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Biomedical subjects

P Di Muro

Publications and source records attributed to P Di Muro.

14 recordsLinked to original sources
PubMed2005

Contribution of the copper ions in the dinuclear active site to the stability of Carcinus aestuarii hemocyanin.

We have investigated the effect of copper binding on the structural properties of hemocyanin (Hc). To this aim, we have studied the holo- and apo-form of the protein, both in the hexameric and in the monomeric state (CaeSS2 subunit), with experimental approaches that report on the protein aggregation and conformational stability. The results of gel-filtration chromatography and small angle X-ray scattering (SAXS) provide evidence that the hydrodynamic and gyration radius (R(g)) of Hc in the hexameric form only slightly increase upon copper removal, whereas a remarkable enhancement in the R(g) value is observed for the CaeSS2 monomer. CD measurements in the far- and near-UV region indicate that removal of copper only marginally affects the conformation of the hexameric Hc. Instead, copper depletion in the CaeSS2 strongly alters the tertiary structure of the monomer (near-UV CD), even though it is almost inconsequential on the secondary structure content (far-UV CD). These findings are fully consistent with the results of limited proteolysis experiments showing that the hexameric Hc is similarly resistant to proteolysis by trypsin both in the holo- and apo-form. Conversely, the apo-form of CaeSS2 monomer is much more susceptible to proteolytic attack by trypsin than the holo-form. Based on SAXS measurements, the concentration-dependent oligomerization process for apo-CaeSS2 has been analyzed on the basis of a thermodynamic model involving a concentration-dependent equilibrium between a monomer in a native-like and an hexameric aggregate of monomers.

Animals↗
PubMed2004

Structural subunit organization of molluscan hemocyanins.

The quaternary structure of Molluscan hemocyanins is not still defined, in particular the spatial distribution and the structural subunits. It is important to establish the number and the nature of interations between functional units. Here we present two non-proteolytic methods for the depolymerization of hemocyanins. The results suggest that the carbohydrate moieties apparently play a basic role in the organization of the structural subunits.

Animals↗
PubMed2004

Structural role of the copper ions in the dinuclear active site of Carcinus aestuarii hemocyanin.

In this work we show, by a combination of biochemical and biophysical approaches, that the copper ions bound in the binuclear active site of Carcinus aestuarii hemocyanin play a stabilizing role on the tertiary structure of the protein. Upon removal of copper, the monomeric hemocyanin, but not the hexameric oligomer, undergoes changes at the level of tertiary structure while the secondary structure is almost unaffected. By Small-Angle X-Ray Scattering, supported by gel chromatography measurements, it can be concluded that the apo-monomer, but not the holo form or the hexameric form, undergoes a slow time-dependent oligomerization process.

Animals↗
PubMed2004

Comparative analysis of structural properties of Portunid Crab Hemocyanin.

In order to explore the hemocyanin adaptative potential and evolutive dynamics, we have analyzed the structural properties of this oxygen-carrier protein, in some species of portunid Crabs, (Brachyura, Portunida). We have compared the intra- and interspecific subunits patterns, in native and denaturant conditions, to estimate the phenetic relationships and the different stabilities of the protein.

Animals↗
PubMed2004

Resistance to stress and Hc functional modulation in Liocarcinus sp.

This study is included in a project aimed to study the alterations on the structure of the Northern Adriatic Sea ecosystem produced by fishing activity. The indirect or secondary effects of fishery such as the changes of the structure and trophic relationships of the ecosystem are under investigation and we have particularly considered the effects on species such as Liocarcinus depurator that are captured and then rejected because devoid of commercial value. The objective of this study is the Liocarcinus sp. adaptative resistance to stress and the effects of biochemical parameters (allosteric effectors) on Hc functional modulation.

Adaptation, Physiological↗
PubMed2000

Interaction of lactoferrin with ceruloplasmin.

When added to human blood serum, the iron-binding protein lactoferrin (LF) purified from breast milk interacts with ceruloplasmin (CP), a copper-containing oxidase. Selective binding of LF to CP is evidenced by the results of polyacrylamide gel electrophoresis, immunodiffusion, gel filtration, and affinity chromatography. The molar stoichiometry of CP:LF in the complex is 1:2. Near-uv circular dichroism spectra of the complex showed that neither of the two proteins undergoes major structural perturbations when interacting with its counterpart. K(d) for the CP/LF complex was estimated from Scatchard plot as 1.8 x 10(-6) M. The CP/LF complex is found in various fluids of the human body. Upon injection into rat of human LF, the latter is soon revealed within the CP/LF complex of the blood plasma, from where the human protein is substantially cleared within 5 h.

Animals↗
PubMed2000

Low-resolution structure of the proteolytic fragments of the Rapana venosa hemocyanin in solution.

Rapana venosa hemocyanin (Hc) is a giant oxygen-binding protein consisting of different subunits assembled in a hollow cylinder. The polypeptide chain of each subunit is believed to be folded in several oxygen-binding functional units of molecular mass 50 kDa, each containing a binuclear copper active site. Limited proteolysis with alpha-chymotrypsin of native R. venosa hemocyanin allows the separation of three functional proteolytic fragments with molecular masses of approximately 150, 100, and 50 kDa. The functional fragments, purified by combining gel filtration chromatography and ion-exchange FPLC, were analyzed by means of small-angle X-ray scattering (SAXS). The gyration radius of the 50-kDa Rapana Hc fraction (2.4 nm) agrees well with that calculated on the basis of the dimensions determined by X-ray crystallography for one functional unit of Octopus Hc (2.1 nm). Independent shape determination of the 50- and 100-kDa proteolytic fragments yields consistent low-resolution models. Simultaneous fitting of the SAXS data from these fragments provides a higher-resolution model of the 100-kDa species made of two functional units tilted with respect to each other. The model of the 150-kDa proteolytic fragment consistent with the SAXS data displays a linear chain-like aggregation of the 50-kDa functional units. These observations provide valuable information for the reconstruction of the three-dimensional structure of the minimal functional subunit of gastropod hemocyanin in solution. Furthermore, the spatial relationships among the different functional units within the subunit will help in elucidation of the overall quaternary structure of the oligomeric native protein.

Animals↗
PubMed2000

Saccharose solid matrix embedded proteins: a new method for sample preparation for X-ray absorption spectroscopy.

In this study, solid samples of hemoglobin and hemocyanin have been prepared by embedding the proteins into a saccharose-based matrix. These materials have been developed specifically for specimens for X-ray absorption spectroscopy (XAS). The preservation of protein conformation and active site organization was tested, making comparisons between the solid and the corresponding liquid samples, using resonance Raman, infra red, fluorescence and XAS. The XAS spectra of irradiated solid and liquid samples were then compared, and the preservation of biological activity of the proteins during both preparation procedure and X-ray irradiation was assessed. In all cases, the measurements clearly demonstrate that protein solid samples are both structurally and functionally quite well preserved, much better than those in the liquid state. The saccharose matrix provides an excellent protection against X-ray damages, allowing for longer exposure to the X-ray beam. Moreover, the demonstrated long-term stability of samples permits their preparation and storage in optimal conditions, allowing for the repetition of data collection with the same sample in several experimental sessions. The very high protein concentration that can be reached results in a significantly better signal-to-noise ratio, particularly useful for high molecular weight proteins with a low metal-to-protein ratio. On the bases of the above-mentioned results, we propose the new method as a standard procedure for the preparation of biological samples to be used for XAS spectroscopy.

Animals↗
PubMed2000

Molecular heterogeneity of the hemocyanin isolated from the king crab Paralithodes camtschaticae.

Native Paralithodes camtschaticae hemocyanin is found as a mixture of dodecamers (24S; 80%) and hexamers (16S; 20%). Removal of Ca2+ ions by dialysis against EDTA-containing buffer solution at neutral pH induces complete dissociation of the 24S form into the 16S form. Under these conditions, a further increase in pH to 9.2 produces complete dissociation of the hexamers into monomers (5S). In both cases, the dissociation process is reversible. The dodecamer (24S) is composed of two different hexamers which can be discriminated only by ion-exchange chromatography in the presence of Ca2+ ions. At alkaline pH and in the presence of EDTA, two major monomeric fractions can be separated by ion-exchange chromatography: ParcI (60%) and ParcII (40%). The reassociation properties of the two fractions were studied separately to define their ability to form hexamers and dodecamers. The oxygen-binding properties of the different aggregation states were investigated. Native hemocyanin binds O2 co-operatively (nH = 3) and with low affinity (p50 approximately 103 Torr). The two monomeric fractions, ParcI and ParcII, are not co-operative and the affinity is twice that of the native protein (p50 approximately 65 and 52 Torr). Oxygen-binding measurements of native hemocyanin carried out at different pH values indicate a strong positive Bohr effect within the pH range 6.5-8.0 and an increase in oxygen affinity at pH below 6.5.

Animals↗
PubMed1998

Subunits composition and allosteric control in Carcinus aestuarii hemocyanin.

Carcinus aestuarii hemocyanin (Hc) exists in two aggregation forms at pH 7.5 and 20 mM Ca2+: 24S accounting for 90% of total hemocyanin and 16S accounting for 10%. Removal of metal cations by EDTA at neutral pH causes the complete dissociation of 24S hemocyanin into two different 16S. At pH 9.2, 24S hemocyanin dissociates into a pH stable 16S and a 5S component. The 5S component consists of three monomeric fractions named CaeSS1 (10%), CaeSS2 (50%) and CaeSS3 (40%); the latter fraction consisting of two isoforms. The fractions CaeSS1, CaeSS2 and CaeSS3 have been studied as far as their reassociation properties to form hexamers are concerned. We investigated the oxygen-binding properties of the native form (24S), the mixture of the two 16S forms, the pH-stable 16S alone and of purified subunit fractions to define the role of each species on the expression of the allosteric behaviour of the 24S aggregate. The analysis of O2-binding data reveals that 24S-Hc can be well described by the modified Monod Wyman and Changeaux-model (nested MWC-model), while the half-molecules (16S) bind oxygen according to the simple MWC-model. The two hexameric 16S within the dodecameric 24S hemocyanin can be regarded as nested allosteric units. They behave as being functionally coupled in the T-states (tT and rT). In the R-states (tR and rR) the two half-molecules seem to be functionally uncoupled since they have the same values of oxygen binding constants as deduced for isolated 16S hexamers.

Allosteric Regulation↗
PubMed1996

The o-diphenol oxidase activity of arthropod hemocyanin.

Arthropod hemocyanin (isolated from the crab Carcinus maenas and the lobster Homarus americanus) is usually referred to as an oxygen transport-storage protein. The protein, however, also catalyses with low efficiency the oxidation of o-diphenol to quinone, similarly to tyrosinase (monophenol, o-diphenol:oxygen oxidoreductase). The enzymatic parameters of hemocyanin are affected by the aggregation state of the protein; namely V(max) exhibited by a dissociated subunit is one order of magnitude greater than that of aggregated species. The reaction velocity is increased by the presence of perchlorate, an anion of the Hofmeister series. The results are also discussed on the basis of active site accessibility in comparison with tyrosinase.

Animals↗
PubMed1994

An oxygenation-sensitive dye binding to Carcinus maenas hemocyanin.

The interaction of 4',6-diamidino-2-phenylindole (DAPI) with Carcinus maenas hemocyanin has been investigated by steady state fluorescence, dynamic fluorescence and circular dichroism measurements. The dye binds to apohemocyanin (without copper) as well as to oxygenated hemocyanin and to deoxygenated hemocyanin with very similar affinities (kd approximately equal to 1 microM ) and number of binding sites (one per subunit). In contrast, the fluorescence quantum yield enhancement of DAPI bound to oxygenated hemocyanin is nearly 60% lower than that observed for deoxygenated and apo forms. The decrease of fluorescence of the dye bound to deoxygenated hemocyanin is a sigmoidal function of the oxygen partial pressure, specular to that observed by following the absorbance of the copper-oxygen charge transfer band at 340 nm. This result provides preliminary evidence that DAPI may be used as a functional probe to monitor the cooperative binding of oxygen to the protein. The higher fluorescence quantum yield of DAPI bound to either apohemocyanin or deoxygenated protein is characterized by a single fluorescence decay with lifetime of about 3 ns, while with the oxygenated protein two components of about 1 ns and 3.0 ns are observed. This result is interpreted assuming the existence of two rotamers of DAPI in solution (Szabo et al. Photochem. Photobiol. 44 (1986) 143-150) both able to interact with oxygenated hemocyanin but only one to deoxygenated and apo forms. We conclude that the different fluorescence behaviour of the dye induced by the presence of oxygen bound to the protein is probably due to a structural change of hemocyanin in cooperative interaction with oxygen. Furthermore, the interaction is confirmed by the induced negative ellipticity of DAPI bound to apohemocyanin and deoxy-hemocyanin and by the increase of fluorescence anisotropy of DAPI bound to all forms of protein investigated.

Journal Article↗
PubMed1993

Structural properties of Rapana thomasiana grosse hemocyanin: isolation, characterization and N-terminal amino acid sequence of two different dissociation products.

1. The native Rapana thomasiana grosse hemocyanin is dissociated under mild conditions and fractionated into two dissociation products, RHSS1 and RHSS2, with an apparent molecular mass of approximately 250 and approximately 450 kDa, respectively. The two species are present in approximately equivalent amounts. SDS-PAGE analysis reveals that the latter component is a dimer of approximately 250 kDa polypeptide chains. 2. The amino acid compositions, as well as some spectroscopic properties of RHSS1, are very similar to those of RHSS2. After dissociation under mild conditions of the native hemocyanin both species preserve their capability of binding reversibly molecular oxygen. 3. RHSS1 and RHSS2 are sequenced directly from the amino-terminus for 15 and 20 steps, respectively. These parts of the two polypeptide chains are highly homologous but with microheterogeneity associated with some positions. They also exhibit high homology with the N-terminal region of subunits or functional domains of other gastropod Hcs.

Amino Acid Sequence↗