Rapana thomasiana hemocyanin (RtH): dissociation and reassociation behavior of two isoforms, RtH1 and RtH2.

Rapana thomasiana hemocyanin (RtH) is a mixture of two hemocyanin isoforms, termed RtH1 and RtH2. The two subunit types, purified by ion exchange chromatography, were used for macromolecular reassociation studies. In vitro reassociation was achieved with Tris-saline stabilizing buffer at pH 7.4, containing 100mM calcium and magnesium chloride at 4 degrees C. The relatively slow progress of reassociation was monitored, and the different oligomeric forms of RtH1 and RtH2 were studied by transmission electron microscopy, using samples negatively stained with 1% (w/v) uranyl acetate or 5% (w/v) ammonium molybdate containing 1% (w/v) trehalose at pH 7.0. The two subunits reassociate to produce characteristic didecamers, oligomeric and polymeric forms depending on the dissociated material and the reassociation conditions (i.e. divalent ion concentration, duration). In contrast to the didecamers of the freshly isolated RtH preparations, RtH1 and RtH2 show after 2 weeks' reassociation a clear tendency to generate multidecameric structures. The behavior of the native RtH1 and RtH2 during reassociation in the presence of 100mM calcium and magnesium chloride corresponds to the reported common oligomerization characteristics of KLH1/HtH1 and KLH2/HtH2, respectively. It is important to note that during the reassociation of the RtH isoforms: (I) no smaller diameter tubular polymers (ca. 25-27nm) were formed from the subunits as well as from the decamers; (II) multidecamers with one or more 'nucleating' didecamers were detected in addition to the multidecamers, composed of didecamers with associated decamers at one or both ends.

Preliminary X-ray diffraction studies of the external functional unit RtH2-e from the Rapana thomasiana.

The 'external' oxygenated functional unit RtH2-e of the Rapana hemocyanin subunit RHSS2 was isolated and crystallized. X-ray intensity data to 3.3 A resolution have been collected at 100 K and the structure has been solved using the molecular-replacement method. The space group is assigned to be the tetragonal P4(3)2(1)2, with unit-cell parameters a = b = 105.5, c = 375.0 A.

Penaeus monodon (tiger shrimp) hemocyanin: subunit composition and thermostability.

Penaeus monodon (class Crustacea, order Decapoda) is one of the largest shrimps of the Penaeidea family from the Indo-West Pacific region. The dioxygen-transporting protein hemocyanin, isolated from the hemolymph of this invertebrate, is composed of three 75-76 kDa structural/functional subunits designated as Pm1, Pm2 and Pm3. The N-terminal sequences of the chains were determined and compared with those of other decapodan hemocyanin subunits. Pm2 and Pm3 are highly homologous and electrophoretically undistinguishable polypeptides. In comparison to Pml, they have an extension of six residues. Pm1 is closely related to the subunit Pv2 of the Penaeus vannamei hemocyanin. Probably, subunits like Pm1 and Pv2 are family-specific for the Penaeidea hemocyanins and the other subunits are species-specific. Comparison of N-terminal sequences of respiratory proteins from the sub-orders Natantia and Reptantia demonstrated family- and sub-order-specific sequences. A melting point of 69 degrees C, lower than those for the di-hexameric decapodan hemocyanins, was determined from the temperature dependence of ellipticity of the mono-hexameric Penaeus monodon hemocyanin. Thermostability of decapodan hemocyanins depends on their aggregation state.

Arrangement of functional units within the Rapana thomasiana hemocyanin subunit RtH2.

For the determination of the number and linear sequential arrangement of functional units (FUs) within the polypeptide chain of the Rapana hemocyanin subunit RtH2, a panel of mono-, di-, tri- and penta-FU fragments was generated by limited proteolysis of the purified subunit with four different enzymes. The individual cleavage products were isolated, characterized by SDS-PAGE and N-terminally sequenced. Most of the information about the FU sequential arrangement within RtH2 was obtained after limited proteolysis of the subunit with plasmin. Overall correlation of the data revealed the sequential order of the eight FUs within the polypeptide chain of RtH2, termed RtH2-a to RtH2-h. The sites, most sensitive to proteolytic cleavage with plasmin, are located at the C-terminus, between the FUs ef, fg and gh. A second main cleavage site was observed between the FUs cd. Endoproteinase GluC hydrolyzes these sites, too, but produces exclusively a mixture of mono-, di- and tri-FU fragments. The most stable fragments, the trimer abc and the dimer gh, are found in all cleavage mixtures of RtH2 studied. RtH2-h is compared with the corresponding h-FUs of the gastropodan hemocyanins of Pila leopoldvillensis, Helix pomatia, Megathura crenulata and Haliotis tuberculata, and a remarkable similarity is observed between them: an increased M(r) of approximately 65000 instead of approximately 50000, estimated for an average FU, suggesting that the sequence of RtH2-h is elongated by about 95 amino acid residues at the C-terminal part of the molecule, as found for beta(c)-HpH, HtH1 and HtH2.

Fluorescence properties and stability of dioxygen--binding functional units from the Rapana thomasiana hemocyanin subunit RHSS2.

Molecular aggregates of the Rapana thomasiana hemocyanin are composed of two structural subunits, RHSS1 and RHSS2, each of which contains eight functional units (FUs) reversibly binding dioxygen. Multiunit fragments and individual 50-60 kDa FUs from RHSS2 were isolated and characterized by electron and fluorescence spectroscopy. The units have similar fluorescence parameters demonstrating that the tryptophyl side chains are located in the hydrophobic core of the globular folded regions. The copper-dioxygen system at the binuclear active site stabilizes considerably the native protein structure and quenches the indole emission. The removal of this system decreased the 'melting points' drastically Tm by 13-20 degrees C and increased 2-4 times the fluorescence quantum yields. The individual FUs differ considerably in their thermostability. The activation energy for the thermal deactivation of the excited tryptophyl residues of the apo-FUs is lower compared to that of the whole molluscan apo-Hcs.

Complete amino acid sequence of dioxygen-binding functional unit of the Rapana thomasiana hemocyanin.

The complete amino acid sequence of the Rapana thomasiana hemocyanin N-terminal functional unit Rta was determined by direct sequencing and matrix-assisted laser desorption ionization mass spectrometry of the protein and peptides obtained by cleavage with EndoLysC proteinase, TPCK-trypsin and cyanogen bromide. The single polypeptide chain consists of 407 residues. This is the first report on the primary structure of a dioxygen-binding unit from a marine gastropod hemocyanin and of an N-terminal domain from a molluscan dioxygen carrier. Comparison with the sequences of other molluscan hemocyanin functional units shows an average identity of 48 +/- 5 %. Inspection of the Rta sequence revealed residues 27 and 250 as carbohydrate attachment sites. Conclusions about the molecular evolution of the molluscan hemocyanin dioxygen-binding functional units are made.

Amino-terminal oxygen-binding functional unit of the Rapana thomasiana grosse (gastropod) hemocyanin: carbohydrate content, monosaccharide composition and amino acid sequence studies.

The amino-terminal oxygen-binding unit Rta of the Rapana thomasiana hemocyanin is a glycoprotein with a carbohydrate content of 4.8% (w/w). Sugar analysis revealed as monosaccharide constituents xylose, fucose, 3-O-methylgalactose, mannose, galactose, N-acetylgalactosamine and N-acetylglucosamine residues. On subtracting the carbohydrate contribution from the molecular mass of 49,698 Da, determined by laser desorption mass spectrometry for Rta, an M(r) value of 47,318 Da was determined for the polypeptide part of the functional unit. The Rapana hemocyanin oxygen-binding unit Rta contains 400 residues in a single polypeptide chain. The nearly complete amino acid sequence (about 90%) is determined. This is the first report on a sequence of a marine gastropod oxygen-binding unit and also on a molluscan hemocyanin amino-terminal unit. Comparison of the Rta sequence with those of other molluscan hemocyanin units, localized in the C-terminus or in the middle of the respective multidomain polypeptide chains, revealed 42-46% homology (52-55%, including isofunctional residues). Probably, all molluscan oxygen-binding units evolved from a common ancestral gene.

Functional unit of the Rapana thomasiana (Grosse) (marine snail, gastropod) hemocyanin.

The amino terminal functional unit (domain a) of the Rapana hemocyanin "heavy" structural subunit, designated as Rta, was obtained after limited trypsinolysis of the whole polypeptide chain. Mass spectrometric analysis showed a molecular mass of 49,698 daltons for the electrophoretically homogeneous fragment. Twenty-five amino acid residues were sequenced directly from the N-terminus of Rta, which allowed the location of the domain in the polypeptide chain of the subunit. Physicochemical parameters were determined by absorption and fluorescence spectroscopy and circular dichroism. Comparison with the respective parameters of the whole Rapana hemocyanin showed that the polypeptide backbone folding, binuclear active site and capability of oxygen binding of the isolated functional unit are identical to those of the native hemocyanin. Comparison of N-terminal sequences of functional units from different molluskan hemocyanins and located at different positions revealed some evolutionary relationships.

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.

Photoreactivity of histidyl residues in subtilisins Novo and DY. Photooxidation of subtilisins.

Subtilisins Novo and DY were photoinactivated in the presence of methylene blue according to first order kinetics. The competitive inhibitor N alpha-benzoyl-L-arginine protected significantly against inactivation. Under the conditions employed in this study a selective photooxidation of the active site histidine 64 was achieved. Rate constants of 0.32 X 10(-2), s-1 and 0.35 X 10(-2), s-1, were calculated for the Novo enzyme and subtilisin DY, respectively. Apparent pKa values of the catalytically important imidazole group of 7.0 +/- 0.1 (s. Novo) and 7.1 +/- 0.1 (s. DY) were directly determined. The histidyl residues in the two proteases, except the active site histidine, which is the first target of photooxidation, are "buried" in the interior of the protein globule. Conformational studies suggested that the photoreactive histidine is not involved in the stabilization of the protein conformation.

Modification of arginine residues in subtilisins Novo and Carlsberg. Effect on the protein structure and enzymatic activity.

The modification of arginine 186 and arginine 247 in subtilisin Novo as well as the four guanidino groups in subtilisin Carlsberg decreased the catalytic activity. The inactivation proceeded by 60-70% toward casein and by 80% toward p-nitrophenyl acetate during 4 h of incubation with glyoxal. No decrease in the lysyl content was found. The modification had little effect on the fluorescence and circular dichroism properties of the two subtilisins. It was deduced that the inactivation of subtilisins was due to changes in the catalytically active conformation of the active sites, induced by the modification of the arginyl residues. The role of guanidino groups in structure and function of the subtilisins Novo, Carlsberg, DY and mesentericopeptidase is quite similar.