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The carbohydrate content and composition of hemocyanins (Hcs) of three prosobranchs (gastropods), Rapana thomasiana, Megathura crenulata and Haliotis tuberculata, were compared. The analyses were performed by gas-liquid chromatography after methanolysis, re-N-acetylation and trimethylsilylation. The two structural subunits of R. thomasiana Hc, RtH1 and RtH2, both showed 2.6% (w/w) carbohydrate content with very similar monosaccharide composition, indicative for N-glycosylation. The two isoforms of M. crenulata Hc (KLH), KLH1 and KLH2, on the other hand, definitely differed in glycosylation: KLH2 (3.4% carbohydrate, w/w) comprised relatively less mannose and more N-acetylgalactosamine than KLH1 (3.0% carbohydrate, w/w), in agreement with the fact that O-glycosylation has been observed in a functional unit (FU) of KLH2. For the Hc of the abalone H. tuberculata, with 4.5% (w/w) carbohydrate, appreciable amounts of 3-O-methyl-d-mannose and 3-O-methyl-d-galactose were detected, showing that the occurrence of methylated sugars is not restricted to the Hcs of pulmonates. From the structural subunit RtH2 of Rapana Hc the FUs RtH2-b and RtH2-d were isolated. On the basis of amino acid sequence analysis and matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) of the respective native and PNGase-F-treated glycopeptides, one N-glycosylation site was found for each FU. This site was located at Asn-405 for RtH2-b and at Asn-394 for RtH2-d; the carbohydrate moiety corresponded to GlcNAc2Man6 and GlcNAc2Man5, respectively. A comparison was made with the N-glycosylation sites of other FUs of Rapana Hc.

The hemocyanin (Hc) from Buthus sindicus, studied in the native state, demonstrated to be an aggregate of eight different types of subunits arranged in four cubic hexamers. Both, the 'top' and the 'side' views of the native molecule have been identified from the negatively stained specimens using transmission electron microscopy. Out of these, eight different polypeptide chains, the partial primary structure (68%) of a subunit Bsin1 (Mr = 72422.7 Da) was established using a combination of automated Edman degradation and mass spectrometry. A multiple sequence alignment with other closely related cheliceratan Hc subunits revealed average identities of ca. 60%. Most of the structurally important residues, i.e. copper and calcium-binding ligands, as well as the residues involved in the presumed oxygen entrance pathway, proved to be strictly conserved in Bsin1. Sequence variations have been observed around the functionally important chloride-binding site, not only for the B. sindicus subunit Bsin1, but also for the subunit Aaus-6 of the scorpion A. australis and the subunit Ecal-a from the spider Eurypelma californicum Hcs. Deviation in the primary structure related to the chloride-binding site suggest that the effect of chloride ions may vary in different hemocyanins. Furthermore, the secondary structural contents of the Hc subunit Bsin1 were determined by circular dichroism revealing ca. 33% alpha-helix, 18%, beta-sheet, 19% beta-turn, and 30% random coil composition. These values are in good agreement with the crystal structure of the closely related Hc subunit Lpol-II from horseshoe crab L. polyphemus. Electron microscopic studies of the purified Hc subunit under native conditions revealed that Bsin1 has self aggregation properties. Results of these studies are discussed.

A neutral proteinase (NPS) was purified from the culture broth of Saccharomonospora canescens sp. novus, strain 5, using DEAE cellulose and a POROS HQ/M 4.6 x 100 mm column. The stability towards thermal and chemical (guanidine hydrochloride, Gdn.HCl) denaturation of NPS was investigated by kinetic and equilibrium studies. The unfolding processes were monitored by circular dichroism and fluorescence spectroscopy. The free energy of stabilization in water was calculated to be 2.1 kcal mol-1. The thermostability was determined by the critical temperature Tc from fluorescence measurements (69 degrees C) and the melting temperature Tm (70 degrees C) from (1) measurements. Quenching with acrylamide, iodide and cesium gives information about the microenvironment of intrinsic protein fluorophores. The Ksv constant for NPS is 4.6 and classifies the emitting tryptophans as 'buried' in the hydrophobic interior of the investigated protein.

Fourteen structural subunits of hemocyanins from two different infraorders of crustacea, brachyura (Maia squinado, Carcinus maenas) and astacidea (Homarus americanus) were isolated and characterized. N-terminal amino acid sequences were determined and compared with known sequences of crustacean and cheliceratan hemocyanins. Relationships between the investigated polypeptide chains were established. The results demonstrate that the degree of identity, calculated from the amino terminal sequences, is lower than that determined from the complete sequences and can be used for reliable characterization of the whole individual subunits. Independent evolution is possible not only for members of different subphyla, but also for those from one infraorder or from the same hemocyanin.

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.

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.

The hemocyanin of Rapana thomasiana grosse (marine snail, gastropod) is a glycoprotein with a carbohydrate content of 8.9% (w/w) and monosaccharide constituents xylose, fucose, 3-O-methylgalactose, mannose, galactose, N-acetylgalactosamine and N-acetylglucosamine residues. The two structural subunits of this oxygen carrier, RHSS1 and RHSS2, are unevenly glycosylated. On subtracting the carbohydrate contribution from the M(r) values of 250 and 450 kDa attributed to the two subunits, values of 2.18 x 10(5) daltons and 4.30 x 10(5) daltons were calculated for the polypeptide part of the "light" and "heavy" subunits, respectively. Comparison of the monosaccharide compositions of gastropodan hemocyanins revealed qualitative similarities, as well as relationships between the quantities, of the individual monosaccharides: Man > or = 3MeGal > GlcNAc > or = GalNAc and Fuc > or = Xyl.