Aldo Focesi

Dimer-tetramer transition in hemoglobins from Liophis miliaris--I. Effect of organic polyphosphates.

Hemoglobin from the water-snake Liophis miliaris in the stripped form presents high oxygen affinity of about P50 = 1 mmHg and Hill coefficient of about 1.0 at pH from 6.8 to 8.5. In the presence of ATP such values become P50 = 20 mmHg and nH about 2.0, respectively, at low pH from 6.5 to 7.5. When the pH increases an abrupt decrease of both P50 and nH values occurs falling close to those found for the stripped hemoglobin. Gel-filtration in Sephadex G-100 equilibrated with 0.05 M Tris-HCl buffer containing 1 mM EDTA of the stripped hemoglobin show the presence of only one component of mol. wt of about 32,500 dt similar to the dimer of human hemoglobin A. The deoxy form of the dimer previously treated with ATP and placed on Sephadex column in the same condition but containing 1 mM IHP emerges as tetramer with mol. wt similar to that found for human hemoglobin, i.e. of about 65,000 dt. Results of the multiplicity of the snake hemoglobin, as well as the large alkaline Bohr effect in the presence of ATP previously reported, seems to be inconsistent due to the dimer-tetramer transition that occurs when ATP is bound to the stripped hemoglobin. A molecular mechanism involving the dimer-tetramer transition is proposed to described the oxygen transport in these animals.

Separation and characterization of the hemoglobin components ofPterygoplichthys pardalis, the acaribodo

Abstract 1. The four main hemoglobin components of the hemolysate ofPterygoplichthys pardalis have been isolated and characterized. 2. The functional properties investigated for the isolated components comprise the effect of pH and ATP on (i) the O2 equilibrium, (ii) the O2 dissociation kinetics, (iii) the CO combination kinetics. 3. Component I, corresponding to approx 50% of the total hemoglobin, is characterized by functional properties which are distinctly different from those of Components II, III and IV, which are alike 4. Thus it is shown, once more, that multiple components in an hemolysate fall into categories of hemoglobins characterized by distinct and complementary functional properties

Oxygen dissociation constants in haemoglobins of Helicops modestus and Liophis miliaris, two water-snakes with different morphological adaptations to their aquatic environment☆

1. 1.Both Helicops modestus and Liophis miliaris. Brasilian water snakes with different degrees of aquatic adaptations, present 3 haemoglobin components identified by starch-gel electrophoresis and separated by DEAE-Sephadex A-50 and CM-cellulose chromatography respectively. 2. 2.Some functional properties of their haemoglobins were determined. Both the oxygen affinity 1.0mmHg at pH 7) and the alcaline Bohr effect value (−0.07) of the stripped haemoglobins of the more aquatic species H. modestus were higher than those of L. miliaris (1.4mmHg and −0.30 respectively). 3. 3.The ATP effect on both haemoglobin-oxygen affinity and Bohr effect was more pronounced in L. miliaris haemoglobins. The values found were 17.8mmHg at pH 7 and −0.9; in H. modestus. they were 8.93 mmHg and −0.55. 4. 4.Nucleotide triphosphate concentrations were determined as 2.84 mmHg in H. modrstus and 2.65 mM in L. miliaris (whole blood); haemoglobin concentrations were 6.14mM and 4.21 mM (as haem), respectively. The physiological implications of these results are discussed.

Content of organic polyphosphates and their allosteric effects on haemoglobins from the water-snakes Helicops modestus and Liophis miliaris

Abstract 1. 1. The ion-exchange Chromatographie analysis of the red cell nucleotides of two water-snakes with different degrees of adaptation to water showed a ratio ATP/GTP of about 2.3 in the more aquatic species Helicops modestus and of about 0.7 in Liophis miliaris. 2. 2. The haemoglobin oxygen affinity assayed in the presence of ATP, GTP, DPG and IHP in haemolysate from both snakes were determined as a function of pH. The Bohr effect values were always found to be increased in comparison with the stripped haemolysate, becoming about 1.5 times higher in L. miliaris compared with H. modestus by the nucleotide treatment. 3. 3. The Hill coefficient calculated from the slope of the haemoglobin binding curves was consistent with an increase in the protein cooperativity in the presence of the nucleotides assayed. The very low nH values of about 1.2 in absence of the effectors became close to 2.0 in their presence. 4. 4. The data of nucleotide Chromatographie analysis were compared to those found in fishes with bimodal respiration.

Dimer-tetramer transition in hemoglobins from Liophis miliaris. II, Evidence with the stripped proteins

Abstract 1. 1. Whole Liophis miliaris hemolysate stored under refrigeration at pH 7 becomes progressively deoxygenated and presents two hemoglobin forms of mol. wts 32,500 and 64,000, consistent with oxydimers and deoxytetramers respectively. 2. 2. The Hill plot determined at pH 6.5 considering oxygen pressure capable to saturate up to 50% of the hemoglobin shows a coefficient value of nH = 2.06, whereas that found at higher oxygen pressure was nH = 0.85. 3. 3. The apparent Bohr effect of about ΔH+ = −0.4 in the stripped hemoglobin was explained by the increase of co-operative deoxytetramer T-state/non-co-operative ratio as a function of proton concentration.

Effect of pH on the kinetics of oxygen and carbon monoxide reactions with hemoglobin from the air-breathing fish,Loricariichthys

Abstract 1. Loricariichthys sp., an air-breathing fish from the Amazon River has one major hemoglobin component. 2. Quantitative studies on the kinetics of O 2 dissociation and CO combination to the protein were performed by stopped-flow experiments at different pH values and a constant ATP concentration of 1.25 mM. 3. The oxygen dissociation shows a simple first order behavior and a strong pH dependence. 4. The CO combination kinetics, on the other hand, were homogeneous and fast at higher pH values and slow and clearly autocatalytic at pH values below 7.0.

Functional properties of Aplysia brasiliana myoglobin

Abstract 1. 1. The radular myoglobin of the Brazilian sea hare, Aplysia brasiliana, has been isolated and its ligand binding properties have been characterized. The myoglobin is similar to the myoglobin of Aplysia limacina (a Mediterranean species), and has a lower oxygen affinity than does myoglobin of the sperm whale, Physeter macrocephalus. 2. 2. Aplysia brasiliana myoglobin is a monomer and binds oxygen and other heme ligands non-cooperatively. There is no evidence of pH sensitivity in these processes. 3. 3. The kinetics of ligand binding and dissociation are simple processes. The lower oxygen affinity of Aplysia brasiliana myoglobin relative to sperm whale myoglobin is associated with a much higher rate of oxygen dissociation, with first order rate constants of 125 sec−1 and 10 sec−1 respectively.

FUNCTIONAL PROPERTIES OF THE HEMOGLOBIN FROM THE SOUTH AMERICAN SNAKE MASTIGODRYAS BIFOSSATUS

The hemolysate of Mastigodryas bifossatus shows two major hemoglobins with very close isoelectric points, and four different globin chains. The stripped hemolysate exhibits a low alkaline Bohr effect (delta log P50/delta pH = -0.30 between pH 7 and 8) and a decrease of the co-operativity from 2.3 to unity when the pH increases from 6.15 to 8.5. In the presence of ATP, large changes in the oxygen affinity and co-operativity are observed. The Bohr effect rises to -0.46 and the n50 values stay at around 3 in the pH range 6-9. An increase in temperature induces a large decrease in the oxygen affinity for the stripped hemolysate. In the pH range between 7.5 and 8.5, the values of delta H in kcal/M are around 10 fold larger for the stripped protein than for the protein in the presence of ATP. Measurements of rapid kinetics of oxygen dissociation and carbon monoxide binding reflect the ATP sensitivity observed in equilibrium experiments.

Dimer-tetramer transition in hemoglobin from Liophis miliaris. III: The phenomenon in snake species of different evolutionary levels

1. 1. The primitive snake Boa constrictor presents in the stripped hemolysate one component which from O2, equilibrium curves produced the following values: log P50 ± −0.1 and Hill coefficient nH = 1.1, independent of pH. 2. 2. Bothrops alternatus, an evolved snake, presents two components in the hemolysate with functional parameters: log P50 = 0.4 and nH = 1.7 at pH 7.1, decreasing to log P50 = −0.1 and nH ± 1.4 at pH 8.2. 3. 3. In the presence of ATP, an increase to log P50 ± 0.9 and nH ± 2 is found in both snake hemolysates, up to pH 7.8 4. 4. Molecular weight determinations by gel filtration at pH 8 show only the dimeric form (mol. wt 32,000) in B. constrictor hemolysate, and dimers and tetramers (mol. wt 64,500) in that of B. alternatus. 5. 5. The Hill plots as a function of O2 tension show for B. alternatus hemolysate a shift from deoxytetramer nH = 2.02 to oxydimer nH = 0.88 as occurs in L. miliaris. For B. constrictor hemoglobin the nH remains 1.0 independent of the O(in2) tension.

Some larval properties of Pipa carvalhoi adult hemoglobins

1. 1. Pipa carvalhoi. an anuran from northeast Brazil that lives in an aquatic environment even as an adult, has four hemoglobin components which have been identified by starch-gel electrophoresis and separated by CM-cellulose chromatography. 2. 2. Some structural and functional properties of its hemoglobins were determined and compared with those of terrestrial anurans. The oxygen affinity, Bohr effect and rate of methemoglobin formation in the presence of alkali, urea and sodium benzoate have features similar to those of tadpole hemoglobins already described. 3. 3. The number of reactive thiol groups are 1.5–2 per tetramer of hemoglobin, in contrast to the hemoglobins of tadpoles, which lack thiol groups.