Maurizio Gattoni

The unusual co-assembly of H- and M-chains in the ferritin molecule from the Antarctic teleosts Trematomus bernacchii and Trematomus newnesi

Ferritins from the liver and spleen of the cold-adapted Antarctic teleosts Trematomus bernacchii and Trematomus newnesi have been isolated and characterized. Interestingly, only H- and M-chains are expressed and no L-chains. The H-chains contain the conserved ferroxidase center residues while M-chains harbor both the ferroxidase center and the micelle nucleation site ligands. Ferritins have an organ-specific subunit composition, they are: M homopolymers in spleen and H/M heteropolymers in liver. The M-chain homopolymer mineralizes iron at higher rate with respect to the H/M heteropolymer, which however is endowed with a lower activation energy for the iron incorporation process, indicative of a higher local flexibility. These findings and available literature data on ferritin expression in fish point to the role of tissue-specific expression of different chains in modulating the iron oxidation/mineralization process.

The dimer-tetramer equilibrium of recombinant hemoglobins. Stabilization of the α1β2 interface by the mutation β(Cys112 → Gly) at the α1β1 interface

Abstract The dimer-tetramer association constants of several recombinant human hemoglobins (in the CO form) have been measured by differential gel filtration. Recombinant human hemoglobin prepared from recombinant β-chains, and mutant hemoglobins where the substitution was on the surface, β(Thr4 → Asp), in the heme pocket, β(Val67 → Thr), at the 2,3-DPG binding site, β(Val1 → Met + His2del), had a twofold smaller association with respect to natural hemoglobin. In a mutant at the α 1 β 2 1 interface, β(Cys93 → Ala), the association constant was decreased three-fold. Conversely, in a mutant at the α 1 β 1 interface, β(Cys112 → Gly), the association constant was two- and four-fold increased with respect to natural and recombinant human hemoglobin. These differences are energetically very small, consistent with the correct folding of the recombinant hemoglobins. The stabilization of the tetrameric structure by a mutation at the α 1 β 1 interface indicates that structural changes at this interface can be propagated through the protein to the α 1 β 2 interface and, thereby, exert an effect on the allosteric equilibrium.

Immobilized hemoglobin in the purification of hemoglobin-based oxygen carriers

Chemically modified hemoglobins can be used as oxygen carriers in cell-free fluids provided that they have a low oxygen affinity and are stable towards dissociation into subunits. The latter species are undesirable because they are filtered rapidly through the kidneys, have renal toxicity and are characterized by a high oxygen affinity. A most important step in the preparation of hemoglobin-based oxygen carriers is therefore their purification from any dissociable material. Hemoglobin immobilized as alpha beta dimers on Sepharose lends itself naturally to this purpose as it is able to interact in a specific and reversible way with soluble alpha beta dimers. Hemoglobin affinity columns are very effective in the purification of cross-linked and pseudo-cross-linked human and bovine hemoglobin. The applicability of the technique is enhanced by the ease with which alpha beta dimers from different species cross-interact to yield hybrid alpha 2 beta 2 tetramers. It is shown that hemoglobin affinity columns may provide analytical information on the cross-linking reaction itself.

Immobilized apo-myoglobin, a new stable reagent for measuring rates of heme dissociation from hemoglobin

Apo‐myoglobin covalently linked on CNBr‐activated Sepharose 4B is proposed as a new heme acceptor for investigating the heme transfer reaction from hemoproteins. Immobilized apo‐myoglobin has the desirable properties of an ideal heme acceptor in that it is characterized by a high affinity for ferric heme, a high stability towards denaturation even at physiological temperatures and can be lyophilized for long‐term storage. The study of heme release from myoglobin at pH 5.0 and 37°C indicates that heme affinity is increased at least 10‐fold relative to the soluble protein. Experiments with human hemoglobin allowed the estimation of the heme release rates from both α and β chains and brought out the greater temperature sensitivity of the α chain heme‐globin linkage.

[43] Use of immobilized subunits for the purification of oligomeric and self-associating proteins

Publisher Summary Oligomeric proteins can be purified in an effective and simple way by exploiting two properties of subunit interactions––namely, the high specificity of subunit recognition and the nature of the forces stabilizing the oligomeric structure. The purification of an oligomeric protein from a tissue homogenate, for example, can be effected in principle by a two-step procedure: in the first step, soluble subunits are extracted from the medium and are bound to those immobilized under conditions that favor subunit association; subsequently, the protein is eluted under conditions that promote its dissociation into subunits. In this latter step, the immobilized subunits are regenerated and become ready for a new purification cycle after equilibration with the associating buffer. The same approach can be used also for the isolation of homologous proteins capable of forming hybrids with the immobilized subunits and for the purification of self-associating systems.

Subunit interactions in the dimeric and tetrameric hemoglobins from the mollusc Scapharca inaequivalvis

The dissociation behaviour of oxygenated Scapharea inaequivalvis HbII, the tetrameric hemoglobin component contained in the erythrocytes of this bivalve mollusc, has been compared with that of oxygenated human hemoglobin, HbA. At neutral pH the molluscan protein dissociates reversibly into dimers as does HbA, although dissociation is less marked; moreover the dimer-tetramer association constant is not sensitive to the presence of inorganic phosphates and high salt concentrations. HbII dimers hybridize with HbA dimers in solution, pointing to an overall similarity of the dimer interfaces in these hemoglobins from distantly related species. The gel filtration behaviour of the dimeric hemoglobin component of the erythrocytes. HbI, indicates that at neutral pH the protein has very little tendency to dissociate into monomers even at micromolar concentrations. Hb I was found to contain small amounts (2-4%) of bound carbohydrates.

On the identity of the dissociation-linked chloride binding sites in human hemoglobin. Studies with hemoglobin modified with 4-isothiocyanatobenzenesulfonic acid and 4-isothiocyanatobenzenesulfonamide

The binding of chloride ions to specific sites on the human hemoglobin molecule has well-known effects on the oxygen equilibrium and on the stability of the tetrameric structure. Several lines of evidence suggest that the oxygen-linked and the dissociation-linked chloride binding sites differ. Direct evidence for this difference has been obtained from the chloride dependence of the dimer-tetramer equilibrium of oxyhemoglobin modified with 4-isothiocyanatobenzenesulfonic acid, in which all the oxygen-linked chloride binding sites are blocked, or with 4-isothiocyanatobenzenesulfonamide, in which the linkage between chloride and oxygen is unperturbed. Thus, the chloride dependence of the dimer-tetramer assembly is unaffected by the chemical modification in both proteins and resembles that of unreacted hemoglobin. It is suggested that histidines alpha-103, alpha-122 and beta-97 may constitute, at least in part, the dissociation-linked chloride binding sites.

The effect of isoflurane on erythrocyte membranes studied by ATR-FTIR

The effect of isoflurane on erythrocyte membranes has been investigated by means of attenuated total reflection infrared spectroscopy. Infrared spectra were measured on sonicated erythrocyte ghosts layered upon a ZnSe crystal covered with D(2)O saline solutions containing increasing amounts of isoflurane. At clinically relevant anesthetic concentrations and 37 degrees C, significant changes in the structural and dynamic properties of the membrane phospholipid bilayers are observed. Both the acyl chain methylene symmetric and asymmetric stretching modes and the carbonyl ester stretching band displayed frequency shifts interpreted as transitions toward disordered liquid-like structure accompanied by dehydration of the phospholipid polar heads. In turn, no secondary structure-linked changes are observed in the amide I region of membrane proteins. Higher anesthetic concentrations (500-900 microM), resulted in progressive detachment of the multilayers from the ATR crystal and irreversible formation of denatured protein. Polarization studies in correspondence of the acyl lipid methylene stretching bands indicated that isoflurane decreases the dichroic ratio thus inducing disorder in the orientation of the lipid acyl chains.

Subunit exchange chromatography: A tool for the purification of oligomeric and self-associating protein systems

Abstract Subunit exchange chromatography exploits the observation that subunits immobilized on a solid matrix retain the capacity to recognize the subunits in solution in a highly specific way. Hence, oligomeric and self-associating proteins can be extracted in a simple and efficient way from a multicomponent mixture under conditions that favour subunit association; subsequently, the protein is eluted under conditions that favour its dissociation into subunits. The method lends itself also to the isolation of homologous proteins capable of forming hybrids with the immobilized subunits.

Immobilized human hemoglobin, a versatile matrix for analytical and biotechnological applications

The analytical and biotechnological applications of human hemoglobin immobilized covalently on CNBr-Sepharose 4B are reviewed. Hemoglobin is bound to the matrix as alphabeta dimers via either chain. The immobilized alphabeta dimers maintain the capacity to interact reversibly with soluble ones under conditions where the soluble protein is in self-association equilibrium. Under these conditions, therefore, immobilized dimers bind part of the soluble protein. In turn, the binding process can be used to assess the specific features of the equilibrium on solid-phase and to extract selectively hemoglobin from a variety of biological specimens of practical interest. A different application of immobilized alphabeta dimers concerns their use in the determination of the equilibrium and kinetic stability of the heme-globin linkage, a property that is directly correlated with the stability of the hemoglobin molecule. The advantages and limitations attendant the use of the immobilized protein relative to the soluble one are discussed.