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 beta chains (CD2 Glu-43 --> Thr and G3 Glu-101 --> Val), which result in the loss of two negative charges at the alpha1beta2 interface and favors the dissociation into dimers. The ATP-dependent dimer left arrow over right arrow 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.

Tobacco mosaic virus disassembly by high hydrostatic pressure in combination with urea and low temperature.

We investigated the effect of low temperature and urea combined with high pressure on tobacco mosaic virus (TMV). The evaluation of its aggregation state and denaturation process was studied using gel filtration, transmission electron microscopy, and spectroscopic methods. The incubation at 2.5 kbar induced 18% dissociation, and decreasing of temperature to -19 degreesC promoted additional dissociation to 72%, with stabilization of the dissociation products. Under such conditions, extensive denaturation did not occur. The apparent enthalpy and entropy of dissociation (Delta and TDelta) were -9.04 kcal/mol subunit and -15.1 kcal/mol subunit, respectively, indicating that the TMV association is an entropicly driven process. The apparent free energy of stabilization given by the presence of RNA is at least -1.7 kcal/mol subunit. Urea-induced dissociation of TMV samples and incubation at high-pressure promoted a higher degree of dissociation. The volume change of dissociation decreased in magnitude from -16.3 to -3.1 mL/mol of dissociated subunit, respectively, in the absence and presence of 2.5 M urea, suggesting exposure of the protein-protein interface to the solvent. High-pressure induced remarkable TMV denaturation in the presence of 2.5 M urea, with a volume change of -101 mL/mol of denatured subunit. The apparent enthalpy and entropy of denaturation (Delta and TDelta) by 1.75 M urea at 2.5 kbar was -11.1 and -10.2 kcal/mol subunit, respectively, demonstrating that the TMV protein coat presents an apparent free energy of denaturation by urea close to zero. Although the processes could not be assumed to be pure equilibria, these thermodynamic parameters could be derived by assuming a steady-state condition.

Further evidence of dimer-tetramer transition in hemoglobin from Liophis miliaris.

Liophis miliaris hemoglobins in the stripped form exhibit a high oxygen affinity, a small alkaline Bohr effect, a pronounced organic polyphosphate effect and a pH-dependent Hill coefficient, close to 2 below pH 7.5 and near 1 at higher pHs. Molecular weight determinations indicate 2 forms, a dimeric form of MW 32000 d. and a tetrameric form of about 64000 d. The deoxyhemoglobin is tetrameric. Ion-exchange chromatography shows two components which may bind to each other being eluted as a single component of MW 32000 d. The oxygen alkaline Bohr effect observed for the stripped hemoglobin may be explained by the transition from the tetramer to the dimer during oxygen addition experiments. The phenomenon appears to be unique among animals. beta-Chain sequencing determinations show that abnormal human hemoglobin with similar properties has a glutamic acid residue substituted at the alpha 1-beta 2 interface by valine in snake hemoglobin.

Further evidence of dimer-tetramer transition in hemoglobin from Liophis miliaris

Liophis miliaris hemoglobins in the stripped form exhibit a high oxigen affinity, a small alkaline Bohr effect, a pronounce organic polyphosphate effect and a pH-dependent Hill coefficient, close to 2 below pH 7.5 and near 1 at higher pHs. Molecular weight determinations indicate 2 forms, a dimeric form of MW 32000 d. and a tetrameric form of about 64000 d. The deoxyhemoglobin is tetrameric. Ion-exchange chromatography shows two components which may bind to each other being eluted as a single component of MW 32000 d. The osygen alkaline Bohr effect observed for the stripped hemoglobin may be explained by the transition from the tetramer to the dimer during oxygen addition experiments. The phenomenon appears to be unique among animals. ß-Chain sequencing determinations show that abnormal human hemoglobin with similar properties has a glutamic acid residue substituted at the alfa1-ß2 interface by valine in snake hemoglobin