amino acid composition, tryptic peptide patterns, and the amino acid sequence of the NH 2 -terminal segment. The oxygen equilibrium of a dimeric fraction has been determined at pH 7.5 and 20 °C; the pressure of half-saturation is 2.3 mm Hg.

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).