Paola Vecchini

Dimeric and tetrameric hemoglobins from the mollusc Scapharca inaequivalvis: Structural and functional properties

The bivalve mollusc Scapharca inaequivalvis contains in the coelomic fluid erythrocytes with a dimeric (HbI) and a tetrameric (HbII) hemoglobin like the other members of the arcid family. The tetrameric protein is made up from two types of polypeptide chain, while the dimeric protein is made from a single type of chain which differs from the other two in terms of molecular weight and isoelectric point. The optical and circular dichroism spectra show that the heme environment in HbI and HbII resembles that of vertebrate hemoglobins, although distinctive features are present in the deoxygenated derivative. p]The dimeric HbI in the pH range 6 to 9 does not change its association state upon deoxygenation, while the tetrameric HbII polymerizes as indicated by the appearance of a fast peak in the sedimentation velocity patterns. The dependence of the areas and sedimentation coefficients of the fast and slow peaks on protein concentration is characteristic of a rapidly established association-dissociation equilibrium between tetramers and polymers higher than octamers. The pH, ionic strength and temperature dependence of polymer formation indicate that both hydrophobic and ionic interactions stabilize the polymers. The functional properties of HbI and HbII differ. HbI shows co-operative oxygen binding (h = 1·5) and a constant oxygen affinity (p12 = 7.8 mm Hg) over the pH range 5.5 to 9.5. HbII likewise shows co-operativity in oxygen binding (h = 2·0). Its oxygen affinity at neutral and alkaline pH values is slightly lower (p12 = 9.1 mm Hg) than that of the dimeric protein, but becomes higher at pH values below 6.5 due to the presence of an acid Bohr effect. At high protein concentrations, under conditions of extensive polymerization of the deoxygenated derivative, the oxygen affinity is lowered and co-operativity slightly increased. Both phenomena require that the oxygen affinity of the polymer be lower than that of the tetramer, consistent with the predictions of linkage theory.

Studies on erythrocruorin. I. Physicochemical properties of earthworm erythrocruorin.

Abstract Erythrocruorin has been purified by preparative ultracentrifugation from the blood of Lumbricus. The protein is homogeneous in the ultracentrifuge and in gel electrophoresis. The sedimentation coefficient is 59.6, and the molecular weight by light scattering is 3.45 × 106 daltons. The protein has 2.67% heme iron corresponding to a minimum molecular weight of ~23 × 103 daltons. The full protein molecule thus contains 144 hemes arranged in a subunit structure similar to that of other erythrocruorins and of Spirographis chlorocrun.

Studies on erythrocruorin: II. Dissociation of earthworm erythrocruorin

Earthworm erythrocruorin (Lumbricus terrestris) dissociates into subunits at alkaline pH values. The dissociation products have been correlated to definite molecular structures on the basis of electron microscope and minimum molecular weight data. The final state in dissociation is approached by complex kinetics as determined in rapid mixing and static light-scattering experiments. On the other hand, the final state reflects only in part an equilibrium situation, since the intact molecules at any given pH are not in equilibrium with the dissociated components. Between the dissociated subunits a reversible equilibrium is established which is sensitive to the presence of ligand. The results give clear evidence of molecular microheterogeneity in the system.

On the mechanism of action of glutamic-aspartic transaminase: intermediate steps in the reaction.

Abstract Substrate amounts of highly purified GAT react with glutamate, aspartate and, at a much slower rate, with alanine, but not with glycine, arginine, serine and isoleucine, to give the corresponding ketoacids. The absorption spectrum of the enzyme changes during the reaction and the coenzyme passes from the aldehydic (enzyme-PLP) to the aminic (enzymic-PMP) form. Enzyme-PMP was isolated from the reaction mixture and its spectrum was studied at different pH values. The coenzyme/protein ratio and the specific activity of enzyme-PMP were the same as those of enzyme-PLP. The aminic form of the enzyme reacts with ketoglutarate, oxalacetate and, at a slower rate, with pyruvate, to give the corresponding amino acids and the aldehydic form of the enzyme.

A reinvestigation on the quaternary structure of ascorbate oxidase from Cucurbita pepo medullosa

SummaryThe optical properties, copper content, catalytic activity and quaternary structure of many preparations of ascorbate oxidase purified with two different methods were examined.Fresh samples appeared identical and were characterized by optical ratios A280/A610 = 25 ± 1 and A330/A610 = 0.8 ± 0.05, by specific activity toward ascorbate of 3.48 ± 0.05 mol g−1 min−1 and by a copper content of 8 ± 0.3 mol/145 000 Mr.The enzyme is composed of two non-covalently linked subunits of slightly different molecular mass (75 000 and 72 000 respectively). These subunits cannot be further resolved by reduction of disulfide bonds. Proteolytic cleavage of the protein chains was observed during purification and storage in the absence of the protease inhibitor 6-amino caproic acid.Ascorbate oxidase exists as a monomer at neutral pH and undergoes reversible association into higher molecular weight species at slightly acid pH values. Association is not accompanied by spectroscopic or catalytic changes.

CALCIUM- AND PH-LINKED OLIGOMERIZATION OF SORCIN CAUSING TRANSLOCATION FROM CYTOSOL TO MEMBRANES

Sorcin, a cytosolic calcium‐binding protein containing a pair of EF‐hand motifs, undergoes a Ca2+‐dependent translocation to the cell membrane. The underlying conformational change is similar at pH 6.0 and 7.5 and consists in an increase in overall hydrophobicity that involves the aromatic residues and in particular the two tryptophan residues which become less exposed to solvent. The concomitant association from dimers to tetramers indicates that the tryptophan residues, which are located between the EF‐hand sites, become buried at the dimer–dimer interface. Ca2+‐bound sorcin displays a striking difference in solubility as a function of pH that has been ascribed to the formation of calcium‐stabilized aggregates.

Studies on erythrocruorin: VI. Ferric derivatives of earthworm erythrocruorin

Abstract Oxidation of the ferrous derivatives of earthworm erythrocruorin with potassium ferricyanide at pH 7 gives rise to met-erythrocruorin with the characteristic spectral properties of aquo-met-haem proteins. Met-erythrocruorin maintains the same overall conformation ( s 20,w , α - helical content ) as the native protein. However, its stability is limited to a very narrow pH range around neutrality; outside this range it is converted rapidly and irreversibly to another spectral species (hemichrome). The erythrocruorin-hemichrome undergoes a reversible pH-dependent transition ( pK = 9.2) which is accompanied by a decrease in α-helical structure. On reduction the met-form and the hemichrome yield the deoxy and oxy derivatives. Hemichrome formation is accompanied by a drastic change of the quaternary structure; thus the sedimentation coefficient drops from 60 S to ~4 S and the α-helical content decreases. By addition of ligands (CN − , N 3 − ) or by reduction to the ferrous form, the hemichrome reassociates into 10 S subunits.

Studies on iron uptake and micelle formation in ferritin and apoferritin

SummaryIron uptake and micelle formation in ferritin and apoferritin have been followed both spectrophotometrically and by means of sedimentation velocity experiments. Information was thus obtained on the molecular weight distribution of the reconstitution product.To achieve incorporation ‘native’ ferritin (whole ferritin as purified from horse spleen), ‘native’ apoferritin (apoferritin prepared by fractionation of ferritin preparations) and ‘reduced’ apoferritin (apoferritin prepared by reduction of ferritin by dithionite or ascorbic acid) have been incubated with ferrous salts in the presence of oxidizing agents under different experimental conditions.Although some iron is incorporated in ‘native’ ferritin, full saturation is not achieved and the molecular weight distribution of the incubated products remains heterogeneous.‘Native’ and ‘reduced’ apoferritin show a similar iron incorporation, but the reconstitution products markedly differ in terms of their iron distribution.Ferritin reconstituted from ‘native’ apoferritin has a broad molecular weight distribution, while that reconstituted from ‘reduced’ apoferritin is characterized by a narrow, homogeneous molecular weight distribution. However treatment of apoferritin with reducing or oxidizing agents prior to the incubation alters the characteristics of the iron distribution without changing the iron incorporation properties.These results point to a role of the protein moiety not only in iron oxidation, but also in micelle formation.

Site-directed mutagenesis in hemoglobin. Effect of some mutations at protein interfaces.

The role of selected amino acid residues in the monomer‐monomer contacts of Hb A has been studied by site‐directed mutagenesis of the α chain bearing substitutions in the subunit surfaces. Mutation α 38Thr → Trp induced a stabilization of tetrameric Hb‐CO with a decrease of the K d for the equilibrium α2β2 ⇌ 2αβ, but had no effect on ligand binding. Mutation α40Thr→Arg resulted in a complete loss of cooperativity in ligand binding. Mutation α103His→Val had no noticeable effect. We also studied the behaviour of isolated, mutated α chains with respect to self association: compared to wt α chains, mutant α38Thr→Trp showed stabilization of the dimeric state and (at high protein concentration) a detectable amount of tetramers. Mutant α103His→Val showed only a minor stabilization of the α2 dimer.

Purification of the human urinary glycoprotein with gastric antisecretory activity by ‘subunit exchange chromatography’

A glycoprotein with marked gastric antisecretory activity (human urinary gastric inhibitor) has been isolated in our laboratory from human urine [ 1,2]. This inhibitor is made of identical subunits whose molecular weight was evaluated to be -15 000 by gel-filtration and disc-gel electrophoresis in the presence of sodium dodecyl sulphate (SDS) and by C-terminal analysis [3]. Preliminary experiments have suggested that the molecular weight of the glycoprotein depends on pH due to a reversible associationdissociation process. It has been shown recently [4-61 that, in the case of an associating-dissociating protein system, the protein subunits immobilized on a solid matrix will interact with the subunits in solution. This interaction can be measured quantitatively and can be used to analyze the association-dissociation properties of the system under various conditions. It also provides a powerful purification tool because of its specificity. The process which has been called ‘subunit exchange chromatography’ has been already applied to various proteins [4-61; the results obtained with the gastric antisecretory glycoprotein are reported here.