Influence of limited proteolysis, detergent treatment and lyophilization on the phenoloxidase activity of Rapana thomasiana hemocyanin.

The intrinsic and inducible phenoloxidase (PO) activity of Rapana thomasiana hemocyanin (RtH) and its substructures were studied. With catechol as substrate, a weak o-diPO activity was measured for the didecameric RtH and its subunits. Some activation of the o-diPO activity of RtH was achieved by limited treatment with subtilisin and by incubation of RtH with 2.

Involvement of glycans in the immunological cross-reaction between alpha-macroglobulin and hemocyanin of the gastropod Helix pomatia.

By tandem-crossed immunoelectrophoresis and ELISA experiments an immunological relationship was observed between alpha-macroglobulin (alphaM) and hemocyanin (Hc) of the terrestrial snail Helix pomatia. Both glycoproteins occur in the hemolymph: alphaM (minor component) as a specific proteinase inhibitor, Hc (consisting of three components: alpha(D)-HpH, alpha(N)-HpH and beta-HpH) as oxygen transport protein. The cross-reaction was found to be correlated with glycosylation.

Involvement of glycan chains in the antigenicity of Rapana thomasiana hemocyanin.

Functional unit (FU) RtH2-e from Rapana thomasiana hemocyanin (Hc) was degraded into small fragments with chymotrypsin. The glycopeptides were separated from the non-glycosylated peptides by chromatography on Concanavalin-A-Sepharose and characterized by mass spectrometry. The glycan part of the glycopeptides (all with common peptide stretch of 14 amino acids) consists of the classical trimannosyl-N,N-diacetylchitobiose core for N-glycosylation, predominantly extended with a unique tetrasaccharide that is branched on fucose.

Conformational stabilization at the active site of molluskan (Rapana thomasiana) hemocyanin by a cysteine-histidine thioether bridge A study by mass spectrometry and molecular modeling.

In some type-3 copper proteins (molluskan hemocyanin, catechol oxidase and fungal tyrosinase) one of the histidine residues, liganding the Cu(A) atom of the dinuclear copper active site, is covalently linked to a cysteine residue by a thioether bridge. The purpose of this study was to disclose the function of this bridge. Mass spectral analysis of a peptide, isolated from Rapana thomasiana (gastropodan mollusk) hemocyanin, indicated a stabilization of the peptide structure in the region of the bridge.

Location of intrinsic and inducible phenoloxidase activity in molluscan hemocyanin.

The phenoloxidase (PO) activity of the hemocyanins (Hcs) from two molluscan species, the gastropod Helix pomatia (Hp) and the cephalopod Sepia officinalis (So), was studied. With catechol as substrate the Hcs showed a weak o-diPO activity, which was moderately enhanced on limited proteolysis with subtilisin. The sites in the Hc molecules mainly responsible for this activity were identified.

Fluorescence properties and conformational stability of the beta-hemocyanin of Helix pomatia.

The beta-hemocyanin (beta-HpH) is one of the three dioxygen-binding proteins found freely dissolved in the hemolymph of the gastropodan mollusc Helix pomatia. The didecameric molecule (molecular mass 9 MDa) is built up of only one type of subunits. The fluorescence properties of the oxygenated and apo-form (copper-deprived) of the didecamer and its subunits were characterized.

Mass spectral evidence for N-glycans with branching on fucose in a molluscan hemocyanin.

Glycopeptides, isolated from a trypsinolysate of functional unit (FU) RtH2-e of Rapana thomasiana hemocyanin subunit 2, were analysed by electrospray ionization mass spectrometry and MS/MS. From the molecular mass observed after deglycosylation, it was inferred that all glycopeptides shared the same peptide stretch 92-143 of FU RtH2-e with a glycosylation site at Asn-127. Besides the core structure Man(3)GlcNAc(2) for N-glycosylation, structures with a supplementary GlcNAc linked to either the Man(alpha1-3) or the Man(alpha1-6) arm and/or an additional tetrasaccharide unit connected to the other Man arm were observed, indicating the existence of microheterogeneity at the glycan level.