No abstract

In functional units d and g from the P,-haemocyanin of the gastropod Helix pornatia, aminoacid analysis in the presence of dimethyl sulfoxide showed the occurrence of seven cysteine residues. Titration with 5,5'-dithiobis(2-nitrobenzoic acid), however, did not reveal any free thiol group. Pepsinolysis at pH 2.0 followed by amino acid analysis and partial sequencing of the cysteine-containing peptides showed that six cysteine residues are involved in the formation of three disulfide bridges at corresponding positions. The results indicated that the remaining cysteine residue is linked by a thioether bridge to a histidine residue located two positions further in the sequence (H. pomatia d m i s 6 2 ; H. pomatiu g m H i s 6 8 ) . This residue corresponds to one of the three histidine residues considered to be involved in the coordination of the copper A atom of the dinuclear copper group of the functional units.The presence of the thioether bond was further evidenced by an absorption band at 255 nm and by molecular mass determinations with electrospray mass spectrometry on a peptic peptide from H. pomatiu d and on peptides obtained by proteolysis of carboxymethylated H. pomutiu d with trypsin and pronase. Upon sequence analysis the cysteine-histidine thioether bridge was cleaved into didehydroalanine (polymers) and 2-thiolhistidine.A peptide with a cysteine-histidine thioether bridge was isolated from pepsinolysates of functional units c, e, ,f, g and h of Sepia officinalis and unit g of Octopus vulgaris, obtained from haemocyanin of the cephalopods S. ofiicinalis and 0. vulgaris. A cysteine-histidine thioether bridge, which was reported previously for tyrosinase of Neumsporu crassa, thus seems to be a general feature for functional units of molluscan haemocyanins.