Functional properties of the two major hemoglobin components from Leporinus friderici (Pisces)

• 1.1. The hemoglobins of Leporinus friderici were separated by liquid chromatography on DEAE-Sepharose in order to isolate the two major electrophoretic components.
• 2.2. The chromatographic fraction I (electrophoretically slow anodic) showed no Bohr effect and no nucleoside triphosphate modulation.
• 3.3. The chromatographic fraction III (electrophoretically fast anodic) showed a normal Bohr effect and addition of nucleoside triphosphate decreased oxygen affinity but did not alter the Bohr effect.
• 4.4. The whole hemolysate showed a normal Bohr effect and phosphate modulation altered both Bohr effect and oxygen affinity.
• 5.5. No or little difference between the effect of adenosine or guanosine triphosphates on hemoglobin function was observed.

     

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 P₅₀2DpH> 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.

     

Multiple hemoglobins in Triturus cristatus: their study by analytical isoelectrofocusing

• 1.1. Electrophoretic separation of the hemoglobin of healthy adult Triturus cristatus reveals four components.
• 2.2. Isoelectric focusing of the samee hemolysates in various commercial ampholytes of different chemical composition and pH range results in the separation of eight individual hemoglobin bands.
• 3.3. The bands obtained by electrophoresis are not homogeneous as revealed by individual gel electrofocusing. They finally separate into the same eight components, as in the whole hemolysate.
• 4.4. From the above findings it is concluded that this species has not four but eight individual hemoglobin molecular forms.
• 5.5. Our results demonstrate lack of hemoglobin polymorphism in this species.

     

Study of the soluble lens proteins from fifteen sparidae species

• 1.1. Soluble eye lens proteins of fifteen different Sparidae species were analysed.
• 2.2. Species-specific electrophoretic and isoelectric focusing patterns were found.
• 3.3. Significant differences in the distribution of β and γ-crystallin protein components were noted for all species.
• 4.4. These data suggest that the Sparidae family may be a heterogenenous taxonomic group encompassing considerable genetic diferences and with different evolutionary histories.

     

Effects of erythrocytic nucleoside triphosphates on oxygen equilibria of composite and fractionated hemoglobins from the facultative air-breathing amazonian catfish,Hypostomus andPterygoplichthys

• 1. Oxygen equilibria ofHypostomus andPterygoplichthys hemoglobins and their sensitivities to the erythrocytic nucleotide triphosphates (NTP), ATP and guanosine triphosphate (GTP) are studied to investigate the mechanisms by which blood adapts to air- and water-breathing (cf. Weberet al., 1979).
• 2. Hemoglobins of both species are heterogeneous. All hemoglobin fractions isolated by iso-electric focusing reveal a high sensitivity to NTP, but GTP depresses O₂ affinity about twice as effectively as ATP. A cathodal hemoglobin component with a reversed Bohr effect was found inPterygoplichthys but not inHypostomus.
• 3. The data are discussed in relation to thein vivo cofactor modulation of blood O₂ affinity and the adaptive significance of functional heterogeneity of fish hemoglobins.

     

The isolation and characterization of the hemoglobin ofBrachyplatystoma sp.: A tropical catfish

• 1. The single hemoglobin component ofBrachyplatystoma sp. has been isolated. The CO-hemoglobin has an apparent molecular weight of 69,000 as determined by gel filtration.
• 2. The hemoglobin displays both acid and alkaline Bohr effects, as organic phosphate effect and no Root effect. The whole bloodp_1/2 for oxygen shifts from 10.7 mm Hg in air equilibrated solutions to 25.1 mm Hg after the addition of 5.6% CO₂ to the equilibration gas. Thep_1/2 of purified hemoglobin varies from 0.3 mm Hg at pH 8.4 to 4.5 mm Hg at pH 5.9. The Bohr effect measured for stripped hemoglobin between pH 8.0 and 7.0 isΔlog p_1/2/ΔpH= −0.23. Additions of 1 mM ATP induce a shift in the Bohr effect toΔlog p_1/2/ΔpH= −0.58 over the same pH range.
• 3. Then value of stripped hemoglobin solutions varies from 1 at pH 5.9 to 1.7 at pH 7.0. Additions of 1 mM ATP shift the variation inn to higher pH values, and cause an increase in then value (n = 2 at pH 7.4).
• 4. The kinetics of carbon monoxide binding and oxygen dissociation are pH dependent. The CO_on rate becomes autocatalytic as the pH is lowered, indicating positive subunit interactions. The O_2off rate was homogeneous at all pH values.
• 5. The Bohr effects ofBrachyplatystoma hemoglobin and other pimelodid hemoglobins are greater than those determined for the unfractionated hemoglobins of more sedentary species from other catfish families such as the Loricariidae and Callichthyidae.

     

Adaptative features of amazon fishes: Hemoglobins,hematology, intraerythrocytic phosphates and whole blood Bohr effect of Pterygoplichthys multiradiatus (Siluriformes)

• 1.1. Hemoglobin, hematological parameters, intraerythrocytic phosphates and whole blood Bohr effect of Pterygoplichthys multiradiatus, from the Amazon river, were studied in three different conditions: in their natural environment, acclimated to normoxia and acclimated hypoxia conditions.
• 2.2. Nine anodal hemoglobin fractions were detected on starch gel electrophoresis. No qualitative differences in the Hb electrophoretic patterns were detected in the three studied groups.
• 3.3. Hematocrit, hemoglobin concentration, MCV, MCHC and MCH were different among studied conditions.
• 4.4. GTP was almost absent in the blood of animals in natural conditions and acclimated to hypoxia, but was present at a concentration similar to ATP in normoxic acclimated animals.
• 5.5. There is a tendency for higher Hb-O₂ affinity for hypoxic acclimated/acclimatized animals.

     

Comparative examination of the soluble muscle proteins of two species of angler-fish (Lophiidae): Lophius piscatorius (L.) and Lophius budegassa (Spinola)

• 1.1. A comparative examination of sarcoplasmic proteins of the two nominal European species of angler-fish, Lophius piscatorius and L. budegassa was carried out using isoelectric focusing techniques.
• 2.2. Two protein bands differing in isoelectric point proved diagnostic for L. budegassa (pI 4.40 and pI 5.75) while a third characterized L. piscatorius (pI 4.65).
• 3.3. These species-specific protein profiles provide a method of species discrimination independent of morphological criteria.
• 4.4. Within-species heterogeneity of banding pattern suggested the presence of polymorphic gene loci of potential use in studies of population structure.

     

Isolation and characterization of the hemoglobin from Paramecium caudatum

• 1.1. The hemoglobin of Paramecium caudatum has been purified to a state of homogeniety on polyacrylamide disc electrophoresis.
• 2.2. The hemoglobin appears as a single molecular species with a mol. wt of 13,500 daltons as determined by SDS disc electrophoresis.
• 3.3. An isoelectric point of 4.27 was calculated from isoelectric focusing experiments using ampholines in a pH range of 3–6.
• 4.4. The amino acid composition is: lys₅, his₂, arg₃, asp₁₂, thr₁₁, ser₉, glu₁₆, pro₂, gly₁₈, ala₂₁, cys₀, val₁₂, met₂, ile₂, leu₈, tyr₂, phe₆.
• 5.5. The spectra of three ferrous and the ferric cyanmethemoglobin derivatives are presented.

     

Root effect hemoglobins in marine fish

• 1.1. The Root effect was evaluated in hemolysates from 26 species of bony fish and 20 species of cartilaginous fish found on the Brazilian southeastern coast.
• 2.2. Teleost Root shifts, with a single exception, are correlated with the presence of the choroid rete mirabile but not with its counterpart in the swimbladder.
• 3.3. Five ray species displayed weak and moderate Root effects despite the absence of choroid and swimbladder rete.
• 4.4. The presence and intensity of the Root effect is probably primarily related to the high oxygen demand of the retina and with the importance of visual perception in fish.
• 5.5. In marine teleosts the magnitude of the Root effect seems to be associated with the presence and size of both the choroid rete and the pseudobranch.
• 6.6. An antioxidant protection of the fish eyes can be advocated for the pseudobranch.

     

Oxygen binding of intact coelomic cells and extracted hemoglobin of the echiuran Urechis caupo

• 1.1. The oxygen affinity of Urechis caupo coelomic cells is the same in normoxic and in hypoxic animals. There is no Bohr effect between pH 6.8 and 8.0.
• 2.2. The oxygen affinity of intact coelomic cells is the same as that of extracted, stripped hemoglobin. The oxygen binding properties of stripped hemoglobin are not affected by 1 mM ATP, IMP, or hydrogen ions between pH 6.8 and 8.0, nor do they clearly show cooperativity. The heat of oxygenation. ΔH, = −13.1 kcal/mol between 10 and 25 C.
• 3.3. Although U. caupo coelomic cell hemoglobin is tetrameric and intracellular, it apparently exhibits neither heterotropic nor homotropic interactions.

     

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

• 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)

     

Disparity of native oxyhemoglobin components isolated from Paramecium caudatum and Paramecium primaurelia

• 1.1. Native oxyhemoglobin components were isolated chromatographically from Paramecium caudatum and Paramecium primaurelia, and some properties of the isolated components were investigated.
• 2.2. P. caudatum was endowed with one homogeneous hemoglobin component, while the hemoglobin in P. primaurelia was resolved into three heterogeneous components being two main and one minor.
• 3.3. Spectral properties of the isolated hemoglobin components were quite similar to each other. The isolated components, however, were distinctly different in electrophoretic mobilities.
• 4.4. Molecular weight of the isolated hemoglobin components was estimated to be about 11,000.

     

Equilibria and kinetics of oxygen and carbon monoxide binding to the haemoglobin of the teleostSynbranchus marmoratus

• 1. The haemoglobins of the air-breathing fishSynbranchus marmoratus have multiple components and demonstrate polymorphism.
• 2. The equilibrium of oxygen with whole cell and isolated haemoglobin has been compared. There is a considerable alkaline Bohr effect and this is more marked in stripped haemoglobin to which 1 mM ATP has been added.
• 3. The kinetics of oxygen dissociation from oxyhaemoglobin and of carbon monoxide combination to deoxyhaemoglobin have been studied, and show surprisingly uniform kinetic time courses for a system composed of multiple components.
• 4. These findings are discussed within the framework of the requirements of the animal in its mixed aerial-aquatic environment.

     

Respiratory function and nucleotide composition of erythrocytes from tropical elasmobranchs

• 1.1. Oxygen carrying capacity and parameters of erythrocyte-oxygen binding are similar for a range of elasmobranchs with markedly different swimming behaviour.
• 2.2. Erythrocyte nucleotide components in sharks include the allosteric hemoglobin modifiers GTP and ATP in similar ratios, and the total pool appears independent of locomotory activity. A rhinobatoid ray had no detectable erythrocyte trinucleotides, but had an appreciable pool of AMP together with IMP.
• 3.3. There was no evidence for either urea or NaCl modulation of hemoglobin function in erythrocytes from a carcharhinid shark.
• 4.4. These observations lead to the conclusion that parameters of the oxygen transport system in elasmobranchs are highly conserved.

     

Respiration in the eastern water dragon,Physignathus lesueurii (agamidae)

• 1.1. Some aspects of the gas exchange system of a diving lizard, Physignathus lesuewii were studied.
• 2.2. Breathing patterns were analysed.
• 3.3. Breathing rate increases logarithmically with temperature and Q₁₀ = 1.8. LogBR = −0.237 + 0.0256 T.
• 4.4. Gas tensions in lung air and arterial and venous blood were measured. Arterial pH declines with increasing temperature.
• 5.5. Temperature has a marked effect on oxygen affinity of the blood (ΔH = −10.1 kcal mol⁻). A Bohr effect was also noted.
• 6.6. CO₂ equilibrium curves were drawn.
• 7.7. The results are considered with a view to anticipating the efficiency of the gas exchange system of this species under conditions of variable temperature and during diving.

     

Purification and characterization of hatching enzyme of the pike (Esox lucius)

• 1.1. A choriolytic enzyme was isolated from the hatching medium of the pike, Esox lucius.
• 2.2. The enzyme is defined as hatching enzyme.
• 3.3. The molecular weight of the enzyme is 24,000.
• 4.4. The enzyme is a glycoprotein containing 2% carbohydrate.
• 5.5. Its isoelectric point is 6.5.
• 6.6. The pH optimum is around pH 8.
• 7.7. The enzyme molecule contains two disulfide bonds but no free cysteine.
• 8.8. Inhibitor studies and metal analysis show that the enzyme is a zinc-metalloprotease.

     

Ultrastructure of maturing fish (Oreochromis niloticus) and snake (Waglerophis merremii) erythroid cells with regard to hemoglobin biosynthesis

• 1.1. Fish and snake immature erythrocytes were submitted to a comparative ultrastructural study, analysing changes in organelles involved in hemoglobin (Hb) biosynthesis.
• 2.2. Iron uptake occurs probably via transferrin, and ferruginous compounds accumulate as siderosomes, taken as iron sources for heme biosynthesis, later on caught by a double lamella.
• 3.3. Mitochondrial membrane of the inner camera differentiates to lamellated bodies that, sucessively, give rise to expansions for ferruginous material and globin chains captation, constituting prehemosomal vesicles, which become condensed vesicles, followed by prohemosomes.
• 4.4. Through an internal membrane rearrangement, prohemosomes change to hemosomes wherein, hypothetically, heme and the globin chains assembly may occur.
• 5.5. In both fish and snake erythroid cells, all stages for hemosomegenesis are similar to the stages found in erythroid cells of other vertebrate species, including humans, except that fish cells often present single organelles of still unknown function, void of internal membrane.
• 6.6. Through electrophoresis of the respective supernatants obtained after osmotical lysis of the organellar fractions, it was shown that fish hemosomes contain three Hb patterns, while snake hemosomes present two patterns.

     

Erythrocyte supply from the spleen of exercised carp

• 1.1. Weight and hemoglobin content of the spleen of 3-year-old carp under the resting condition showed seasonal change, and those in male fish were larger than those in females at any season.
• 2.2. Weight of the spleen of 3-year-old fish of both sexes (examined in August and October) and immature fish (examined in September) showed a decrease of more than 50% during severe exercise for 60 min. Hemoglobin content per unit body weight of the same fish showed a decrease of more than 80% with the same exercise.
• 3.3. Hematocrit value and hemoglobin concentration of the blood elevated 41 and 25%, respectively, in the 3-year-old male fish, and 20 and 11%, respectively, in the 3-year-old females during the exercise for 60 min.
• 4.4. Fifty percent and 30% of the hematocrit elevations are considered to have been caused by erythrocyte supply from the spleen in the male and female fish, respectively, and 80% of the elevations in the fish of both sexes can be explained by erythrocyte supply, erythrocyte swelling and water shift out of the blood vessel.