Maria Sumiko Arita Matsuura

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.

ATP-induced Tetramerization and Cooperativity in Hemoglobin of Lower Vertebrates

The importance of intraerythrocytic organic phosphates in the allosteric control of oxygen binding to vertebrate hemoglobin (Hb) is well recognized and is correlated with conformational changes of the tetramer. ATP is a major allosteric effector of snake Hb, since the absence of this nucleotide abolishes the Hb cooperativity. This effect may be related to the molecular weight of about 32,000 for this Hb, which is compatible with the dimeric form. ATP induces a pH-dependent tetramerization of deoxyHb that leads to the recovery of cooperativity. This phenomenon may be partially explained by two amino acid replacements in the β chains (CD2 Glu-43 → Thr and G3 Glu-101 → Val), which result in the loss of two negative charges at the α1β2 interface and favors the dissociation into dimers. The ATP-dependent dimer ↔ tetramer may be physiologically important among ancient animal groups that have similar mutations and display variations in blood pH that are governed by these animals′ metabolic state. The enormous loss of free energy of association that accompanies Hb oxygenation, and which is also observed at a much lower intensity in higher vertebrate Hbs, must be taken into consideration in allosteric models. We propose that the transition from a myoglobin-like protein to an allosteric one may be of evolutionary significance.

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.

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.

Interactions of adenosine triphosphate with snake hemoglobins. Studies in Liophis miliaris, Boa constrictor and Bothrops alternatus

The hemoglobins of three snake species: Liophis miliaris, Bothrops alternatus and Boa constrictor present a single ATP binding site per tetramer. The ATP association constant values for the deoxyhemoglobins at pH 7.5 were about KD ≅ 106 M−1 (107 M−1 for B. contrictor), three to four orders of magnitude higher than the respective values for oxyhemoglobin of about KO ≅ 102 M−1. The deoxyhemoglobin constant values markedly decrease as a function of pH, becoming, at pH 8.5, about KD ≅ 103 M−1 whereas for the oxyhemoglobin the constants remain of about the same, KO ≅ 102 M−1, at the pH range studied. The high ATP binding affinity constants, compared to those of human hemoglobin A, were explained from a molecular structural standpoint, considering L. miliaris hemoglobin, whose complete primary sequence is known. Two distinct amino acid residue differences were found in the β-chain, one being Trp (NA3) (more hydrophobic) in the snake hemoglobin which substitutes the Leu (NA3) in human hemoglobin, and the second being Val 101 β (G3) instead of Glu 101 β (G3). The substitutions could provide an un-neutralized, positively charged, residue Lys-104β and, taking into account its high pK value, the pH dependence of ATP binding affinity for the snake hemoglobin would originate from pH-dependent ionization of phosphate groups of the allosteric effector. The physiological implications of the high ATP binding constant, as well as the possible protective role of the nucleotide binding against the effect of high environmental temperatures on the oxygen dissociation curves, are discussed.

Bothrops alternatus hemoglobin components: Oxygen binding properties and globin chain hydrophobic analysis

Bothrops alternatus oxyhemolysate showed two components by DE-52 cellulose ion- exchange chromatography and polyacrylamide gel electrophoresis: Hb I representing 70% of the bemolysate and Hb II (30%); both are dimeric in the stripped form (mol.wt 32,500 Da) and tetrameric in the presence of IHP (64,000 Da). 2. Hb I, Hb II and whole hemolysate showed functionally similar properties to those ofLiophis miliaris, i.e. for the stripped form over the pH interval 7.2-8.9: log Ps0 values decreasing from +0.1 to + -0.15 (thereby an alkaline Bohr effect); AH + = -0.38 and Hill coefficient values decreasing from n a = 1.5 to 1.0. In the presence of ATP, an abrupt decrease in 02 affinity occurs and the log Ps0 values change from 1.0 to 0.05; the Bohr effect increases to AH + = -0.7 whereas the n n values decrease from I>2 to values dose to unity. 3. For B. alternatus, at a physiological pH range (7.8-9.0) the hemoglobin Bohr effect becomes apparent only in the presence of ATP and this seems to be fundamental for the 02 uptake of the snake. 4. HPLC analysis of the globins shows eight different chains instead of four, as found in L. miliaris hemoglobin, which corroborates the presence of Hb I and Hb II components in B. alternatus, and also shows that the unique tetramer formed from different ct and fl chains is also consistent in this snake.

Multiplicity and immunological characterization of the haemoglobin components from Liophis miliaris and Helicops modestus

Abstract 1. 1. The haemoglobins from Helicops modestus and Liophis miliaris , water-snakes with different degrees of aquatic adaptation, present 3 and 5 components respectively, separated by isoelectrofocusing methods. 2. 2. The haemolysates of both snakes when assayed against antiserum from H. modestus haemoglobin, by double immunodiffusion test, showed complete identity. The haemolysates assayed against the antiserum obtained from L. miliaris presented “cross-reaction”. 3. 3. The L. miliaris and H. modestus haemoglobins presented differences in antigenic determinants, according to cross-reaction and absorption experiments.

Functional and dissociation properties and structural organization of the hemocyanin of Ampullaria canaliculata (gastropoda, mollusca)

Abstract 1. 1. The molecular weight of the native hemocyanin from Ampullaria canaliculata determined by gel filtration has a molecular mass of 7.5 × 10 6 Da. Dialysis against buffer free of Ca 2+ and Mg 2+ ions dissociated the hemocyanin into molecular species of 1.0 × 10 6 and 3.2 × 10 5 Da. 2. 2. The oxygen-binding curves of hemocyanin were sigmoidal with Hill coefficient of about 2. A positive Bohr effect was demonstrable at pH values ranging from 6.5 to 9.0. 3. 3. Differences between associated and dissociated hemocyanin as well as the effect of the divalent cations on the aggregation of hemocyanin molecule were illustrated by immunoelectrophoresis analysis. 4. 4. Negatively stained preparations of hemocyanin observed by TEM showed the cylindrical configuration of molluscan hemocyanins molecules. Its inner structure, enhanced by the negative contrasting, reveals a regular periodicity as a result of the alignment of serial subunits.